Cell-Mediated Immunity and Serum Blocking Factors in Cancer Patients During Chemotherapy and Immunotherapy1

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[CANCER RESEARCH 37, 2473-2480, August 1977] Cell-mediated Immunity and Serum Blocking Factors in Cancer Patients during Chemotherapy and Immunotherapy1

Frances P. Noonan, William J. Halliday, Darryl R. Wall, and Gordon J. A. Clunie2

Departments of Microbiology (F. P. N., W. J. H.J and Surgery (D. A. W., G. J. A. C.), University of Queensland. Brisbane, Australia

SUMMARY creasing humoral antibodies that were antagonistic to cell mediated reactions (11, 13, 27). Systemically administered The leukocyte adherence inhibition test was used to mon C. parvum, although a potent reticuloendothelial stimulant itor tumor-specific cell-mediated immunity in 15 patients 19) and an effective antitumor agent in animals (37) and hu who had a variety of malignant tumors and were undergoing mans(25),depressedT-ceIl-dependentimmunity (4, 22) and chemotherapy alone or in combination with immunotherapy had an adjuvant effect on non-thymus-derived lymphocyte by Corynebacterium parvum. function (19, 22). The route of administration appears to be A rapid and prolonged loss of cell-mediated immunity in important in demonstrating these effects (33). blood leukocytes was observed after treatment in all but one It is obvious, therefore, that agents used in cancer ther of the patients studied. Abolition of reactivity was due to the apy may have diverse effects, some of them undesirable. lack of production of the soluble lymphokmne-like factor There is thus a clear need to monitor both cell-mediated affecting leukocyte adherence to glass. A new phenomenon immunity and humoral factors that may interfere with this of adherence stimulation by antigen, also mediated by a immunity, particularly as related to tumor antigens, in pa soluble factor, was observed after treatment in some pa tients undergoing chemotherapy or immunotherapy. tients. Immune responses related to tumor-associated antigens A drop in titer or total abrogation of serum blocking have previously been monitored during therapy, and in factors occurred in six of six patients tested following some cases correlations have been found between in vitro chemotherapy or immunotherapy. The lowered levels of determinations and clinical status (10, 28, 31, 34). However, blocking activity persisted during treatment and, with the the technique of lymphocyte cytotoxicity used in such in possible exception of one patient, were not correlated with vestigations has been criticized for lack of specificity, and clinical benefit. data obtained from individual patients may be difficult to interpret(7,20). In this study, tumor-specific parameters of immunity were INTRODUCTION monitored with the LAI test during the course of chemother apy and immunotherapy in 9 patients with melanoma, 4 Considerable interest has recently been expressed in the patients with colon carcinoma, 1 patient with gastric carci combination of chemotherapy and immunotherapy (usually noma, and 1 patient with osteogenic sarcoma. The LAI test referred to as chemoimmunotherapy) in the treatment of has previously been used to determine specific cell-me malignant tumors in man (16, 23, 33). Combinations of diated immunity in blood leukocytes and specific serum chemotherapeutic agents with an immunostimulant such as blockingfactorsina varietyofhuman tumors (17,18). Bacillus Calmette-GuÃ©rin,C. parvum, or levamisole are su perior to treatment with either alone (6, 9, 15, 23, 24, 32). All drugs used in chemotherapy exert at least some immu MATERIALS AND METHODS nosuppressiveeffect (1). However,such drug-induced mod ulations of the immune response may be selective in experi Patients mental animals. For example, cyclophosphamide de pressed anti-tumor-blocking factors in serum but not antitu Details of the patients are recorded in Table 1, together mor cytotoxicity of lymph node cells or peripheral blood with the treatments administered. These patients were leukocytes (36). Cyclophosphamide, 1-f3-D-arabinofurano tested on an average of 4 to 5 occasions, and selected data sylcytosine, and a combination of 5FU3and methotrexate arepresentedbelow. enhanced delayed-type hypersensitivity responses by de

LAI Test I This investigation was supported by the Anti-Cancer Council of the Queensland Cancer Fund and the Cancer Research Fund of the University of Queensland. The methods are based on those previously published a To whom requests for reprints should be addressed, at Department of Surgery, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, (17, 18, 29). Australia. Leukocytes. Twenty ml of blood were collected by veni @ The abbreviations used are: 5FU, 5-fluorouracil; T-cell, thymus-derived puncture; 15 ml were mixed with approximately 500 units of lymphocyte; LAI, leukocyte adherence inhibition; DTIC, imidazole-4-carbox amide. 5-(3,3-dimethyl-1 -triazeno); i a., intraarterial. heparmn in a glass bottle and allowed to sediment under Received December 6, 1976; accepted April 28, 1977. gravity at 37Â°.The leukocyte-rich plasma was removed by

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Table1 givenAgePretreatment---â€”â€”Details of patientsstudied and treatments condi PahentSex(yr)TumorPrevioustherapytionTherapyResponseJ. mos.T.A.M24MelanomaWide excision and parvum; 5FUNil; died after 3 block dissectionDisseminateddiseaseC. H.M35MelanomaWide excision and MelphalanPartial regression; alive at mos.K. block dissectionDisseminateddiseaseDT1C; 5 M.M27MelanomaWide excision and parvum; 5FU; regression with block dissectionDisseminateddiseaseC. DTICPartial later extension; alive at 8 mos.C. 0.F31MelanomaWide excision; change in lesions; alive mos.F. craniotomyDisseminated diseaseDTICNo at 5 mos.E.R.F55MelanomaWide excision and parvum; DTIC; died after 5 block dissectionDisseminateddiseaseC. cyclophospha mideNil; S.M59MelanomaWide excision and parvum; DTIC; regression, but later block dissection; diseaseC. MelphalanInitial extension; died after 11 mos.J. amputationDisseminated W.M28MelanomaWide excision and parvum; DTIC; regression, but later block dissectionDisseminateddiseaseC. radiotherapyInitial extension; alive at 8 mos.A. mos.B.Y.M69MelanomaWide excision and parvumDied after 6 block dissectionDisseminateddiseaseC. moV.R.M23MelanomaWide excision and parvum; DTICWell at 1 block dissectionDisseminateddiseaseC. B. i.p. died after 2 mos. of colon spread G.mos.M. C.F F64 49CarcinomaCarcinoma LaparotomyExtensive Disseminated 5FUNil; Nil; died after 4 of colonHemicolectomy disease5FU H.F54Carcinoma parvum; 5FUInitial regression, but later of colonDisseminated diseaseC. extension; died after 8 mos.D. T.M39Carcinoma hemicolectomyPeritoneal spread5FU? complete regression; mos.P. of alive at 9 appendixRight mos.D.T.M70Carcinoma parvum; 5FUNil; died after 3 of diseaseC. stomachDisseminated C.F13Osteogenic apparent at 6 mos.; free from sarcomaAmputationNo metastasisMethotrexateWell disease

Pasteur pipet into plastic tubes and centrifuged at 200 x g tracts, 1 extract of gastric cancer, and 1 extract of osteo for 5 mm. The cell pellet was suspended in 1 ml of 0.15 M genic sarcoma were used in this study. LAI reactivity was NH4CIat room temperature for 5 mm to lyse erythrocytes; 4 monitored by testing leukocytes against extracts of their ml of Eagle's basal medium, containing 10% fetal calf serum own tumor type, but autologous systemswere not used. and 0.28 mg of sodium bicarbonate (Commonwealth Serum Method of Direct LAI Test. Duplicate mixtures were set Laboratories, Melbourne, Australia) per ml, were then up as follows in plastic tubes: control, 0.10 ml (1 x 106cells) added, and the cells were washed twice by centrifugation. of leukocyte preparation, 0.05 ml of medium, and 0.05 ml of The final cell concentration was adjusted to 1 x 10@leuko normal serum; reactivity, 0.10 ml of leukocyte preparation, cytes/mI. 0.05 ml of tumor extract (diluted 1/5 in medium), and 0.05 Serum. The remaining 5 ml of blood were allowed to clot, ml of normal serum; blocking, 0.10 ml of leukocyte prepara and the serum was collected. Serum samples were kept at tion, 0.05 ml oftumor extract (diluted as above), and 0.05 ml â€”20Â°.Controlsera were obtained from healthy laboratory oftestserum. staff members. The tubes were incubated with intermittent shaking for 30 Tumor Extracts (Antigens). Tumor tissue, either fresh or mm at37Â°ina waterbath.The mixtureswerethen intro stored at â€”50Â°,waschopped finely, passed through a stain duced with a Pasteur pipet into hemacytometer chambers less steel mesh, and homogenized in 4 volumes of cold and incubated in a humid atmosphere for 1 hr at 37Â°. phosphate-buffered saline (equal parts of 0.9% NaCI solu Method of Indirect LAI Test. The mixtures were made up tion and 0.15 M potassium phosphate solution, pH 7.4) in a as for the direct LAItest, but at the end of the 1Stincubation motor-driven glass homogenizer (B. Braun Apparatebau, the tubes were centrifuged (1000 x g for 5 mm). The super Melsungen, West Germany). The homogenate was centri natants were removed and stored at â€”20Â°.Fordetermina fuged in the cold, first at 1,000 x g for 30 mm and then at tion of the activity of these supernatants, mixtures of 0.1 ml 20,000 x g for 30 mm. The resulting clear supernatant was of supernatant and 0.1 ml (1 x 106cells) of a suspension of frozen and thawed, briefly centrifuged, and stored in 0.1-mi normal mouse peritoneal cells were incubated in plastic aliquots. Six melanoma extracts, 3 colon carcinoma ex tubes at 37Â°for 30 mm. These mixtures were then intro

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. Tumor !mmunity during Chemoimmunotherapy duced into hemacytometers as for the direct LAI test. It was Laboratories, West Haven, Conn.), 3 mg/kg/day i.v. for 5 necessary to assay the supernatants within 2 weeks of prep days, repeated at monthly intervals; cyclophosphamide aration and not to freeze and thaw them more than once if (Mead Johnson, Crow's Nest, Sydney, Australia), 50 mg p.o. activity was to be retained. daily; methotrexate (Lederle Laboratories, Pearl River, Counting. Cells were counted in a pattern of 5 squares in N.Y.), 500 mg of methotrexate i.v. over 16 hr, then leucovo the RBC area on each side of the hemacytometer, with a nfl (Lederle), 100 mg i.v. initially, followed by 6 doses of 15 total of 20 squares for each determination. The coverslip mg i.v. every 5 hr; melphalan (Burroughs Weilcome, Rose was then floated off the slide by slowly immersing it in a 15- bery, Sydney, Australia), i.a. perfusion of 75 to 100 mg over cm 0.9% NaCI solution-filled Petri dish. The hemacytometer 1 hr; C. parvum (killed vaccine; Wellcome Research Labora was removed from the dish and held vertically by 1 hand tories, Beckenham, England), initial dose of 21 mg i.v., while being immersed in a 200-mi beaker of 0.9% NaCi followed at monthly intervals by 7 mg i.m. solution. It was then removed, inverted, reimmersed, re moved, and placed on the bench; a drop of 0.9% NaCI RESULTS solution was placed on each counting area, and a coverslip was gently lowered into place. The remaining adherent cells None of the tumor extractsreducedthe adherence of were counted in the samesquares as for the 1st count. The leukocytes from normal individuals or from patients with mean percentage of adherence (Â±SE.) for pairs of hema unrelated conditions (Table 2). cytometers was calculated from the percentage of adher Ten patients were tested prior to treatment, and all were ence of each of the 20 squares. Student's t test was used to reactive in the LAI test (Table 3). Two patients (G. C. and T. obtain the degree of significance of differences between H.) were tested only after a preliminary course of treatment means. but were also reactive at that stage. Reactive (+) leukocytes had a significantly lower adher The 3 remaining patients (C. 0., tested on the 1st day of a ence (p < 0.05) in the presence of antigen extract and 2nd course of DTIC; P. T., tested on the 3rd day of a 1st normal serum than in control mixtures without antigen. course of 5FU; and M. H., tested on the 4th day of a 1st Nonreactive (0) leukocytes showed an adherence not sig course of 5FU) were not reactive and are thus not included nificantly different in the presence or absence of antigen, inTable3. with normal serum. Alteration of LAI Reactivity by Chemoimmunotherapy. A Adherence stimulation (â€”)was recorded when the leuko change from LAI reactivity in patients' leukocytes to either cyte adherence in the presence of antigen increased sign ifi nonreactivity or adherence stimulation could be effected by cantly compared to the control value. C. parvum administered either i.v. or i.m. and by DTIC, 5FU, Serum blocking was determined by comparing adher methotrexate, or melphalan perfusion, as shown in Table 3. ences in the presence of antigen and normal serum and in All patients were tested repeatedly during treatment (a the presence of antigen and test serum. A significantly total of 78 direct LAI tests). In 14 of 15 patients, a pattern of greater adherence in the presence of test serum indicated rapid and prolonged abolition of LAI reactivity following the presence of serum blocking factors. Blocking was not treatment was established. Only 4 showed recovery of Ieu usually measured with autologous cells. kocyte activity during ongoing treatment; 3 of these epi Blocking titers were determined by testing 4-fold dilutions sodesoccurred following DTICtreatment (PatientsJ. W., E. oftest serum, diluted in normal serum. The highest dilution S. , and F. A.), and the other (Patient V. B.) occurred 5 days giving significant blocking was designated the blocking after commencement of 5FU treatment. In 1 patient (J. A.) titer. Specificity of blocking activity in sera was determined there was no loss of reactivity following treatment with C. with leukocytes and antigens from patients with unrelated parvum and 5FU. tumors. Adherence stimulation occurred in the leukocytes of both Coding of Samples. All LAI tests, including direct, mdi chemotherapy and immunotherapy patients, most fre rect, and blocking assays, were performed with coded mix quently during or following 5FU treatment (7 instances) and tures. Up to 5 subjects/day were bled. The relevant LAI also following i.v. C. parvum (1 instance), i.m. C. parvum (2 mixtures were prepared, and then each tube was assigned a instances), and DTIC (2 instances). Antigenic specificity was different number according to a random code. All counting not investigated, but the effect appeared to be mediated by was thus done â€œblind,â€•andprocedures, up to the calcula release of a soluble factor, as defined below. tion of mean percentage of adherence, were performed Comparison of Direct and Indirect LAI Tests. Since the without reference to the code. direct LAI test depends on antigenic stimulation of lympho cytes to produce a soluble lymphokmne that diminishes leu Serum Immunoglobulins kocyte adherence to glass (21, 29), direct and indirect LAI tests on leukocytes from 6 patients were performed to de Serum IgG levels were determined using Diffu-Gen immu termine whether the changes were due to alterations in the nodiffusionplates(OxfordLaboratories,FosterCity,Calif.).reactivity of lymphocytes or of adherent cells. The results shown in Table 4 demonstrate a complete correlation be Drug Treatments tween the 2 tests for reactive, nonreactive, and adherence stimulated leukocytes. These results indicate dependence Administration of therapeutic agents was as follows: 5FU on the production or absence of lymphocyte factors and not (Hoffmann-LaRoche, Basel,Switzerland),15mg/kg/day on any deficiency in the adherent cells. i.v. for 5 days, repeated at monthly intervals; DTIC (Dome Reduction in Serum Blocking Activity by Chemoimmu

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. F. P. Noonan et a!. Table2 Lack of reactivity against tumor extracts in LA! of leukocytes from normal donors and from patients with unrelated conditions Comparisonof adherencesin presenceand absenceof antigen showed no significant inhibition. SourceadherenceControlAntigenJ. of Ieukocytes (Patient)Clinical conditionAntigen% 4.5F.R.Normal subjectMelanoma75.8 Â±3.8â€•73.8 Â± 5.6A.N.Normal subjectMelanoma72.5 Â±4.569.3 Â± 3.5P.M.Normal subjectMelanoma58.9 Â±3.954.7 Â± 4.5F.J.Normal subjectMelanoma53.4 Â±4.650.4 Â± 6.2A.N.Normal subjectMelanoma34.8 Â±6.435.5 Â± 3.2F.M.Normal subjectMelanoma36.9 Â±2.742.0 Â± 4.1A.N.Normal subjectMelanoma39.6 Â±4.954.7 Â± 4.1F.V.Normal subjectMelanoma65.9 Â±3.661 .4 Â± 4.1A.N.Normal subjectColon carcinoma72.5 Â±4.564.8 Â± 3.0J.V.Normal subjectColon carcinoma65.9 Â±3.671 .4 Â± 4.2F.R.Normal subjectColon carcinoma75.8 Â±3.881.7 Â± 4.8F.N.Normal subjectColon carcinoma63.2 Â±4.258.6 Â± 5.4A.N.Normal subjectColon carcinoma51 .7 Â±5.345.8 Â± 4.4D.V.Normal subjectOsteogenic Â±3.671.4 Â± sarcoma65.9 3.2A.T.Colon carcinomaStomach Â±3.474.3 Â± carcinoma80.1 3.5M.W.Pigmented basal cell Â±3.756.9 Â± carcinomaMelanoma58.8 5.0H.E.Diverticular diseaseColon carcinoma71 .4 Â±6.264.0 Â± 3.7G.H.Squamous cell carci carcinoma39.6 Â±4.343.7 Â± noma of the anusColon 3.4aS.Giant cell Â±3.560.3 Â± osteoblastomaOsteogenicsarcoma59.6

Mean Â±SE. notherapy. Serum blocking factors were titrated in 6 pa blocking in Patient E. S. 2 days after C. parvum), and the tients. Examples of the titration of blocking factors are lowered or undetectable levels of blocking activity persisted given in Table 5. Pretreatment serum from Patient D. T. even when positive ieukocyte reactivity was temporarily re blocked the reactivity of leukocytes from another colon stored(Table6). carcinoma patient. A 1/4 dilution of this serum also had Serum lgG during Chemoimmunotherapy. Serum lgG significant blocking activity, but a 1/16 dilution did not. The levelsbeforeand duringtreatmentweredeterminedinPa blocking titer was therefore designated 1/4. Identical titers tientsF.R.,D.T.,V.B.,J.W.,A.Y.,J.A.,andE.S.They were obtained for blocking activity when titrations were remained unchanged within the normal range during the repeated on leukocytes from different patients. Sera from courseoftherapy. Patients V. B. and F. R. were each titrated against leuko cytes from 2 donors, with reproducible results for blocking titers. DISCUSSION Serum blocking activity was specific for tumor type. Pre and posttreatment blocking sera from Melanoma Patients The specific inhibition of leukocyte adherence in mice is a E. S. , J. A. , and F. R. did not block the reactivity of leuko T-celI-dependent immunological phenomenon mediated by cytes from a colon carcinoma patient, whose reactivity was the production of a soluble factor with lymphokmne proper blocked by serum from Patient D. T. Similarly, sera from ties (21, 29). Studies in man have revealed a monocyte Colon Carcinoma Patients V. B. and D. T. did not block the relatedreactionleadingtoadherenceinhibitionina test reactivity of leukocytes from a melanoma patient, whose tube modification of the LA1technique (14). In this study we reactivity was, however, blocked by serum from Melanoma have demonstrated depression of specific tumor-directed PatientA.Y. host responses not only by cytotoxic drugs but also by the The results of titrations of blocking activity in 6 patients reticuloendothelial stimulant C. parvum. are shown in Table 6. The pretreatment titers of blocking Nonreactivity or adherence stimulation in the LAI test were not simply a function of tumor mass.PatientsF. R.and could conceivably be caused by lack of suitable iympho E. S. , with larger volumes of tumor, had initial blocking cytes (immunologically specific) or lack of adherent cells titers of 1/1024 and 1/16, respectively, and Patients D. T. (immunologically nonspecific). The disappearance of LAI and J. A., with relatively small volumes of tumor, had initial reactivity was due to the lack of production of the soluble titers of 1/4. However, Patient V. B., who had very extensive LAI factor and not to any deficiency in adherent properties tumor, had a pretreatment titer of 1/1. of the patients' blood leukocytes. Thus, the cells affected in In all patients tested, serum blocking activity decreased this system either directly or indirectly by cytotoxic drugs during treatment (although there was a transient rise in and C. parvum are the lymphocytes. Some of the rapid

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Table3 Alteration of direct LA! reactivity by drug treatment % adherence Days after Patient treatment Control Antigen interpretation parvwml.v.J. C. W. (melanoma)0 Â±1.8@ 56.5Â±18b Reactive 4.7E. 278.4 64.7 Â± 81.9Â±3.1'@ Adherence stimulation S. (melanoma)0 Â±4.2 41.4 Â±4.6c Reactive 4.6A. 258.9 67.4 Â± 58.9Â±4.0 Not reactive Y. (melanoma)0 Â±3.3 47.4Â±30b Reactive 51.4Â±4.6F. 2667.6 57.2 Â±4.4 Not reactive A. (melanoma)0 Â±3.3 54.0Â±4@0b Reactive 3.3B. 3177.0 80.9 Â± 78.3Â±3.3. Not reactive R. (melanoma)0 Â±4.1 61.1 Â±47d Reactive 473.9 78.0 Â±4.4 85.8Â±3.2 Not reactive 5FU D. T.@(colon carcinoma) 0 80.1 Â±3.4 58.8Â±54b Reactive Day 3 of course 77.9 Â±2.8 86.5Â±2.3-' Adherence stimulation G. C. (colon carcinoma) Day 1 of 2nd course 53.9Â±5.0 39.1Â±4.1d Reactive Day5 of2ndcourse 42.9 Â±5.5 78.5Â±2.8* Adherencestimulation V. B. (colon carcinoma) 0 79.5Â±3.4 55.8Â±2.8'@ Reactive Day 2 of course 47.9Â±4.9 58.9Â±4.3 Not reactive Day5 ofcourse 71.5 Â±8.5 54.3Â±4â€¢5d Reactive 30 days after end of 45.6Â±4.6 44.4Â±4.2 Not reactive course 6daysafterendof2nd 59.2 Â±3.4 74.2 Â±4.2c Adherence stimulation course Methotrexate 0. C. (osteogenic 0 69.6Â±5.5 51.9 Â±54d Reactive sarcoma) After methotrexate 80.1 Â±3.7 84.7 Â±2.5 Not reactive After leucovorin 73.8Â±4.0 83.5 Â±2.8 Not reactive 28 days after end of 77.9 Â±4.1 68.9Â±4.6 Not reactive 2nd treatment course Melphalan 1. H. (melanoma) 0 (30 days after 1 42.5 Â±7.8 25.6Â±33d Reactive course of DTIC) 50 41.0 Â±2.6 33.4 Â±4.2 Not reactive

C. parvum and 5FU K.M. (melanoma) 0 58.6Â±6.0 41.2 Â±39d Reactive 102 (after 2 courses of 63.8Â±4.2 61.7 Â±3.8 Not reactive C. parvum; 2 courses of 5FU) J. A. (melanoma) 0 67.1 Â±3.7 51.0 Â±34b Reactive 36 (after2coursesof 76.0 Â±4.6 62.7Â±4.2â€• Reactive C. parvum; 1 course of 5FU) C. parvumi.m. F. R.' 0 80.3 Â±4.8 60.9Â±4.1 Reactive 30 42.1 Â±4.8 43.7 Â±4.7 Not reactive DTIC E.S.' Day1 ofcourse 85.5Â±3.2 67.2 Â±44C Reactive Day5 ofcourse 58.2Â±5.4 77.8 Â±4@5C Adherencestimulation J. W.f 0 89.9Â±2.4 79.8 Â±2.4c Reactive Day5 ofcourse 62.3Â±4.5 62.9 Â±4.6 Notreactive

a Mean Â± SE. lip < 0.001.

C p < 0.01.

d ,@ < 0.05. e Leukocytes from Patient 0. T. (carcinoma of appendix) reacted with extracts of colon carcinoma. I These patients regained reactivity during treatment, which was then abolished as shown.

changes observed in LAI reactivity within 1 to 3 days of curred following administration of DTIC, which lends sup treatmentmay be a reflectionofdepressionofthe total porttoprevioussuggestionsthatDTICisonlyweaklyimmu number ofcirculatinglymphocytes.Threeofthe4 instances nosuppressive (3). of regained LAI reactivity during ongoing treatment oc Previousworkershaveshown thatcertainformsofcancer

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. F. P. Noonan et a!. Table4 Comparison of direct and indirect LA! tests onpatient leukocytes Days after % adherence com mence- Direct Indirect ment of initialPatienttreatment InterpretationE. Control Antigen Control Antigen S. (melanoma)123 81.2 Â±2.6â€• 82.1 Â±2.7 87.6 Â±3.8 77.4 Â±3.8 Not reactive 154 85.5 Â±3.2 67.2 Â±44b 78.2 Â±2.8 58.3 Â±53@) Reactive 158 58.2 Â±5.4 77.8 Â±45b 23.9 Â±2.5 41.5 Â±2.8e Adherence stimulationJ. W. (melanoma)68 71.4 Â±4.2 84.1 Â±2.5â€• 69.3 Â±4.4 90.4 Â±2.3- Adherence stimulation NotreactiveC. 138 71.6Â±3.9 76.6Â±3.9 53.8Â±5.1 56.1Â±6.0 0. (melanoma)34 50.6 Â±3.8 53.8 Â±3.2 66.6 Â±5.7 74.4 Â±4.2 Not reactive reactiveG. 37 40.8Â±4.6 34.5Â±6.3 34.2Â±5.9 44.9Â±5.5 Not Reactivecarcinoma)34C. (colon31 53.9 Â±5.0 39.1 Â±4.5â€• 78.8 Â±3.9 56.6 Â±59b 42.9 Â±5.5 78.5Â±2.9r 19.8 Â±2.6 37.9 Â±3.V Adherence stimulationV. reactivecarcinoma)5B. (colon2 47.9 Â±4.9 58.9 Â±4.4 38.9 Â±4.8 41.3 Â±7.3 Not ReactiveD. 71.5 Â±8.5 54.3Â±4.5â€• 47.2 Â±4.8 33.3 Â±3.5â€• reactivecarcinoma)aT. (colon37 84.8 Â±3.3 78.6 Â±2.9 76.4 Â±2.9 73.8 Â±4.4 Not

SE.bpMean Â± <0.01.Cp <0.001.d ,@ < 0.05.Table 5 activityLeukocyte Effect of blocking serum dilutions on LA!: titration of blocking adherence

Test serum dilutions do Serum TiterA. norâ€•% Control Antigen 1/1 1/4 1/16 donorb 1/4carcinoma)(pS. (colon61.5 Â±7.2 34.7 Â±4.0 76.4 Â±4.6 61.5 Â±5.4 30.8 Â±5.2 D. T. (NS)@A. < 0.01) (p < 0.001)â€• (p < 0.01) 1/1carcinoma)(pC. (colon72.1 Â±4.4 55.1 Â±5.1 84.1 Â±2.8 58.8 Â±4.0 V. B. (NS)V. < 0.05) (p < 0.001) 1/1carcinoma)(p0. (colon75.1 Â±5.2 59.3Â±3.7 78.9Â±3.4 56.8Â±5.3 V. B. (NS)S. < 0.02) (p < 0.01) J. (melanoma)76.2 Â±7.4 36.8 Â±4.8 71.1 Â±4.8 43.0 Â±4.1 F. R. 1/1 (NS)A. (p < 0.001) (p < 0.001) T. (melanoma)62.3 Â±4.8 31.9 Â±2.3 53.1 Â±4.0 40.2 Â±2.9 F. R. 1/1 (p < 0.001) (p < 0.01) (NS) Â°Leukocytesfromdonors not undergoing therapy b Sera from Patients V. B. and F. A. each tested against 2 leukocyte donors.

C Mean Â± SE.

d Significance compared to adherence in the presence of normal serum and antigen. e NS, not significant. therapy may selectively affect in vitro imm unological reac functions unrelated to tumor antigens, e.g. , lymphocyte tions. O'Toole et a!. (31) demonstrated that, following abla blastogenesis (5), delayed hypersensitivity to dinitrochloro tion of tumor-specific lymphocyte reactivity by radiother benzene, and primary humoral response to Vi antigen (3), apy, patients with good prognosis (mostly with local dis were little affected by chemotherapy or chemoimmuno ease) regained reactivity rapidly but patients with poor therapy. prognosis did not. Sinkovics et a!. (34) found that lympho All patients in the present series had disseminated dis cyte cytotoxicity was unaffected by 1-f3-D-arabinofurano ease, and there is evidence that patients with advanced sylcytosine treatment in 3 patients, while Currie (8) reported tumors already have a major depression of immunological that lymphocyte cytotoxicity appeared during immunotherapy reactivity, at least to nonspecific challenge. Since the with irradiated tumor cells, probably due to a diminution in mechanism of loss of LAI reactivity is not clear, in some blocking factors. Sequential assays during DTIC treatment circumstances a measurement of blood-borne cell-me of melanoma patients were reported by Cummings et a!. (7); diated immunity may not reflect reactivity at the tumor site. there was no consistent depression of cell-mediated re C. parvum may cause sequestration of specifically reactive activity or serum blocking factors. Difficulties in interpret lymphocytes in the tumor (4); this mechanism would pre ing microcytotoxicity assay results from individual patients sumably benefit the patient, in spite of apparently reduced were stressed by these same investigators (7, 20). Immune immunoreactivity as judged by in vitro tests.

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Adherence stimulation by antigen, mediated by produc clearance of immune complexes, including blocking fac tion of soluble factor, has not previously been reported in tors. Alternatively, there may be an alteration in the relative the LAI test, but an analogous stimulation of macrophage amounts of circulating tumor antigen and specific antibody migration by a soluble factor has been observed (12). The required to form the blocking factor complex. A sudden biological significance of these observations is not clear. increase in circulating tumor antigens as a result of tumor Serum blocking factors as measured in the LAI test are cell killing without a corresponding increase in specific considered to be immunological in nature. Antigen alone antibody could cause the formation of complexes in antigen does not cause blocking under the conditions used in the excess, which are not blocking. Similarly, either a decrease LAI tests in this study. Although it is possible that blocking in specific antibody production as a result of immuno may be caused by antigen-specific T-cell-dependent sup suppression or an increase in antibody production as was pressive factors (30), data on the specificity of blocking in reported to occur following C. parvum administration (26) the LAI test and on the abolition of blocking activity by could change the composition of antigen-antibody corn antiimmunoglobulin (29) lend support to the concept of plexes and possibly diminish blocking activity. blocking factors as antigen-antibody complexes of a defi The presence of serum blocking activity in LAI tests on nite composition (2, 35). The drop in serum blocking titers cancer patients appears to correlate with the presence of following treatment in the 6 patients tested in this study is in progressing tumors (18). During courses of chemotherapy agreement with the findings in patients treated with 1-f3-D- and immunotherapy, however, this correlation does not arabinofuranosylcytosmne (34) and in patients undergoing always hold. In the 1 patient in this series who has unequiv immunotherapy with irradiated tumor cells (8). ocally benefited from chemotherapy (D. T.), blocking activ There are 2 possible explanations for this fall. Firstly, C. ity was lost rapidly (Table 6). This patient presented 80 days parvum as a reticuloendothelial stimulant may increase the after the suspension of treatment, with LAI ieukocyte reac tivity restored but no serum blocking activity, and 67 days Table 6 later the patient showed neither leukocyte reactivity nor factorsDays Titers of serum blocking serum blocking factors. He has no clinical evidence of aftercom tumor at present. On the other hand, Patients V. B., F. A., ofmence Result and E. S., who obviously maintained large masses of tumor, Blockingmentdirect LAI had no serum blocking activity at varying intervals during ofinitialpatient'softest on titer treatment. In these patients the abolition of serum blocking patient'streatmentleukocytes serum Drug treatment activity is presumably an effect of the treatment itself rather than of tumor removal. (melanoma)0+Patient F. A. In conclusion , chemotherapy or chemoimmunotherapy 1/lO24None310 C.parvum51NTâ€• 1/1 as described in this study resulted in a rapid and prolonged Cyclophosphamide64NT1/4 suppression of antitumor cell-mediated immunity. Serum parvum780 1/4 C. blocking factors were also greatly suppressed or abolished. DTIC82+ 1/1 The clinical results of such therapy must be a balance <1/1bPatient between direct killing of tumor by drugs and the adverse carcinoma)0+ D. T. (colon effects of immunosuppression. Immune mechanisms other None30 1/4 than those determined in the LAI test, e.g., antibody-de 5FU39â€” <1/1 pendent cell-mediated cytotoxicity, direct killing by acti 5FU144+ <1/1 treatment2110 <1/1 80 days after last vated macrophages, and killing by cytotoxic antibodies, treatment3020 <1/1 147daysafter last may also operate in vivo. The complexity of the clinical treatmentPatient<1/1 238daysafter last variables and of the immunological events makes the draw carcinoma)0+ V. B. (colon ing of general conclusions difficult, but it is clear that chem None20 1/1 otherapy or immunotherapy with C. parvum can bring about 5FU5+ <1/1 a depression of 1 aspect of cell-mediated immunity, as 5FU46â€” <1/1 monitored by an in vitro assay. 5FUPatient <1/1 (melanoma)0+ E. S. None20 1/16 ACKNOWLEDGMENTS parvum31â€” 1/64 C. C.parvum550 <1/1 we thankmanysurgicalandmedicalcolleaguesforcooperationandA. Meiphalan154+ 1/4 Visona and B. Morris for excellent technical assistance. DTIC158â€” <1/1 we aregratefultoDr. A. GriffithsandDr. Z. SolomonofBurroughs

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Frances P. Noonan, William J. Halliday, Darryl R. Wall, et al.

Cancer Res 1977;37:2473-2480.

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