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Inhibition of the phytohaemagglutinin response of normal human lymphocytes by serum from patients with… Lymburner, Kathleen Mary Harper 1982

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INHIBITION OF THE PHYTOHAEMAGGLUTINTN RESPONSE OF NORMAL HUMAN LYMPHOCYTES BY SERUM FROM PATIENTS WITH METASTATIC BREAST CANCER by KATHLEEN MARY HARPER LYMBURNER B.Sc., U n i v e r s i t y of Western On t a r i o , 1968 M.D., U n i v e r s i t y of Western On t a r i o , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1982 "c\ Kathleen Mary Harper Lymburner, 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f fV) [ r.v-o \> \ n\ n. 5rH The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT The e f f e c t of sera from p a t i e n t s w i t h m e t a s t a t i c breast cancer on the response of normal lymphocytes to phytohaemagglutinin (PHA) was studied. A p r e l i m i n a r y experiment i n d i c a t e d that d i a l y s i s of sera against t i s s u e c u l t u r e medium RPMI-1640 revealed d i f f e r e n c e s between p a t i e n t and c o n t r o l sera which were not apparent us i n g undialysed sera. The a b i l i t y of a d i a l y s e d serum sample to support the PHA response of normal lymphocytes, as measured by 3H-thymidine i n c o r p o r a t i o n , was c a l l e d the serum support of lymphocyte s t i m u l a t i o n (SSLS). Sera having an SSLS value l e s s than 30% of the c o n t r o l mean i n the same experiment were c a l l e d i n h i b i t o r y sera. D i a l y s e d sera from a p r o p o r t i o n of p a t i e n t s w i t h m e t a s t a t i c breast cancer on v a r i o u s treatments were found to i n h i b i t the response of normal lymphocytes to PHA compared w i t h c o n t r o l sera. This e f f e c t was not due to c y t o t o x i c i t y of the i n h i b i t o r y sera. The SSLS of i n h i b i t - . ory sera remained lower than that of most c o n t r o l s when experimental c o n d i t i o n s such as co n c e n t r a t i o n of reagents and d u r a t i o n of in c u b a t i o n were v a r i e d . The a b i l i t y of undialysed sera to support a mixed lymphocyte r e a c t i o n was s i g n i f i c a n t l y c o r r e l a t e d w i t h the SSLS of the same sera when d i a l y s e d . The presence of i n h i b i t o r y sera was apparently r e l a t e d mainly to treatment. In the e a r l i e r (Phase 1) experiments, treatment w i t h chemo-therapy or oestrogens was as s o c i a t e d w i t h i n h i b i t o r y s e r a , whereas treatment w i t h androgens or c o r t i c o s t e r o i d s was ass o c i a t e d w i t h sera which supported PHA s t i m u l a t i o n w e l l . In l a t e r (Phase 2) experiments, there was a higher p r o p o r t i o n of persons w i t h i n h i b i t o r y sera among i i i patients receiving one or both of the newer hormonal agents, Tamoxifen and Aminoglutethimide, than among controls or untreated patients. In the phase 1 experiment, advanced disease and disease °of long duration appeared to be associated with i n h i b i t o r y sera, but these associations were not confirmed i n Phase 2. Higher patient age i n both phases and greater tumour d i f f e r e n t i a t i o n i n Phase 1 were associated with i n h i b i t o r y sera, but i n Phase 1, the presence of i n h i b i t o r y sera was unrelated to prognosis. Prognosis and tumour d i f f e r e n t i a t i o n were not studied i n Phase 2. Lymphocytes from breast, cancer patients responded to PHA s i g n i f i c -antly less well than did control lymphocytes. However, there was no correlation between the PHA response of patients' lymphocytes and the SSLS of sera from the same patients. A low molecular weight (10,000 dalton) i n h i b i t o r y substance appeared to be present i n a pool of two very i n h i b i t o r y patients' sera i n much greater concentration than i n control sera. However, no similar sub-stance could be demonstrated i n other less i n h i b i t o r y sera. Measurement of several serum proteins revealed a borderline s i g -n i f i c a n t negative l i n e a r relationship between the serum concentration of a i - a n t i t r y p s i n and the logarithm of the SSLS. V a r i a b i l i t y i n the concentration of a i - a n t i t r y p s i n could account for less than 10% of the v a r i a b i l i t y i n log SSLS. Thus, apparently more than one substance i s involved i n determining SSLS. iv TABLE OF CONTENTS Page ABSTRACT . i i LIST OF TABLES x' LIST OF FIGURES. . x i i i LIST OF ABBREVIATIONS . x i v ACKNOWLEDGEMENT S x v i i CHAPTER I - INTRODUCTION 1 I. AIMS OF THE RESEARCH. . . . . . . . . . I I I . BIOLOGY AND TREATMENT OF HUMAN BREAST CANCER... 2 1. S t a t i s t i c s 2 2. Pathology 2 3. Sites of Metastases 3 4. Staging 3 5. Factors Determining Prognosis.... 4 6. Treatment 9 I I I . DETECTION AND MECHANISMS OF ANTI-TUMOUR IMMUNITY 16 A. Cell-mediated Immunity to Tumours 17 1. Cy t o t o x i c i t y Assays 17 2. Leukocyte Migration I n h i b i t i o n Tests 34 3. Leukocyte Adherence I n h i b i t i o n Tests 37 4. Others 43 B. The Role of Humoral Antibody i n Tumour Immunity. 43 IV. LYMPHOCYTE STIMULATION BY PHYTOHAEMAGGLUTININ.......... 46 V Page V. THE IMPORTANCE OF THE IMMUNE RESPONSE TO BREAST CANCER.., 48 VI , IMMUNE COMPETENCE IN BREAST CANCER P A T I E N T S , 5 2 V I I , TMMUNOSUPPRESSIVE SUBSTANCES IN SERUM AND PLASMA 54 A. Evidence For- and Against Serum and Plasma Immuno-suppressive Factors i n Cancer.............. 54 1, Immunosuppression by Serum or Plasma i n Cancers Other Than Breast C a n c e r . 5 4 2, Immunosuppression by Serum or Plasma i n Human Breast Cancer ........................... 57 B. The Chemical Nature of. Tmmurioregulatory Substances i n Serum and Plasma......................................... 59 1. Normal Serum Components w i t h A l t e r e d Concentrations i n Cancer. 60 2. Abnormal Substances i n Serum or Plasma of Cancer PatxentS' ..,,.•,••,..•••,•.••,»••..,<•,#,*. 70 3. I n t e r a c t i o n of PHA w i t h Serum P r o t e i n s , 7 7 4. Summary ............................ r 77 VIT.I. THE EFFECT OF SEX HORMONES ON THE IMMUNE SYSTEM , 78 A. Normal PlasmaLLevelsrarid"-Transport of- O e s t r o g e n s and Androgens , 78 B. Sex D i f f e r e n c e s i n Immune Responses 79 C. E f f e c t s of C a s t r a t i o n and Sex Hormone A d m i n i s t r a t i o n on Immune Responses, . , 82 1. E f f e c t s of Sex Hormones on the Development or Recovery of Immune Competence, 82 2. E f f e c t s of Oestrogen A d m i n i s t r a t i o n on the Mature Immune System., 83 3. E f f e c t s of Androgen A d m i n i s t r a t i o n on the Mature Immune System,............... 86 v i Page D, In V i t r o E f f e c t s of Sex Hormones,,,,.,,,,,,«,.,,,,,.,. 89 1, E f f e c t s of Qestrogens i n V i t r o . 8 9 2, E f f e c t s ©f Androgens i n V i t r o 9 1 E, E f f e c t of Sex Hormones on Serum P r o t e i n L e v e l s . , , , , , . , 91 1, E f f e c t s of Oestrogens on Serum P r o t e i n L e v e l s , , , , , , 91 2. E f f e c t s of Androgens on Serum P r o t e i n L e v e l s . , , . , , . 94 F, E f f e c t s of Hormonal Treatment of Breast Cancer on Immune Responses .......................... 96 1 CHAPTER I I - THE EFFECT OF VARYING EXPERIMENTAL CONDITIONS ON THE RESPONSE OF NORMAL LYMPHOCYTES TO PHA IN CONTROL AND BREAST CANCER PATIENTS1' SERA. ,,,,,,, 97 I. INTRODUCTION' f , • • ^ • • ^ • t t * * r « * * . 97 I I . MATERIALS AND METHODS,,,,.,..,,.«.,,,,,,,.,,,«,,«,,.,, 97 1 • 1?3.t XGxifc s~ • • «. > •«t • • • f 11 97 2, C o n t r o l s 9 8 3, Normal Lymphocyte. D o n o r s . 9 9 4, P r e p a r a t i o n of S e r a " . 9 9 5, P r e p a r a t i o n of Leukocytes and Lymphocytes,,,,,,,,,. 100 6. Lymphocyte S t i m u l a t i o n w i t h P H A . 1 0 1 7. Te s t i n g D i a l y s e d Sera f o r C y t o t o x i c i t y Toward Lympho-cytes , . . , , « 104 8. Mixed Lymphocyte R e a c t i o n 1 0 4 9. S t a t i s t i c a l M e t h o d s . 1 0 5 :IIT. RESULTS . . , 106 1, Determination of the Duration of Incubation to be Employed Routin e l y i n Lymphocyte S t i m u l a t i o n Exp&i?xtn6.n t s • •«••*•• « • # • • • • • ( • * • • • • • • « • • • • • 106 2. P r e l i m i n a r y Experiments Suggesting the Value of Using D i a l y s e d Serum. , , , . . 108 v i i Page 3. Determination of the PHA Concentration and the Number of Lymphocytes per Well to be Employed Ro u t i n e l y i n Lymphocyte S t i m u l a t i o n Experiments , 108 4. Background 3H-TdR I n c o r p o r a t i o n i n t o Lymphocytes i n the Absence of PHA,., , 110 5. E f f e c t of P a t i e n t s ' D i a l y s e d Sera on the Response of Normal Lymphocytes to P H A . 1 1 4 6. E f f e c t of I n h i b i t o r y D i a l y s e d Sera on the V i a b i l i t y of Normal Lymphocytes..., 116 7. Dose-Response E f f e c t of D i a l y s e d Sera ............... 116 8. Dose-Response E f f e c t of Undialysed Sera.,,.,..,..,... 119 9. Dose-Response E f f e c t of PHA 123 10. E f f e c t of the Duration of Incubation on SSLS at D i f f e r e n t PHA Concentrations...................... 126 11. R e p r o d u c i b i l i t y of 'Measurements of S S L S t , f t . 131 12. R e l a t i o n s h i p Between Serum Support of Lymphocyte Stimulation.by PHA and i n MLR. . , , 138 13', Response of P a t i e n t s ' Lymphocytes to PHA,,,,,,,,.,,,, 138 IV.". DISCUSSION , 142 CHAPTER I I I - THE EFFECT 0F CLINICAL VARIABLES ON THE ABILITY OF SERUM FROM BREAST CANCER PATIENTS TO SUPPORT NORMAL LYMPHOCYTE STIMULATION BY PHA ,, 146 I . INTRODUCTION ,. 146 I I . MATERIALS AND METHODS , , . , . 146 1. P a t i e n t s . 146 2. C o n t r o l s . 147 3. Normal Lymphocyte D o n o r s . 1 4 7 4. P r e p a r a t i o n of S e r a 1 4 7 5 . P r e p a r a t i o n of Lymphocytes-, . , , t ,..........., 147 v i i i Page 6. Lymphocyte S t i m u l a t i o n w i t h PHA . 147 7. E s t i m a t i o n of Extent of Disease i n P a t i e n t s 148 8. Measurement of Serum C o r t i s o l 148 9. S t a t i s t i c a l Methods -.- 148 I I I . RESULTS 150 1. R e l a t i o n Between C l i n i c a l V a r i a b l e s and SSLS-I 150 2. SSLS f o r P a t i e n t s on Current Hormonal Therapies f o r Breast Cancer Compared w i t h SSLS f o r Controls and P a t i e n t s on No Treatment 158 IV. DISCUSSION. 182 V. SUMMARY. . . . ... .. . . 189 CHAPTER IV - THE CHEMICAL NATURE OF THE IMMUNOSUPPRESSIVE SUBSTANCE(S) IN THE SERA OF SOME BREAST CANCER PATIENTS........ •;.. .... .. .............. 192 I. INTRODUCTION. . . . . . . . .. .. . . ................... 192 I I . MATERIALS AND METHODS. . . . 192 1. P a t i e n t s 192 2. Controls 192 3. Normal Lymphocyte Donors....... 193 4. Pr e p a r a t i o n of Sera f o r SSLS Measurement 193 5. P r e p a r a t i o n of Lymphocytes. 193 6. Lymphocyte S t i m u l a t i o n w i t h PHA.. 193 7. F r a c t i o n a t i o n of Serum on Sephadex G-150 Columns 193 8. - F r a c t i o n a t i o n of Serum by Ammonium Sulphate P r e c i p i t a t i o n . 194 9. I s o l a t i o n of Low Molecular Weight Substances Bound to the SAS F r a c t i o n s . . . . . . . . 196 ix Page 10, Radioiodination of 33% SAS ppt, MW 2,000-10,000 First c t ion • • • • ( • • • • • • • • • t * * * * ( » » » * f f » » » » * f » » » » » » » * » * * » 197 11, Sodium Dodecyl Sulphate Polyacrylamide Gel Eleetrophor^sia C§DS^PAGE) and Autoradiography. , , . . , 198 12, Determination of the Concentration of Serum Proteins, 201 13, S ta t i s t i ca l Methods. , , , , , , . 201 III . RESULTS , , 202 1. Fractionation of Serum on Sephadex G-150 Columns., . , 202 2. Fractionation of Three Serum Samples by Ammonium Sulphate Precipitat ion: I n i t i a l E x p e r i m e n t 2 1 3 3. Fractionation of SAS Fractions from Three Serum Samples by U l t r a f i l t r a t i o n on Amieon Membranes, ,, , . . .'.216 4. SDS-PAGE of Radioiodinated 33% SAS Ppt, MW 2,000-10,000 Fractions from a Normal Serum and Pooled Inhibitory Sera 216 5. Fractionation of Other Sera by SAS P r e c i p i t a t i o n , , , . 220 6. Relationship Between Levels of Certain Serum Proteins and SSLS , . , , , 220 IV, DISCUSSION 224 CHAPTER V- CONCLUDING R E M A R K S 2 2 9 REFERENCES , , , , 231 X LIST OF TABLES Table T i t l e Page I System of C l i n i c a l Staging i n Use at the Cancer Control Agency of B r i t i s h Columbia,................. 5 IT System of Pathological Staging i n Use at the Cancer Control Agency of B r i t i s h Columbia. 6 ITT S u r v i v a l of Patients with Breast Cancer Relative to C l i n i c a l Stage............... 7 TV S u r v i v a l of Patients with Breast Cancer Relative to H i s t o l o g i c Stage........................... 8 V Normal Plasma Levels of 17$-0estradiol and Oestrone i n Various Phases of the Menstrual Cycle i n Women, 80 VI E f f e c t of Duration of Incubation on PHA Stimulation of Normal Lymphocytes i n 10% FCS , 107 VII E f f e c t of PHA Concentration and Number of Lymphocytes per Well on PHA Stimulation of Normal Lymphocytes i n Pooled Dialysed Normal Serum ,,, . 110 VITT SSLS With and Without PHA I l l IX SSLS In Dialysed and Undialysed Sera . . . . 121 X E f f e c t of the Duration of Incubation on SSLS at D i f f e r e n t PHA Concentrations 129 XI E f f e c t of D i f f e r e n t Days and D i f f e r e n t Lymphocyte Donors on SSLS.. , 133 XII; Components of Variance Analysis on' the E f f e c t of D i f f e r e n t Patients' Sera, Days, and Lymphocyte Donors on SSLS , 136 XITT Relation Between SSLS-I i n Dialysed Serum and Serum Support of M L R , , , , r , , r , , , , , , , , « ? * « « * t * e ? t t ? ? t r f t i F « e t f t f t t 139 XIV Relation Between % Lymphocyte Stimulation and SSLS-I of Sera from the Lymphocyte Donor............................ 141 XV Results of Step Up and Down Mu l t i p l e Regression Analysis of SSLS-I and Log SSLS-I on Various C l i n i c a l V a riables,.,, 151 x i Table T i t l e Page XVI Proportion of Patients with Inh i b i t o r y Sera Among Patients with D i f f e r e n t Amounts of Disease , 156 XVIT Comparison of Mean Duration of Periods of Disease f o r Patients with Inh i b i t o r y and Noninhibitory Sera........... 159 XVITI Comparison of Mean SSLS f o r Control and Patient Groups.... 161 XIX Proportion of Inhib i t o r y Sera from Control and Patient Groups , 163 XX C o r t i s o l Levels i n Dialysed Sera and t h e i r Relationrto SSLS 164 XXI E f f e c t of Tamoxifen and Aminoglutethimide of Lymphocyte S t i m u l a t i o n t . . . . . 166 XXIT Mean Age and Serum Storage Time of Controls and Patients... 168 XXITT Proportion of Patients with Inh i b i t o r y Sera Among Patients of D i f f e r e n t Ages on Hormonal Treatments 169 XXIV Proportion of Inhib i t o r y Sera Among Sera Having D i f f e r e n t Storage Times from Patients on Hormonal Treatments......... 172 XXV Proportion of Inhib i t o r y Sera i n Control and Patient Groups Including Only Sera Stored -,%%0.: Weeks 173 XXVI Relation Between Storage Time of Serum and SSLS f or Controlr-Serav.. ,,«.«, 175 XXVIT Relation Between Storage Time of Serum and SSLS f o r Patients' Sera. ., , 176 XXVITT Proportion of Inhib i t o r y Sera Among Patients with D i f f e r e n t Amounts of Disease on Hormonal Treatments,...,, ,179 XXIX Proportion of Inhib i t o r y Sera Among Patients of D i f f e r e n t Ages and with D i f f e r e n t Amounts of Disease on Hormonal Treatments ....... 180 XXX Proportion of Inhib i t o r y Sera Among Group IV Patients with D i f f e r e n t Amounts of Disease on Hormonal Treatments... 181 XXXI Comparison of Duration of Disease for Patients with Inhib i t o r y and Noninhibitory Sera., 183 x i i Table Title Page XXXTT lymphocyte Stimulation in Peaks I, IT, and III of Sephadex G-150 Fractionation: A Preliminary E x p e r i m e n t 2 0 4 XXXIII Ability of Normal Human Serum to Overcome'Mhhib?: . ition by 8% Peak I Material from Sephadex G-150 Fractionation , , 205 XXXIV Lymphocyte Stimulation in . Serum or. Peak I.... ..,,, t.. ...... 207 XXXV Lymphocyte Stimulation in Serum or Peak III, ,,,,,,,,,, ,. 210 XXXVI Lymphocyte Stimulation in Serum or SAS Fractions,,.,..., 214 XXXVII Lymphocyte Stimulation in.Ultrafiltration'' Fractions of SAS Fractions, of Serum. ......, ,,,,, ....... 217 XXXVITI Mean SSLS in Individuals with Measurable and Nonmeasurable Levels of C R P 2 2 3 XXXIX Mean Levels of Serum Proteins in Controls and Patients on Various Hormonal Treatments .,,, ,,,,.. .,,,.,. , 225 XL Mean Levels of Serum Proteins in Patients with Different Amounts of Disease........................,, 226 x i i i LIST OF FIGURES Figure T i t l e Page 1 E f f e c t of Control and Patients' Dialysed Sera on the Response of Normal Lymphocytes to PHA, 1:1500 , , ....... 115 2 E f f e c t of Concentration of Dialysed Serum Protein on Normal Lymphocyte Stimulation by PHA, 1:1500....,, 117 3 E f f e c t of Concentration of Undialysed Serum on Normal Lymphocyte Stimulation by PHA, 1:1500 .., 120 4. Response of Normal Lymphocytes to Various Concent-rations of PHA i n the Presence of 14 mg/ml Dialysed Serum Protein, 124 5 Response of Normal Lymphocytes to Various Concent-rations of PHA i n the Presence of 40% Undialysed Serum . ., , , 125 6 Response of Normal Lymphocytes to Various Concent-rations of PHA A f t e r D i f f e r e n t Incubation Times i n the Presence of 14 mg/ml Dialysed Serum Prot e i n , . . , . . 127 7 Response of Lymphocytes from Healthy Controls and Breast Cancer Patients to PHA i n the Presence of 8% FCS 140 8 SSLS-I for Healthy Controls of D i f f e r e n t Ages........ 170 9 T y p i c a l E l u t i o n P r o f i l e of Serum Passed Through a Sephadex G-150 Column 195 10 Plot of the Logarithm of the Molecular Weight of Known Proteins Versus Their Distance of Migration from the Origin i n SDS-PAGE 200 11 Autoradiograph of the Gel from the SDS-PAGE of Radioiodinated 33% SAS Ppt, MW 2,000-10,000 Fractions from a Normal Serum and from Pooled In h i b i t o r y Patients ' Sera 219 x i v LIST OF ABBREVIATIONS Ab Antibody E-ACA.. . . £-amino cap r o i c a c i d ADCC Antibody-dependent cell- m e d i a t e d c y t o t o x i c i t y Ag Antigen CEA... Carcinoembryonic antigen CRBC Chicken red blood c e l l s CRP C-re a c t i v e p r o t e i n DNCB .Dinitrochlorobenzene EAC-rosettes Rosettes of lymphocytes w i t h sheep er y t h r o c y t e s coated w i t h antibody plus complement ER+ Oestrogen receptor p o s i t i v e ER- Oestrogen receptor negative E-rosettes Rosettes of lymphocytes w i t h sheep eryt h r o c y t e s FCS F o e t a l c a l f serum a-FP. a-f o e t o p r o t e i n FSH. . . . F o l l i c l e s t i m u l a t i n g hormone 3H-TdR T r i t i a t e d thymidine IATI I n t e r - a t r y p s i n i n h i b i t o r IRA Immunoregulatory a - g l o b u l i n LAI. Leukocyte adherence i n h i b i t i o n LAIF Leukocyte adherence i n h i b i t i o n f a c t o r LH L u t e i n i z i n g hormone LMI Leukocyte m i g r a t i o n i n h i b i t i o n MAF Macrophage a c t i v a t i n g f a c t o r MHC Major h i s t o c o m p a t i b i l i t y complex XV. MIF Migration i n h i b i t o r y factor MLR Mixed lymphocyte reaction MLV Murine leukaemia virus MMTV Murine mammary tumour virus MW Molecular weight NHS Normal human serum NIP Normal immunosuppressive protein NK c e l l s Natural k i l l e r c e l l s NK-TS Natural k i l l e r target structure PAGE.... ... Polyacrylamide gel electrophoresis ORBC Ox red blood c e l l s PAM Pregnancy-associated macroglobulin PG , Prostaglandin PGE2 Prostaglandin E2 PHA Phytohaemagglutinin Polyl-polyC Polyinosine-polycytosine PPD. P u r i f i e d protein derivative of tuberculin Ppt Precipitate PWM Pokeweed mitogen SAS Saturated ammonium sulphate 20a-SDH 20a-hydroxysteroid dehydrogenase SDS Sodium dodecyl sulphate SE. Standard error of the mean SMAF Specific macrophage arming factor SRBC Sheep red blood c e l l s xv i SSLS Serum support of lymphocyte stimulation SSLS-I Serum support of lymphocyte stimulation index Sup Supernatant x v i i ACKNOWLEDGEMENTS The author wishes to thank Dr. L.G. E l l i s o n and Dr. H. S i l v e r and the s t a f f of the Advanced Therapeutics Department, B r i t i s h Columbia Cancer Research Center f o r p r o v i d i n g serum samples from breast cancer p a t i e n t s , the s t a f f of F i l e s of the Cancer C o n t r o l Agency of B r i t i s h Columbia f o r p r o v i d i n g access to p a t i e n t s ' c h a r t s , Dr. M. Schulzer f o r advice regarding s t a t i s t i c a l a n a l y s i s , Mr. Wing Lau and Mr. Robert Wang f o r computer programming, Ms. Beryl..Jacobson and the D i v i s i o n of C l i n i c a l Chemistry, Vancouver General H o s p i t a l , f o r measurements of serum C o r t i s o l , Dr. Roy P u r s s e l l f o r t e c h n i c a l a s s i s t a n c e , and Miss J o s i e Ramos f o r ty p i n g . She a l s o e s p e c i a l l y wishes to thank the research s u p e r v i s o r , Dr. J u l i a Levy, and the other members of the t h e s i s committee, Dr. S i l v e r , Dr. Schulzer, and Dr. D.G. K i l b u r n f o r many h e l p f u l suggestions, and her f a m i l y f o r t h e i r patience and support. This i n v e s t i g a t i o n was supported by a N a t i o n a l Cancer I n s t i t u t e of Canada operating grant No. 65-6048 to Dr. J u l i a Levy.,The author was supported by a Medical Research C o u n c i l - o f Canada f e l l o w s h i p No. 68-8234. 1 CHAPTER I INTRODUCTION I. AIMS OF THE RESEARCH Tumour-bearing animals- and humans, e s p e c i a l l y those w i t h advanced tumours, o f t e n show a general depression of immune responses (Brooks et a l . 1972; Golub et a l . 1974; Whitney e t a l . 1974; Buda et a l 1975). This may i n c l u d e depression of the p a t i e n t s response t o - h i s own tumour, which, i n t u r n , might be expected to hasten tumour p r o g r e s s i o n . Several s t u d i e s have shown that serum from tumour-bearers may depress the response of normal lymphocytes to mitogens such as phytohaemagglutinin (PHA) (Whittaker et a l . 1972; Whitney and Levy 1975; Buda et a l . 1975). This research p r o j e c t i s a study of t h i s phenomenon i n human breast cancer. The response of normal lymphocytes to PHA i n p a t i e n t s ' sera was compared w i t h the response i n c o n t r o l s e r a . The aims of the p r o j e c t were: (1) To determine whether immunosuppressive sera were found at a higher frequency i n p a t i e n t s w i t h breast cancer .than ; i n ' c o n t r o l : women of com-parable age, and to study the e f f e c t s of v a r y i n g serum c o n c e n t r a t i o n , PHA co n c e n t r a t i o n , and du r a t i o n of i n c u b a t i o n on lymphocyte s t i m u l a t i o n i n d i f f e r e n t . t y p e s of se r a . (2) To determine which, i f any, c l i n i c a l v a r i -a b l e s , such as age, amount of disease present, treatment, s u r v i v a l time and others, might be r e l a t e d to the presence of immunosuppressive serum. (3) To attempt to i d e n t i f y the immunosuppressive substance ( s ) . E x p e r i -ments designed to f u l f i l l these three aims form the subject matter of Chapters IT, I I I , and IV r e s p e c t i v e l y . I t was thought that the presence of immunosuppressive serum might prove to be as s o c i a t e d w i t h a poor prognosis, and that more knowledge of 2 the serum substances responsible, and of the circumstances under which they are produced, might suggest a means of reversing the immuno-suppression f o r the benefit of the patien t . I I . BIOLOGY AND TREATMENT OF HUMAN BREAST CANCER 1. S t a t i s t i c s Cancer of the breast i s by f a r the mo sttcommon cancer among North American women (with the exception of skin cancers, most of which do not metastasize) (Brennan 1973). I t i s the most common cause of death i n women aged 40 to 44, and remains among the f i r s t three causes of death u n t i l age 60 (Brennan 1973). Breast cancer comprises 27.5% of a l l cancer i n white women and 23% of a l l cancer i n black women i n the United States (del Regato and Spjut 1977). From 1935-1970 i n the United States among white women there has been a s l i g h t increase i n the incidence of breast cancer with no change i n mortali t y . This would suggest only a s l i g h t improvement i n cure rate (del Regato and Spjut 1977). 2. Pathology About 90% of carcinomas of the breast a r i s e from the epithelium of the ducts, and about 10% from the epithelium of the terminal ducts or lobules (del Regato and Spjut 1977). Malignant c e l l s f i l l the lumen of the duct, and also break through the basement membrane to invade the connective t i s s u e of the breast ( i n f i l t r a t i n g duct carcinoma) (Brennan 1973). The growth patterns vary. In we l l d i f f e r e n t i a t e d cancers the c e l l s , d i f f e r i n g r e l a t i v e l y l i t t l e from normal duct c e l l s , form glandular structures, whereas i n poorly d i f f e r e n t i a t e d tumours c e l l s are ra p i d l y 3 d i v i d i n g and un d i f f e r e n t i a t e d , and are scattered in-small groups and rows* Of course, the poorly d i f f e r e n t i a t e d tumours have a worse prognosis (Brennan 1973). They also i n c i t e a greater p r o l i f e r a t i o n of fibrous tissue (Brennan 1973). 3. Sites of Metastases Cancer of the breast spreads v i a the regional lymphatics most commonly to a x i l l a r y lymph nodes, but also to the i n t e r n a l mammary and supraclavic-u l a r nodes. Usually the a x i l l a r y nodes are the f i r s t to be involved, but sometimes a large tumour i n the medial h a l f of the breast w i l l have spread to i n t e r n a l mammary nodes only (Brennan 1973). Primary breast cancers and nodal metastases also metastasize v i a the blood stream. Common s i t e s of such d i s t a n t metastases are bone, lung, l i v e r , adrenals, distant lymph nodes, and skin (Brennan 1973; del Regato and Spjut 1977). Local recurrence i n the skin of the chest wall i s also quite common. About one t h i r d of patients with recurrent carcinoma have such a l o c a l recurrence (del Regato and Spjut 1977). 4. Staging Breast cancer patients are c l a s s i f i e d as having Stage I to Stage IV cancer on the basis of a c l i n i c a l assessment of the extent of disease before treatment or of a pathological assessment of the extent of disease at mastectomy. Although the patient's disease may subsequently increase or decrease, her stage does not change. Several systems of staging are i n use, and these d i f f e r i n some de-t a i l s , but are a l i k e i n that prognosis worsens from Stage I to Stage IV (Brennan 1973; del Regato and Spjut 1977; Henderson and Canellos 1980a). 4 The c l i n i c a l and pathological staging systems used at the Cancer Control Agency of B r i t i s h Columbia are shown i n Tables I and I I . 5. Factors Determining Prognosis The various studies reviewed by Brennan (1973) have shown that 40% to 70% of patients treated with modified r a d i c a l mastectomy e v e n t u a l l y s u f f e r recurrence and death. In these cases, microscopic f o c i of tumour c e l l s must have existed at the time of surgery, e i t h e r l o c a l l y or at di s t a n t s i t e s . Much e f f o r t has been devoted to c l a r i f y i n g factors which indi c a t e a high p r o b a b i l i t y of such distant micrometastases, so that patients at high r i s k may be given some add i t i o n a l treatment aimed at e r a d i c a t i n g the micrometastases. The most important prognostic factors are the extent of disease at diagnosis and the inherent aggressiveness or virulence of the tumour (Coppin and Swenerton 1982). The extent of disease i s expressed as the c l i n i c a l or pathological stage, of which the pathological stage i s , of course, more accurate. The prognosis of patients not given systemic adjuvant therapy and s t r a t i f i e d by c l i n i c a l and pathological stage i s shown i n Tables I I I and IV r e s p e c t i v e l y . With respect to the extent of disease, the absolute number of a x i l l a r y lymph nodes containing metast-ases i s the si n g l e most important prognostic factor (Coppin and Swenerton 1982). Tumour diameter i s also an independent prognostic factor (Coppin and Swenerton 1982). Methods of determing the inherent v i r u l e n c e of a tumour from ti s s u e obtained at biopsy are currently under study. A c o r r e l a t i o n has been found between a high rate of c e l l d i v i s i o n as shown by a high l a b e l l i n g index or m i t o t i c index and early relapse (Tubiana e_t a_l. 1981; Coppin 5 TABLE I SYSTEM OF CLINICAL STAGING IN USE AT THE CANCER CONTROL AGENCY OF BRITISH COLUMBIA Stage Extent of Disease as Detected by Physical Examination and/or Preoperative Diagnostic Tests 1 0 No palpable disease. I Primary tumour f r e e l y movable on contracted pectoral muscles or chest w a l l . IT As Stage I, but with mobile lymph nodes, <2.5 cm. i n diameter, palpable i n the i p s i l a t e r a l a x i l l a . I l l E i t h e r (a) skin involvement of the tumour but confined to the breast. (b) F i x a t i o n of tumour to muscle but not to the chest w a l l . A x i l l a r y lymph nodes must be mobile. IV Extension beyond the breast as indicated by: (a) F i x a t i o n of tumour to the chest w a l l . , (b) Skin involvement wide of the breast. (c) A x i l l a r y nodes not mobile or >2.5 cm. i n diameter. (d) Metastases to the opposite breast. (e) Distant metastases. (f) Inflammatory carcinoma. 6 TABLE II SYSTEM OF PATHOLOGICAL STAGING IN USE AT THE CANCER CONTROL AGENCY OF BRITISH COLUMBIA Stage Extent of Disease as Detected by Pathology I 0 Car ciriomanin...situ*. I Cancer confined to the breast. II Cancer confined to the breast and i p s i -l a t e r a l a x i l l a r y nodes, not including the apicalanbdes. II ? Cancer i n the breast and i p s i l a t e r a l a x i l l a r y nodes, l e v e l unknown. III Cancer i n the breast and i n ski n wide of the tumour or i n underlying muscle. IV Cancer i n the breast and i n i n any of the following l o c a t i o n s : (a) In r i b or c a r t i l a g e of the chest w a l l . (b) Beyond the capsule of an a x i l l a r y node. (c) In a p i c a l or i n t e r n a l mammary nodes. (d) In an i p s i l a t e r a l a x i l l a r y node c l i n i c a l l y > 2.5 cm. (e) Distant metastases including supra-c l a v i c u l a r nodes. TABLE I I I SURVIVAL OF PATIENTS WITH BREAST CANCER RELATIVE TO CLINICAL STAGE C l i n i c a l Staging (American J o i n t Committee) Crude 5 Year S u r v i v a l (%) + Range of S u r v i v a l (%) + Stage I Tumours <2 cm i n diameter. Nodes not f e l t to c o n t a i n metastases. Without d i s t a n t metastases. Stage I I Tumours <5 cm i n diameter. A x i l l a r y nodes,, i f p a l p a b l e , not f i x e d . Without d i s t a n t metastases. Stage I I I Tumours >5 cm, or Tumours of any s i z e w i t h i n v a s i o n of s k i n or attachment to chest w a l l , or Nodes i n s u p r a c l a v i c u l a r area. Without d i s t a n t metastases. Stage IV With d i s t a n t metastases. 85 82-94 66 41 10 47-74 7-80 * Reprinted from Henderson and Canellos (1980a). + Mean of s e v e r a l s t u d i e s reported i n the l i t e r a t u r e . "f* Range of s e v e r a l s t u d i e s . TABLE IV * SURVIVAL OF PATIENTS WITH BREAST CANCER RELATIVE TO HISTOLOGIC STAGE H i s t o l o g i c Staging (NSABP) ** Crude S u r v i v a l (%) 5 yr 10 yr 5 yr di s e a s e -f r e e s u r v i v a l (%) A l l P a t i e n t s Negative A x i l l a r y Lymph Nodes P o s i t i v e A x i l l a r y Lymph Nodes 1-3 P o s i t i v e A x i l l a r y Lymph Nodes 4 P o s i t i v e A x i l l a r y Lymph Nodes 63.5 7 8 . 1 4 6 . 5 62.2 3 2 . 0 45 .9 64 .9 24 .9 37 .5 13.4 60.3 82.3 34.9 50.0 21.1 * Reprinted from Henderson and Canellos (1980a), based on a study of p a t i e n t s w i t h C l i n i c a l Stage I and I I disease t r e a t e d by r a d i c a l mastectomy described by F i s h e r et al.(1975). ** NSABP denotes N a t i o n a l S u r g i c a l Adjuvant Breast P r o j e c t ( F i s h e r et a l . 1975). 9 and Swenerton 1982). The l e v e l of oestrogen receptors i n breast cancer, measured as an i n d i c a t o r of the l i k l i h o o d of response to hormonal t r e a t -ments, i s l i n e a r l y r e l a t e d to recurrence-free s u r v i v a l even i n patients r e c e i v i n g no treatment a f t e r mastectomy and radiotherapy (Godolphin et  a l . 1981). Oestrogen receptor l e v e l was almost completely independent of stage. Invasion into intramammary lymphatics, blood vessels, and surrounding breast t i s s u e , tumour necrosis, poor tumour d i f f e r e n t i a t i o n , m u l t i c e n t r i c cancer, and germinal centre predominance i n the regional lymph nodes have a l l been associated with a poor prognosis (Coppin and Swenerton 1982). 6. Treatment (i) Primary Treatment: Surgery i s the usual i n i t i a l treatment of breast carcinoma. The procedure most commonly employed i n B r i t i s h Columbia i s the modified r a d i c a l mastectomy, i n which the e n t i r e breast with i t s a x i l l a r y extension, and the lower and m i d - a x i l l a r y nodes, i . e . the nodes clos e s t to the breast, and t h e i r surrounding fat are removed (Brennan 1973). Unlike the r a d i c a l mastectomy, the pectoral muscles are not removed (Brennan 1973). Some breast cancer patients are considered inoperable, i . e . modified r a d i c a l mastectomy would o f f e r no hope of cure. These include a l l patients with apparent distant metastases, and those whose l o c a l and regional disease i s so advanced that the presence ot microscopic d i s t a n t metastases i s a v i r t u a l c e r t a i n t y (Brennan 1973; del Regato and Spjut 1977). These are the Stage IV p a t i e n t s . In these patients the primary primary cancer and regional lymph nodes are usually treated with radiotherapy and/or systemic treatments (see below). 10 ( i i ) Treatment of Metastases: Once metastases are apparent, the chance of cure i s n e g l i g i b l e . The aims of treatment, therefore, are to r e l i e v e symptoms and prolong l i f e by causing regression of tumour masses. (a) Local Treatment: Sometimes s o l i t a r y metastases can be removed s u r g i c a l l y . More often radiotherapy i s employed. This i s p a r t i c u l a r l y b e n e f i c i a l i n r e l i e v i n g the pain of bone metastases and reducing the r i s k of pathological f r a c t u r e , and i n t r e a t i n g recurrences i n the chest wall and regional nodes (Brennan 1973; del Regato and Spjut 1977). (b) Systemic Treatment: Since metastases are usually m u l t i p l e , even though only one may be apparent c l i n i c a l l y , patients with metastases are often given some systemic therapy, i . e . a drug which acts on cancer c e l l s throughout the body. There are two main types of systemic therapy: chemotherapy, i n which cytotoxic drugs are given to k i l l tumour c e l l s , and hormonal therapy, i n which the hormonal balance of the patient i s al t e r e d i n a manner unfavourable to the growth of the tumour. Tumours d i f f e r i n t h e i r s e n s i t i v i t y to the various chemotherapeutic agents and to hormones. After one agent has been used with good r e s u l t s f o r a v a r i a b l e period of time, thefew tumour c e l l s r e s i s t a n t to that agent may have m u l t i p l i e d to the point that metastases are again progressing. Then the treatment must be changed. Chemotherapy: Several d i f f e r e n t drug combinations are used. These w i l l not be discussed here, as the research project described i n t h i s thesis i s concerned mainly with patients on hormonal treatments. Combinations are usually more e f f e c t i v e than single agents because c e l l s r e s i s t a n t 11 to one drug may be s e n s i t i v e to another (Coppin and Swenerton 1982). Hormonal Treatment: The hormonal treatment of breast cancer has undergone important changes during the course of the research project described herein, i . e . during the years 1973-1981. Only about one t h i r d of premenopausal patients and a somewhat higher proportion of postmenopausal patients have hormone-responsive tumours (Brennan 1973; Henderson and Canellos 1980b). A major advance i n breast cancer treatment has been the improved a b i l i t y to predict response to hormonal treatment by the measurement of oestrogen receptors i n tumours. Such receptors have been measured r o u t i n e l y i n biopsy specimens i n B r i t i s h Columbia since 1975. Among patients with oestrogen receptor p o s i t i v e (ER+) tumours, the response rate to hormonal treatments i s 50% to 60% compared with less than 10% for ER- tumours (Hoge et a l . 1973; Henderson and Canellos 1980b). Tumours which respond to one hormonal treatment but l a t e r relapse are often s e n s i t i v e to another hormonal t r e a t -ment. Nomura e_t a l . (1976) showed that tumours recurring a f t e r an i n i t i a l response to oophorectomy are s t i l l ER+. Patients with ER- tumours are usually treated with chemotherapy because of the low response to hormones. The hormonal treatments employed have also changed over the past decade. Coventional Hormone Therapy: The following treatments were i n use during Phase 1 of the project described i n t h i s t h e s i s , but have now been discontinued. Adrenalectomy 12 and hypophysectomy were not frequently used. Oophorectomy would be the f i r s t treatment for premenopausal patients with metastatic breast cancer. Those who responded i n i t i a l l y ( i . e . showed objective evidence of tumour r e g r e s s i o n ) b u t whose disease subsequently progressed, might be treated with androgens, c o r t i c o s t e r o i d s , adrenalectomy or hypophysectomy (removal of the p i t u i t a r y ) to remove the l a s t traces of oestrogen production, Patients who responded i n i t i a l l y to androgens, but then relapsed, might then be treated with c o r t i c o s t e r o i d s , adrenalectomy or hypophysectomy. A l t e r n a t i v e l y , they might be switched to chemotherapy. F a i l u r e to respond to, or relapse a f t e r i n i t i a l response to, adrenalectomy or hypophysectomy would lead to chemotherapy. Patients f a i l i n g to respond to oophorectomy might be considered unresponsive to hormonal treatments and givencehemotherapy,or might:be given .androgens f i r s t (del Regato arid Spjut 1977). In patients f i v e years or le s s postmenopausal androgens would be the usual f i r s t hormonal treatment (Brennan 1973). A patient who f a i l e d to respond to, or who relapsed a f t e r response to, androgens would then be t r i e d with oestrogens (del Regato and Spjut 1977). Oestrogens i n quite high doses (higher than physiologic) would be the f i r s t treatment of choice i n women more than f i v e years postmenopausal. Patients who r e -lapsed a f t e r an i n i t i a l response to oestrogens might then respond to oestrogen withdrawal (Henderson and Ganellos!1980a), and then could be treated with androgens and subsequently with corticosteroids,, or adrenal-ectomy or hypophysectomy might be used d i r e c t l y a f t e r oestrogens. Patients who f a i l e d to respond to oestrogens might be switched to 13 chemotherapy or might be given a t r i a l of androgens and/or oestrogens plus progestogens (del Regato and Spjut 1977). In general a patient who re s -ponded to the f i r s t hormonal.-: treatment would have a much better chance of responding to others than one who f a i l e d to respond to the f i r s t treatment (del Regato and Spjut 1977; Henderson and Canellos 1980a). Newer Hormone Therapies: In the period 19 76 - 78 there was a change from the above conventional hormone therapies to two new drugs, Tamoxifen and Aminoglutethimide. Like other hormonal treatments, Tamoxifen and Aminoglutethimide are more l i k e l y to be e f f e c t i v e i n patients with ER+ breast cancer. They are e f f e c t i v e only i n postmenopausal pa t i e n t s , i n c l u d i n g those who have been treated f o r metastatic disease by oophorectomy. These agents are administered s i n g l y or together. Usually one, more often Tamoxifen, i s given f o r the treatment of metastatic disease. Then, i f further progression occurs a f t e r an i n i t i a l good response, the other i s added. A patient relapsing on both these drugs at maximum dosage would usually be treated with chemotherapy. Tamoxifen i n h i b i t s the e f f e c t s of oestrogens i n target organs. The mechanism i s unknown, but the drug binds to oestrogen receptors (Santen and Wells 1980). Aminoglutethimide i n h i b i t s the synthesis of hormones of the adrenal cortex in c l u d i n g oestrogens. Its main action i s thought to be a block i n the adrenal conversion of c h o l e s t e r o l to A-5 pregnenolone, but i t may also block the conversion of androstenedione to oestrogen i n the p e r i -pheral tissues (Henderson and Canellos 1980b). Aminoglutethimide 14 must always be given with g l u c o c o r t i c o i d replacement. Without gluco-c o r t i c o i d replacement the concentration of adrenal c o r t i c o t r o p i c hormone (ACTH) would r i s e s u f f i c i e n t l y to restore s t e r o i d production and negate the benefit of the drug (Henderson and Canellos 1980b). Recent Trends i n the Treatment of Metastases: Hormone responsive tumours have usually been treated with hormones f i r s t and with chemotherapy only a f t e r the l a s t a v a i l a b l e hormone treatment has f a i l e d because the side e f f e c t s of hormones are so much less severe than those of chemotherapy. Recent t r i a l s have studied the benefits of combining hormones and chemotherapy. The r e s u l t s have been somewhat contradictory, but several studies show promising r e s u l t s (Coppin and Swenerton 1982). Since treatment delays and greater tumour burden reduce the e f f i c a c y of chemotherapy more than that of hormones, i t might be preferable to treat a l l patients with metastatic disease with chemotherapy f i r s t and to add hormones at l e a s t to those with ER+ tumours (Coppin and Swenerton 1982). ( i i i ) Adjuvant Treatment: Treatment given a f t e r apparently complete resection of the primary tumour, i n the hope of eradicating microscopic metastases and thus prev-enting recurrence, i s known as adjuvant therapy. Adjuvant therapy i s usually used i n patients at high r i s k of recurrence, i . e . those with a x i l l a r y lymph node metastases. Those with ER- tumours with or without a x i l l a r y node involvement should probably be included i n t h i s group (Coppin and Swenerton 1982). Adjuvant Radiotherapy: Radiotherapy to the chest wall and to a x i l l a r y , s u p r a c l a v i c u l a r , and i n t e r n a l mammary node areas has frequently been employed a f t e r surgery, e s p e c i a l l y i n patients with a x i l l a r y node metastases. Such radiotherapy d e f i n i t e l y decreases the incidence of l o c a l and regional lymph node recurrences, but appears to have no s i g n i f i c a n t e f f e c t upon s u r v i v a l , as i t does not a f f e c t distant metastases.(del Regato and Spjut 1977; Henderson and Canellos 1980a). In f a c t , one study has shown a decrease i n 3-year s u r v i v a l i n Stage I breast cancer treated by r a d i c a l mastectomy and radiotherapy compared with surgery alone (Johnstone 1974). This d i f f -erence was not s t a t i s t i c a l l y s i g n i f i c a n t . Adjuvant Hormone Therapy: Oophorectomy as an adjuvant i n premenopausal women has been studied quite extensively beginning i n 1948. One review suggested i t may s i g n i f i -cantly delay the appearance of .metastases but does not prolong s u r v i v a l (Henderson and Canellos 1980b). However, a recent study (Bryant and Weir 1981) showed improved s u r v i v a l a f t e r oophorectomy i n women l e s s than 50 year with a x i l l a r y node metastases. In another study, ovarian r a d i a t i o n was found to prolong s u r v i v a l but not s i g n i f i c a n t l y , whereas ovarian r a d i a t i o n plus prednisone i n premenopausal women over 45 years s i g n i f i c a n t l y delayed recurrence and improved s u r v i v a l (Meakin e_t a l . 1979) . The prednisone might act by i n h i b i t i n g adrenal hormone synthesis. Tamoxifen has been used as an adjuvant with some success i n postmenopausal women (Coppin and Swenerton 1982). Adjuvant Chemotherapy: A recent study (Bonadonna e_t a l . 1978) has shown that monthly courses of cyclophosphamide, methotrexate, and 5 - f l u o r o u r a c i l , continued for one year a f t e r mastectomy i n patients with p o s i t i v e a x i l l a r y nodes, s i g n i f i c -antly reduced recurrence and improved s u r v i v a l at 4 years for premenopausal patients. Other chemotherapy treatment regimens are under i n v e s t i g a t i o n 16 w i t h some promising results.(Henderson and Canellos 1980b; Coppin and Swenerton 1982). t Combined Adjuvant Hormonochemotherapy: E a r l y s t u d i e s have shown an improved d i s e a s e - f r e e i n t e r v a l i n postmenopausal p a t i e n t s t r e a t e d w i t h adjuvant Tamoxifen and chemotherapy compared w i t h e i t h e r alone (Coppin arid Swenerton 1982). A s i m i l a r e f f e c t i s not seen i n premenopausal p a t i e n t s . As a l l tumours probably c o n t a i n a mixture of ER+ and ER- c e l l s , and ER- c e l l s are more s e n s i t i v e to chemotherapy than are ER+ c e l l s , a combination of hormones and chemotherapy would i n theory have the best chance of e r a d i c a t i n g micrometastases (Coppin and Swenerton 1982). At present, a major area of research i s the determination of sub-sets of p a t i e n t s l i k e l y to b e n e f i t most from a p a r t i c u l a r treatment. (Coppin and Swenerton 1982). I I I . DETECTION AND MECHANISMS OF ANTI-TUMOUR IMMUNITY I t has been shown by many i n v e s t i g a t o r s that most tumours i n animals and man have on t h e i r c e l l surfaces tumour-specific antigens which d i s -t i n g u i s h them from normal c e l l s . E a r l y reviews on t h i s subject included those of Old and Blyse (1964), who considered only tumours i n inbred mice, and of Cinader (1972), who considered human as w e l l as animal tumours. Recently, monoclonal a n t i b o d i e s have been used to study the s p e c i f i c i t y of antigens on tumour c e l l surfaces (Koprowski et a l . 1978; Steplewski et a l . 1979; Herlyn et a l . 1979a; Yeh et a l . 1979). E a r l y s t u d i e s showed that mice could be rendered immune to a syn-' geneic tumour. I f an e a r l y tumour were e x c i s e d , and the same animal l a t e r i n j e c t e d w i t h c e l l s of the same tumour l i n e , the tumour would f a i l to grow (Prehn and Main 1957; K l e i n e j t a l . 1960). I n j e c t i o n of i r r a d i a t -17 ed tumour c e l l s could also immunize syngeneic mice (Klein and Sjogren 1960; K l e i n et a l . 1960). I t has.been shown that such tumour-specific immunity may be transferred with c e l l s but not with serum (Klei n and Sjogren 1960; K l e i n et a l . 1960). Thus cell-mediated immunity was consid-ered to be more important than humoral immunity i n resistance to tumours. A. Cell-mediated Immunity to Tumours Since that time a great many studies have attempted to detect and to c l a r i f y the mechanisms of cell-mediated immunity to tumours. A number of d i f f e r e n t assays have been used. Some, the c y t o t o x i c i t y assays, measure the a b i l i t y of leukocytes from tumour bearers to destroy or i n h i b i t growth of tumour c e l l s . Others, such as the leukocyte migration i n h i b i t i o n (LMI) and leukocyte adherence i n h i b i t i o n (LAI) assays, do not measure tumour c e l l destruction, but are i n v i t r o c o r relates of the d e l -ayed h y p e r s e n s i t i v i t y reaction, a cell-mediated immune reaction. 1. C y t o t o x i c i t y Assays A number of d i f f e r e n t procedures have been used to measure the cytotoxic e f f e c t of lymphoid c e l l s or macrophages on cultured tumour c e l l s . C y t o t o x i c i t y includes both c y t o s t a t i c and c y t o l y t i c a c t i v i t i e s . In the colony i n h i b i t i o n test (Hellstrom et a l . 1970) a given number of tumour c e l l s were incubated for 3 to 5 days with lymphocytes from various sources or with medium alone. The number of macroscopic tumour colonies formed was counted. This assay r e a l l y measured c y t o s t a t i c as well as c y t o l y t i c a c t i v i t y of the lymphocytes.. In a v a r i e t y of m i c r o c y t o t o x i c i t y t e s t s , such as that developed by Takasugi et al.. (1972):, the number of adherent v i a b l e tumour c e l l s were counted with the aid of a microscope a f t e r about 2 days incubation of a given number of tumour c e l l s with various lymphocyte populations or with medium alone. C y t o l y s i s can be measured more d i r e c t l y by measuring the release of a previously incorp-18 orated radioactive isotope from v i a b l e tumour c e l l s upon incubation with various lymphocyte populations (Bean et a l . 1973; Oldham et a l . 1973). Probably the most commonly used c y t o t o x i c i t y assay at the present time involves the release of 5 1 C r from cultured tumour target c e l l s (Plata et a l . 1976; Holden and Herberman 1977; Rodef et a l . 1979b). At l e a s t four d i f f e r e n t c e l l types have been i d e n t i f i e d as being cytotoxic i n the various c y t o t o x i c i t y assays. These are T lymphocytes, e f f e c t o r c e l l s of antibody-dependent cell-mediated c y t o t o x i c i t y (ADCC) , natural k i l l e r (NK) c e l l s , and macrophages. ( i ) Immune T Lymphocytes: In some experimental tumour systems i n inbred mice, the c y t o t o x i c i t y of lymphocytes from mice immunized to tumour antigens has been c l e a r l y shown to be due l a r g e l y to T lymphocytes i n that such c y t o t o x i c i t y i s almost completely abolished by treatment of the lymphocytes with a n t i -Thy-1 plus complement ( C e r o t t i n i et a l . 1970; Herberman et a l . 1973; G i l l e s p i e et a l . 1977; Stutman et a l . 1977; Le c l e r c and Cantor 1980). Comparable studies are not possible i n humans as there i s no T ^ c e l l -s p e c i f i c antigen analogous to Thy-1 i n mice. Human T c e l l s form rosettes with sheep erythrocytes (E-rosettes). Human T c e l l s can be removed by the removal of E-rosette forming c e l l s , but t h i s procedure removes much NK and ADCC a c t i v i t y as well (Kay e_t a l . 1977; Herberman and Holden 1978) . There i s curr e n t l y some controversy as to whether T c e l l immunity to tumours i n mice i s H-2 r e s t r i c t e d . Stutman et a l . (1977) showed that T c e l l s from mice bearing murine mammary tumour v i r u s (MMTV)-induced tumours are cytotoxic.to MMTV-induced tumour c e l l s i n v i t r o as shown by a H-proline release assay, and that the c y t o t o x i c i t y i s biphasic with peaks at 6, and 18 to 20 hours. Stutman and Shen (1978) showed that the 19 f i r s t , but not the second, peak i s H-2 r e s t r i c t e d . Therefore, the f i n d i n g of H-2 r e s t r i c t i o n or n o n r e s t r i c t i o n could depend on the length of the c y t o t o x i c i t y assay. Two studies using r e l a t i v e l y long c y t o t o x i c i t y assays demonstrated a lack of H-2 r e s t r i c t i o n (Holden and Herberman 1977; Ting and Law 1 9 7 7 ) . On the other hand, P l a t a et a l . (1976) demonstrated H-2 r e s t r i c t i o n using both a 3-hour and an 18-hour 5 1 C r release assay depending on the target used. Pape et a l . (1977) found that cell-mediated c y t o t o x i c i t y to. a l l o g e n e i c , but not to autochthonous, human bladder cancer c e l l s was eliminated by removal of surface Ig-bearing and Fc receptor p o s i t i v e lymphoid c e l l s from the lymphoid c e l l s of bladder cancer patients. This suggested a d i f f e r e n t mechanism of c y t o t o x i c i t y against allogeneic and autochthonous tumour c e l l s , possibly HLA r e s t r i c t e d , T cell-mediated c y t o t o x i c i t y toward autochthonous c e l l s . De Vries and Rumke(19 76) found that lymphocytes from melanoma patients showed a s p e c i f i c c y t o t o x i c i t y to cultured melanoma c e l l s super-imposed upon the nonspecific c y t o t o x i c i t y to a v a r i e t y of c e l l l i n e s seen i n lymphocytes from many healthy donors and cancer patients. They did not determine the c e l l responsible for t h i s s p e c i f i c c y t o t o x i c i t y . ( i i ) C e l l s Responsible for Antibody-Dependent Cell-Mediated Cytotoxicty: Antibody-coated target c e l l s can be destroyed by normal nonimmune lymphoid c e l l s . This reaction, known as antibody-dependent cell-mediated c y t o t o x i c i t y (ADCC), was f i r s t described by Moller (1965) using mouse tumour c e l l s coated w i t h r a b b i t anti-mouse antibodies as the t a r g e t s , and normal nonimmune a l l o g e n e i c or semi-allogeneic mouse lymphoid c e l l s as the e f f e c t o r s . ADCC has r e c e n t l y been reviewed by Pearson (1978). Target c e l l d e s t r u c t i o n can be detected by 5 1 C r re l e a s e assays or m i c r o c y t o t o x i c i t y assays. The r e l a t i o n s h i p between e f f e c t o r and ta r g e t c e l l s may be syngeneic, a l l o g e n e i c or xenogeneic. The s p e c i f i c i t y i s conferred by the antibody. The e f f e c t o r c e l l must have Fc receptors f o r the antibody. B l o c k i n g of the Fc receptors w i t h u n r e l a t e d antigen-antibody complexes or w i t h normal or heat aggreggated IgG i n h i b i t s ADCC. Antigen-antibody complexes and perhaps f r e e antibody can arm normal lymphoid c e l l s i n v i t r o to act as e f f e c t o r s i n ADCC (Pearson 1978). Jones et a l . (1981) reported the b i n d i n g of antibody to r a t splenocytes and human blood monocytes and polymorphonuclear leukocytes by a s p e c i a l process c a l l e d " f r a n k i n g " , i n v o l v i n g i n c u b a t i o n of c e l l s and antibody w i t h polyethylene g l y c o l and sedimentation of the c e l l s through phthalate o i l s . These e e l ] s 'then could.''act as e f f e c t o r s i n ADCC w i t h uncoated t a r g e t s . The immunoglobulin c l a s s u s u a l l y a c t i v e i n ADCC i s IgG. However, there have a l s o been r e p o r t s demonstrating a c t i v i t y by IgM i n some systems (Pearson 1978). A recent report (Shen et a l . 1981) showed that monoclonal mouse IgM and p o l y c l o n a l r a b b i t IgM against ox red blood c e l l s (ORBC) were i n a c t i v e alone, but could synergize w i t h IgG i n mediating ADCC against ORBC. In mice and humans, ADCC can be e f f e c t e d by more than one c e l l type (Pearson 1978). One c e l l type i n both s p e c i e s , c a l l e d the lymphoid K c e l l , i s a nonadherent, nonphagocytic c e l l bearing Fc receptors f o r I g , 21 but lacking T c e l l markers and surface Ig. In addition, i n mice and humans, ADCC can be effected by a group of adherent Fc bearing c e l l s which include polymorphonuclear leukocytes, immature granulocytes, monocytes, and macrophages. The c e l l type involved depends on the experimental system and, i n p a r t i c u l a r , on the nature of the target c e l l (Pearson 1978). More:recently, subsets of T lymphocytes have been reported to act as ef f e c t o r s i n ADCC, perhaps with IgM as the antibody (Pearson 1978). In a number of virus-induced murine tumours, the t i t e r of antibody capable of mediating ADCC toward cultured tumour c e l l s v aries inversely with tumour burden (Pearson 1978). A number of studies have shownra possible r o l e f o r ADCC i n human tumours. Cell-mediated c y t o t o x i c i t y s p e c i f i c f o r syngeneic or allogeneic human bladder cancer has been demonstrated i n bladder cancer patients (O'Toole et a l . 1973). The e f f e c t o r c e l l s were removed by passage through a column coated with rabbit anti-human Ig (O'Toole et a l . 1973), but not by removal of E-rosette forming c e l l s (O'Toole et a l . 1974; O'Toole 1977), The non-E-rosette forming c e l l s could act as e f f e c t o r c e l l s i n ADCC using a d i f f e r e n t type of human antibody coated tumour target c e l l . Therefore, the e f f e c t o r c e l l i n t h i s system i s a non-T c e l l , and could be an e f f e c t o r c e l l i n ADCC. Other studies (Pape et a l . 1977 a,b) have shown that the lymphoid c e l l s from healthy controls and bladder cancer patients, cytotoxic for various allogeneic c e l l s from bladder and other cancers, have the same surface c h a r a c t e r i s t i c s , i . e . surface Fc receptors but l i t t l e surface Ig, as c e l l s that act as e f f e c t o r s i n ADCC to chicken red blood c e l l s (CRBC). 22 Fur thermore , t h i s c y t o t o x i c i t y was; i n h i b i t e d by r a b b i t Fab a g a i n s t human IgG (Troye et a l . 1977), It had p r e v i o u s l y been shown t h a t ADCC, but not T - c e l l mediated l y s i s , cou ld be i n h i b i t e d by a n t i - I g . Pret reatment of e f f e c t o r c e l l s w i th . Fab a g a i n s t human IgG d i d not i n h i b i t the c y t o t o x i c i t y . T h e r e f o r e , i t was suggested that plasma c e l l s , which have on ly a s m a l l amount of s u r f a c e I g , are present among the e f f e c t o r c e l l s and r e l e a s e a n t i b o d i e s which b i n d to the t a r g e t s d u r i n g the i n c u b a t i o n , ( i i i ) N a t u r a l K i l l e r (NK) C e l l s : NK c e l l s are lymphoid c e l l s which, k i l l t ransformed t a r g e t c e l l s n o n s p e e i f i c a l l y w i thout r e q u i r i n g p r i o r immunizat ion (Hood et a l . 1978) . Perhaps the e a r l i e s t r e f e r e n c e to what a re now known as NK c e l l s was tha t of Herberman et a l . (1973) . These authors were s t u d y i n g c e l l -mediated a n t i - t u m o u r immunity i n mice w i t h regressed murine sarcoma v i r u s -induced tumours by means of a 5"*Cr r e l e a s e assay . A l though most of the c y t o t o x i c a c t i v i t y of immune sp leen c e l l s cou ld be a b o l i s h e d by t reatment w i t h a n t i - 0 p l u s complement, nonimmune sp leen c e l l s showed a low l e v e l of c y t o t o x i c i t y which was not a b o l i s h e d by a n t i ^ 0 p l u s complement. The authors cons idered the p o s s i b i l i t y t h a t the c y t o t o x i c i t y of nonimmune sp leen c e l l s might ber.an example of ADCC w i t h n a t u r a l l y - o c c u r r i n g a n t i -bodies to leukaemia ant igens hav ing armed the lymphocytes from normal m i c e . The f i n d i n g tha t goat ant i -mouse Ig d i d not e l i m i n a t e the c y t o -t o x i c i t y was somewhat a g a i n s t t h i s p o s s i b i l i t y . C h a r a c t e r i s t i c s of NK c e l l s i n m i c e , i . e . c e l l s from nonimmune mice which cou ld k i l l c e r t a i n tumour l i n e s , were d e s c r i b e d by K i e s s l i n g et  a l . (1975a, b ) . They found NK c e l l a c t i v i t y i n the sp leens of n o n -immunized mice of v a r i o u s s t r a i n s toward th ree of f i v e l i n e s of tumours 23 induced by Moloney leukaemia virus. (MLV) ( K i e s s l i n g e_t ajL_, 1975a) . None of three non-MLV induced tumour lines; were s e n s i t i v e to t h i s NK c e l l a c t i v i t y . D i f f e r e n t mouse st r a i n s d i f f e r e d i n t h e i r degree of NK c e l l a c t i v i t y and the order was the same regardless of the, target c e l l s used. H-2 differences between e f f e c t o r and target c e l l s were apparently un-important. Target c e l l s • s u s c e p t i b l e to NK; c e l l l y s i s were e f f e c t i v e i n competitively i n h i b i t i n g the l y s i s of other susceptible target c e l l s . The order of s u s c e p t i b i l i t y to l y s i s was the same as the order of e f f i -cacy i n i n h i b i t i n g l y s i s . When spleen c e l l s showing NK c e l l a c t i v i t y were subjected to a n t i -Thy-1 plus complement, passed through anti - I g columns and subjected to removal of phagocytic c e l l s by carbonyl i r o n , the 1 to 5% ;of c e l l s which remained contained v i r t u a l l y a l l the o r i g i n a l NK c e l l a c t i v i t y ( K i e s s l i n g et a i . 1975b). These c e l l s were not capable of p a r t i c i p a t i n g i n ADCC. K i e s s l i n g et a l . (1976) found that NK c e l l s were not i n h i b i t e d by aggregated IgG or by the removal of rosettes of sheep erythrocytes coated with antibody and complement (EAC r o s e t t e s ) . They concluded that NK c e l l s lack both Fc and complement receptors. As discussed i n the review by Herberman and Holden (1978), a number of studies have shown that NK c e l l s i n mice, or at l e a s t a subpopulation of them, contain low l e v e l s of Thy-1 antigen and contain Fc receptors. However, they lack surface Ig. It i s generally accepted that NK c e l l s are nonadherent and nonphagocytic..• The factors a f f e c t i n g the l e v e l of NK c e l l a c t i v i t y i n mice have been reviewed by K i e s s l i n g and H a l l e r (1978). NK a c t i v i t y i s not detectable u n t i l 3 weeks of age, peaks at 6 to 8 weeks, and then declines 24 to very low l e v e l s by 6 months. I n j e c t i o n of tumour c e l l s leads to an i n i t i a l i n c r e a s e i n NK c e l l a c t i v i t y followed'.'by a decrease of NK c e l l s i n the spleen and p e r i p h e r a l blood.due, at l e a s t i n p a r t , to an accumu-l a t i o n of NK c e l l s , at the tumour s i t e and i t s d r a i n i n g lymph nodes. C e r t a i n normal c e l l s , b a c t e r i a , and v i r u s e s can a l s o increase NK c e l l a c t i v i t y . The o v e r a l l NK r e a c t i v i t y of a mouse s t r a i n i s g e n e t i c a l l y determined. High r e a c t i v i t y to one s e n s i t i v e c e l l l i n e , YAC-1, i s i n h e r i t e d as a dominant gene l o c a t e d on the same chromosome as the H-2 complex but apparently not w i t h i n i t . Superimposed on a general high or low NK r e a c t i v i t y , one s t r a i n may be a high responder to one t a r g e t and a lower responder to another. Therefore there may be a l s o a genetic r e g u l a t i o n of the c l o n a l expression of receptors on NK c e l l s . The c h a r a c t e r i s t i c s of NK c e l l s i n humans are more c o n t r o v e r s i a l (Herberman and Holden 1978). As i n mice there i s general agreement that NK c e l l s are nonadherent and nonphagocytic. The removal of E-rosette forming c e l l s from human p e r i p h e r a l blood lymphocyte populations removed considerable NK a c t i v i t y i n some but not i n a l l s t u d i e s . Apparently NK c e l l s have low a f f i n i t y receptors f o r sheep red blood c e l l s (SRBC). Most human NK c e l l s a l s o have Fc re c e p t o r s . Although s t u d i e s i n mice c i t e d above have c l e a r l y d i f f e r e n t i a t e d ADCC from NK c e l l a c t i v i t y , n a t u r a l a n t i b o d i e s against c e r t a i n v i r u s -induced tumours have been detected i n mice,-and are. r e s p o n s i b l e f o r some n a t u r a l c y t o t o x i c i t y ( K i e s s l i n g and H a l l e r 1978). Thus some, but c e r t a i n l y not a l l , n a t u r a l c y t o t o x i c i t y may be a form of ADCC. In humans there i s some controversy over the d i s t i n c t i o n between e f f e c t o r c e l l s i n ADCC and NK c e l l s . In some s t u d i e s there i s evidence 25 that what appeared to be NK c e l l activity involved antibodies, and thus would be a variant;.of ADCC. A series of papers by Takasugi and his colleagues (Takasugi et a i . 1973; 1974; Takasugi and Mickey 1978) showed that many controls and cancer patients had lymphocytes that were cytotoxic to a variety of human tumour c e l l l i n e s . The cy t o t o x i c i t y was nonselective' i n most cases, i . e . i t could be predicted by the general r e a c t i v i t y of the donor's lymphocytes and the general s e n s i t i v i t y of the target c e l l s . In those cases i n which a part i c u l a r donor showed selective c y t o t o x i c i t y for a part i c u l a r target, the lymphocyte donor was usually not a patient with a tumour of the same h i s t o l o g i c type as the target. Kiuchi and Takasugi (1976) showed that the cells responsible for thi s nonselective cytotoxicity did not form E-rosettes, differed from B c e l l s i n that they lacked complement receptors and could survive incubation at 30°C, and were retained on Ig or anti-Ig columns. Later studies by this group presented evidence for the role of a n t i -bodies i n this nonselective c y t o t o x i c i t y . Takasugi e_t a_l. .(1977) showed that the apparently nonselective- cytotoxicity of one donor's lymphocytes for a pa r t i c u l a r target could be inhib i t e d only by the c e l l s of the same source as that target and perhaps a few other c e l l types, but not by a l l the c e l l s against which that donor's lymphocytes were cytotoxic. This supported the hypothesis that nonselective^ cytotoxicity was r e a l l y s p e c i f i c c y t o t o x i c i t y by subpopulations of effector c e l l s , each s p e c i f i c for a different target c e l l . Akira and Takasugi (1977) found that natural 26 c e l l - m e d i a t e d c y t o t o x i c i t y of human lymphocytes f o r v a r i o u s human tumour c e l l l i n e s was decreased when the e f f e c t o r c e l l s were t r e a t e d w i t h p r o t e o l y t i c enzymes or low pH. C y t o t o x i c i t y was regained when e f f e c t o r c e l l s were incubated i n serum. This suggested that a n t i b o d i e s might be present on the e f f e c t o r c e l l s . When serum was absorbed w i t h a ta r g e t c e l l , r e c o n s t i t u t i o n of t r y p s i n i z e d e f f e c t o r c e l l s w i t h that serum r e s u l t e d i n s e l e c t i v e l o s s of c y t o t o x i c i t y f o r the absorbing t a r g e t . The s t u d i e s of Pape et_ a l . (1977) and Troye et a l . (1977), c i t e d i n the s e c t i o n on ADCC, showed that most of the spontaneous c y t o t o x i c i t y of c o n t r o l and p a t i e n t s ' lymphoid c e l l s f o r c u l t u r e d t a r g e t s was a form of ADCC. However, some of the c y t o t o x i c i t y appeared to be antibody-independ-ent because the i n h i b i t i o n caused by r a b b i t Fab against human IgG was u s u a l l y incomplete even at very h i g h l e v e l s of Fab. Kay e_t a l . (1977) d i s t i n g u i s h e d between human e f f e c t o r c e l l s i n ADCC and NK c e l l s . ADCC was assessed u s i n g Chang l i v e r c e l l s coated w i t h spe-c i f i c r a b b i t anti-Chang a n t i b o d i e s . NK c e l l a c t i v i t y was assessed us i n g the s u s c e p t i b l e human myeloid c e l l l i n e K-562 as a t a r g e t . 5 1 C r - r e l e a s e assays were used f o r both. Both ADCC and NK c e l l a c t i v i t i e s were found i n the E-rosette forming c e l l p o p u l a t i o n , and both c e l l types contained Fc rec e p t o r s , as the a c t i v i t i e s were removed by a monolayer of a n t i g e n - a n t i -body (Ag-Ab) complexes. However, p r o t e i n A,which binds to the Fc p o r t i o n of IgG, i n h i b i t e d ADCC but not NK c e l l a c t i v i t y , whereas t r y p s i n and chymo-t r y p s i n i n h i b i t e d NK a c t i v i t y but not ADCC. The st u d i e s could not r u l e out the p o s s i b i l i t y that NK c e l l s had been armed w i t h antibody i n v i v o , but NK a c t i v i t y was not due to lymphocytes making antibody which then bound to tar g e t c e l l s because i n that case i t would have been i n h i b i t e d by p r o t e i n A. 27 The nature of the t a r g e t s t r u c t u r e or s t r u c t u r e s f o r NK c e l l s i s a subject of recent research, Roder et a l . (1979a) i s o l a t e d three p r o t e i n s , or more probably g l y c o p r o t e i n s , which appeared to be NK t a r g e t s t r u c t u r e s , from detergent s o l u b i l i z e d e x t r a c t s of the N K - s e n s i t i v e lymphoma YAC. Each of these molecules i n h i b i t e d the b i n d i n g of unprimed lymphoid c e l l s to YAC t a r g e t s . These had molecular weights (MW) of 140,000; 160,000; and 240,000 and were p o s s i b l y g l y c o s y l a t e d as they bound to Concanavalin A (ConA)-sepharose columns and could be e l u t e d w i t h a-methyl D-mannoside. YAC.is an MLV-induced lymphoma. However, the p u t a t i v e t a r g e t s t r u c t u r e s (NK-TS) were d i s t i n c t from Moloney c e l l surface a n t i g e n , H-2 antigen, the v i r a l envelope c o n s t i t u e n t gp71, and the v i r i o n core p r o t e i n p30. The q u a n t i t i e s of the NK-TS v a r i e d w i t h the NK s e n s i t i v i t y of YAC c e l l s which i s greater f o r YAC c e l l s grown:".in v i t r o than i n v i v o . Detergent e x t r a c t s of other murine and human tumour l i n e s s e n s i t i v e to NK c e l l s showed peaks on polyacrylamide g e l e l e c t r o p h o r e s i s which i n -h i b i t e d the b i n d i n g of NK c e l l s to the homologous ta r g e t and which were s i m i l a r i n MW to at l e a s t one of the t a r g e t s t r u c t u r e s of YAC c e l l s . E x t r a c t s of tumour l i n e s i n s e n s i t i v e to NK c e l l s lacked peaks which could i n h i b i t the b i n d i n g of NK c e l l s to YAC t a r g e t s . However, i n s t u d i e s of i n h i b i t i o n of NK c e l l b i n d i n g , two mouse l i n e s , YAC and MPC-11, c r o s s -reacted w i t h each other as d i d two .human l i n e s , Molt-4 and K562, but the mouse and human l i n e s d i d not c r o s s - r e a c t . Subsequently, NK-TS from v a r i o u s N K - s e n s i t i v e mouse and human tumour c e l l l i n e s were i s o l a t e d by polyacrylamide g e l e l e c t r o p h o r e s i s of detergent s o l u b i l i z e d c e l l e x t r a c t s (Roder e_t a l . 1979b) . A l l the tumour c e l l l i n e s had a 140,000 MW peak which i n h i b i t e d NK c e l l a c t i v i t y toward any s e n s i t i v e 28 target, murine or human. Other higher molecular weight; NK-TS; c a r r i e d unique, or at l e a s t le,S;S widely- d i s t r i b u t e d , s p e c i f i c i t i e s - . Young et a l . (1981) studied two sublines of the DBA/2 murine lymphoma L5178Y which, d i f f e r e d in. that one was- s e n s i t i v e and the other 'insensitive to NK c e l l l y s i s . The s e n s i t i v e , but not the i n s e n s i t i v e , clone contained the. g l y c o l i p i d a s i a l o GM^  on. i t s c e l l surface. However, i t seemed un-l i k e l y , , although not impossible, that a s i a l o GM,, was the target f o r NK c e l l l y s i s because a n t i - a s i a l o GM,, did not i n h i b i t l y s i s , v a r i a t i o n s i n a s i a l o GM^  -between subclones were not associated with v a r i a t i o n s i n sus-c e p t i b i l i t y to NK c e l l l y s i s , and a s i a l o GM.^ was not found i n a l l NK-s e n s i t i v e c e l l l i n e s . NK c e l l a c t i v i t y i s increased by i n t e r f e r o n . Reynolds and Herberman (1981) found that NK c e l l a c t i v i t y was increased by incubation at 37°C i n the presence, of. macrophages or by incubation with Coryriebac tefium parvum, and both of these procedures are known to be associated with i n t e r f e r o n production. Perussia and T r i n c h i e r i (1981) reported that NK c e l l s were ina c t i v a t e d by contact with target c e l l s , and that t h e i r a c t i v i t y could be restored by i n t e r f e r o n . The i n vivo s i g n i f i c a n c e of NK c e l l a c t i v i t y has been reviewed by Herberman and Holden (1978). Nude mice lack a thymus and thus lack T c e l l -mediated c y t o t o x i c i t y . However, they have high NK c e l l a c t i v i t y . Because of .the absence of T c e l l cytotoxicity,.nude mice can accept a l l o g r a f t s and xenografts. However, while such sk i n grafts are r e a d i l y accepted, some transplanted tumours and bone marrow and thymus grafts are rejected. The tumours- to which nude mice, are r e s i s t a n t have been shown to be s e n s i t i v e to NK c e l l l y s i s . Bone:'marrow and thymus c e l l s are also somewhat s e n s i t i v e 29 to NK c e l l s . On the other hand, tumours a r i s i n g spontaneously i n nude mice have been almost a l l NK c e l l - r e s i s t a n t . The r e s i s t a n c e of nude mice to c e r t a i n tumours i s greater i n young mice which have higher NK c e l l a c t i v i t y than o l d e r mice. Habu et a l . (1981) showed that treatment w i t h r a b b i t a n t i - a s i a l o GM^, which k i l l s NK c e l l s , c a u s e d increased tumour takes and increased r a t e s of tumour growth i n nude mice i n j e c t e d w i t h v a r i o u s mouse and human tumour c e l l l i n e s . In s h o r t , the mechanism of a c t i o n and ta r g e t s t r u c t u r e s of NK c e l l s are not yet e s t a b l i s h e d . Although the st u d i e s i n nude mice suggest that NK c e l l s i n v i v o may act as a f i r s t l i n e of defence against s e n s i t i v e tumours, t h e i r r e l a t i v e importance i n human cancers i s unknown. Their importance i s l i m i t e d because of the f a c t that not a l l tumours are NK c e l l - s e n s i t i v e . ( i v ) Macrophages: The c y t o t o x i c effect, of macrophages f o r tumour c e l l s has been s t u d i e d mainly i n mice, and most experiments have used p e r i t o n e a l macrophages. The relevance of the f i n d i n g s i n mice f o r the human s i t u a t i o n , i n which i n v i t r o c y t o t o x i c i t y of p e r i p h e r a l blood " monocytes f o r c u l t u r e d tumour c e l l s i s measured, i s not c l e a r . However, i n man and mice blood monocytes pass i n t o the t i s s u e s and undergo f u r t h e r maturation to form t i s s u e macrophages ( T e r r i t o and C l i n e 1976) . The monocyte can c a r r y out many of the same fu n c t i o n s as the macrophage but i s l e s s potent i n these f u n c t i o n s . Macrophages and monocytes migrate toward chemotactic f a c t o r s produced as a consequence of immunological r e a c t i o n s or t i s s u e breakdown (Snyderman and Mergenhagen 1976) and thus could be expected to accumulate at tumour s i t e s . The usual, procedure f o r separating p e r i p h e r a l bipod or spleen lympho cytes from red c e l l s and polymorphonuclear leukocytes does not remove monocytes unless these are subsequently removed by carbonyl i r o n and a magnet or by a l l o w i n g adherence of monocytes to a surface. Therefore, i n many i n v i t r o s t u d i e s of c y t o t o x i t y f o r tumour c e l l s monocytes could be present. The c y t o t o x i c i t y of macrophages f o r tumour c e l l s has been reviewed by Evans and Alexander (1976). The review d e a l t w i t h s t u d i e s i n mice. There are two categories of c y t o t o x i c macrophage. Some macrophages r e q u i r e the r e c o g n i t i o n of antigen i n an immunologically s p e c i f i c way before expressing t h e i r c y t o t o x i c i t y , whereas others show completely n o n s p e c i f i c c y t o t o x i c e f f e c t s (Evans and Alexander 1976). (a) S p e c i f i c C y t o t o x i c i t y of Macrophages : E a r l y evidence f o r the importance of macrophages i n tumour immu-n i t y came from an experiment reviewed by Evans and Alexander (1976) . Lymphocytes from mice immunized w i t h i r r a d i a t e d syngeneic lymphoma c e l l s could prevent tumour growth i n v i v o when mixed w i t h a tumour c e l l inoculum. The e f f e c t was immunologically specific.l""However,::the lymphocytes themselves were not c y t o t o x i c . o r o n l y weakly ".cytotoxic ;to c u l t u r e d tumour c e l l s . Treatment of the mice w i t h s i l i c a , a macrophage but not a. lymphocyte poison, allowed the tumour c e l l s to grow. Therefore i t appeared that macrophages were necessary f o r tumour r e j e c t i o n . The macrophages in v o l v e d i n s p e c i f i c syngeneic or a l l o g e n e i c tumour r e j e c t i o n may be e i t h e r c y t o l y t i c or c y t o s t a t i c . C y t o l y s i s i s u s u a l l y measured by r e l e a s e of a r a d i o a c t i v e isotope and c y t o s t a s i s by counting the number of s u r v i v i n g target c e l l s . 31 C y t o l y t i c and c y t o s t a t i c macrophages are formed under d i f f e r e n t circumstances. In mice, i n t r a p e r i t o n e a l i n j e c t i o n of a l l o g e n e i c d i v i d i n g lymphoma c e l l s and perhaps the i n j e c t i o n of syngeneic lymphoma c e l l s i n t o pre-immunized mice y i e l d c y t o l y t i c macrophages. Other routes of tumour c e l l i n j e c t i o n , i n j e c t i o n of i r r a d i a t e d tumour c e l l s , or i n v i t r o i n c u b a t i o n of macrophages w i t h s e n s i t i z e d lymphocytes or t h e i r supernatants produce c y t o s t a t i c macrophages. In f a c t , there i s evidence that even i n " c y t o l y t i c " populations of macrophages, only about 15% of the macrophages are c y t o l y t i c w i t h the r e s t being c y t o s t a t i c . Cooperation between normal macrophages and immune lymphocytes leads to the "arming" of the macrophages against the s p e c i f i c tumour c e l l s (Evans and Alexander 1976). Arming can only occur i f the macrophages and lymphoid c e l l s are i n d i r e c t contact. The supernatants of immune T c e l l s incubated w i t h the s p e c i f i c t a r g e t c e l l s are capable of s p e c i f i c a l l y arming normal macrophages. The f a c t o r r e s p o n s i b l e i s c a l l e d s p e c i f i c macrophage arming f a c t o r (SMAF). SMAF i s c y t o p h i l i c f o r macrophages and a l s o binds s p e c i f i c a l l y to target c e l l s . I t appears to be a product of T c e l l s . SMAF c o n s i s t s of two f r a c t i o n s , one of MW 50,000 to 60,000 and the other of MW 300,000. SMAF i s not a c l a s s i c immunoglobulin but might be an immunoglobulin-like molecule on T c e l l s . The supernatants produced by the in c u b a t i o n of immune T c e l l s and t a r g e t c e l l s may render macrophages n o n s p e c i f i c a l l y c y t o s t a t i c due to the presence of lymphokines (see below). Besides the arming of macrophages by s p e c i f i c a l l y immune T c e l l s , macro-phages may act as e f f e c t o r c e l l s i n ADCC w i t h antibody-coated t a r g e t s i n mice (Evans and Alexander 1976; H a s k i l l et al.1977; Kay and H a s k i l l 32 1981). Monocyte monolayers i n humans may also functioncas e f f e c t o r s i n ADCC (Shaw et a l . 1978). In the mouse, IgG2a i s the main antibody sub-clas s a c t i v e i n ADCC with macrophages as e f f e c t o r c e l l s ( H a s k i l l et a l . 1977) . In a murine tumour system, Langlois et a l . (1981) found that tumour-s p e c i f i c IgG2a antibody bound to macrophages to mediate tumour c e l l k i l l i n g i n vivo and i n v i t r o . (b) Nonspecific Cytotoxicity of Macrophages: N o n s p e c i f i c a l l y cytotoxic macrophages are c y t o s t a t i c rather .than c y t o l y t i c (Evans and Alexander 1976). Although not immunologically spe-c i f i c , these macrophages i n h i b i t cancer c e l l s p r e f e r e n t i a l l y but may have some e f f e c t on r a p i d l y d i v i d i n g normal c e l l s (Evans and Alexander 1976). Normal macrophages may be activated to become n o n s p e c i f i c a l l y cytotoxic i n a v a r i e t y of ways. Certain polyanions such as double stranded RNA, polyinosine-polycytosine (polyl-polyC), endotoxin, and the peptidoglycan of b a c t e r i a l c e l l walls a c t i v a t e macrophages. Contact of s p e c i f i c a l l y cytotoxic macrophages with the s p e c i f i c antigen leads to the development of nonspecific c y t o t o x i c i t y . Heat-aggregated serum and anti-immunoglo-b u l i n also a c t i v a t e macrophages. Since immune macrophages have increased surface immonoglobulin, i t has been proposed that binding of immune complexes to macrophages by the Fc fragment i s one means of a c t i v a t i n g macrophages (Evans and Alexander 1976). Lymphokines produced by s e n s i t i z e d T lymphocytes upon contact with s p e c i f i c antigen ormitogen can a c t i v a t e macrophages (Evans and Alexander 1976; David and Remold 1979). The act i v e material i n lymphocyte super-natants i s c a l l e d macrophage a c t i v a t i n g factor (MAF) (David and Remold 33 1979). It has not been possible to separate MAF from migration i n -h i b i t o r y f a c t o r (MIF) , and i n f a c t they may represent the same molecules. There are two species of MIF which d i f f e r i n ' i s o e l e c t r i c point and mole-cular weight, and both have MAF a c i t i v i t y . In a d d i t i o n , both MIF and MAF a c t i v i t i e s are i n h i b i t e d by L-fucose and by treatment of macrophages with L-fucosidase or neuraminidase. Therefore, MIF and MAF recognition structures are l i k e l y fucogangliosides. A recent paper (Taramelli et a l . 1981) showed that MIF-MAF-contairiirig supernatants could a c t i v a t e macrophages to become cytotoxic i f l i p o p o l y -saccharide (LPS) were added, and, with or without LPS, could induce macro-phages which suppressed various lymphocyte functions i n c l u d i n g lymphokine production. Macrophages from mice s u f f e r i n g from various p e r s i s t e n t i n f e c t i o n s are activated i n vivo (Evans and Alexander 1976; David and Remold 1979). The mechanism could be the contact of s p e c i f i c a l l y immune macrophages with antigen (Evans and Alexander 1976) or a c t i v a t i o n byMymphokines (David and Remold 1979). S i m i l a r l y i n humans, macrophages, derived by i n v i t r o incubation of peripheral blood monocytes "for.. 5 ..days, could be stimulated by a supernatant of immune lymphocytes and antigen to become cytotoxic for a v a r i e t y of human tumour c e l l l i n e s (Cameron and C h u r c h i l l 1979). Mouse macrophages can also be activated by i n t e r f e r o n (Boraschi and Tagliabue 1981). (c) Role of Macrophages i n i n Vivo Tumour Resistance: The induction of n o n s p e c i f i c a l l y cytotoxic macrophages i n mice has been shown by several groups of researchers to be associated with 34 increased tumour r e s i s t a n c e as reviewed by Evans and Alexander (1976) . Eccles and Alexander (1974) showed that the r a t e of m e t a s t a t i c spread of various tumours i n r a t s was i n v e r s e l y p r o p o r t i o n a l to t h e i r macrophage content. The macrophage content of the tumours was p r o p o r t i o n a l to the immunogenicity of the tumours as shown by t r a n s p l a n t a t i o n s t u d i e s , and was g r e a t l y reduced i n r a t s deprived of T c e l l s by t h o r a c i c duct drainage (Eccles and Alexander 1974). Thus i t appeared that macrophages were r e -c r u i t e d into'itumours by the cell-m e d i a t e d immune r e a c t i o n . Of course i t i s unclear whether the macrophages help prevent metastases, or are j u s t i n c i d e n t a l byproducts of a T cell-m e d i a t e d immune response. The macrophage content of human melanomas and breast carcinomas was i n v e s t i g a t e d by Gauci and Alexander (1975). In both types of cancer the macrophage content ranged from 0 to 30%. The macrophage content of metastases and of primary tumours that had metastasized was uniformly low, whereas the macrophage content of tumours that had apparently not metas-t a s i z e d covered a wide range. Mansell and D i Luzio (1975) reported r e g r e s s i o n of subcutaneous . Modules of malignant melanoma and breast cancer, and':tumour i n f i l t r a t i o n by macrophages, upon i n j e c t i o n of glucan, a potent macrophage s t i m u l a n t . In summary, although the r o l e of macrophages i n human cancer i s not c l e a r , t h e r e i s evidence that animal and human macrophages can destroy tumour c e l l s i n v i t r o and probably i n v i v o . 2. Leukocyte M i g r a t i o n I n h i b i t i o n Tests The leukocyte m i g r a t i o n i n h i b i t i o n (LMI) t e s t i s based on the f i n d i n g that s e n s i t i z e d lymphocytes r e a c t w i t h t h e i r s p e c i f i c antigen to produce a substance, m i g r a t i o n i n h i b i t o r y f a c t o r (MIF), which i n h i b i t s the 35 migration of macrophages. Early evidence for t h i s phenomenon was presented by Rich and Lewis (1932)whoJshowed 'that when fragments of s p l e n i c t i s s u e from guinea pigs s e n s i t i z e d to t u b e r c u l i n were cultured i n the presence of t u b e r c u l i n migration of c e l l s from the explants was i n h i b i t e d . Similar findings were obtained i n guinea pigs with streptococcal extracts (Moen 1936) and i n humans with t u b e r c u l i n (Gangarosa et a l . 1955). The early experiments suggested that the c e l l s whose migration was i n h i b i t e d were macrophages (Moen 1936; Gangarosa et a l . 1955), More recent studies have used variants of a method developed by George and Vaughan (1962) i n which the migration of leukocytes-from a c a p i l l a r y tube i n the presence or absence of antigen i s measured a f t e r 24 to 48 hours incubation. A good c o r r e l a t i o n was found between delayed h y p e r s e n s i t i v i t y on skin t e s t i n g with an antigen and LMI i n the presence of that antigen i n animals (George and Vaughan 1962; Bloom and Bennett 1966) and i n humans (Thor 1967). Bloom et a l . (1969) f i r s t reported the use of LMI i n a tumour system. Guinea pigs immunized with a soluble extract of a chemically induced tumour i n complete Freund's adjuvant developed delayed h y p e r s e n s i t i v i t y to the extract and t h e i r p e r i t o n e a l c e l l s showed LMI i n the presence of the extract, In the years following t h i s report, LMI t e s t s using tumour extracts as antigen came into quite widespread use i n tumour: immunology. Studies have shown that a higher proportion of cancer patients than of controls have leukocytes which are i n h i b i t e d by extracts of allogeneic cancers of 36 the same h i s t o l o g i c type. This has been demonstrated i n breast cancer (Andersen et a l . 1970; Cochran et a l . 1973, 1974, 1976; Jones and Turnbull 1974, 1975; Black et a l . 1974a, 1976; McCoy et a l . 1974, 1976; Rieche et a l . 1976), malignant melanoma (Cochran et a l . 1972), c e r v i c a l cancer (Chiang et a l . 1976), thyroid cancer (George et a l . 1976), and leukaemia (Gangal et a l . 1976). Serum blocking factors have also been demonstrated using the LMI test i n animal (Halliday 1971; Halliday 1972) and i n human (Rieche et_ a l . 1976) studies. Bloom and Bennett \ (1966) and David (1966) showed that the i n h i b i t i o n of macrophage migration was mediated by a soluble nondialysable f a c t o r , probably a p r o t e i n , released from s e n s i t i z e d lymphocytes upon contact with the s p e c i f i c antigen. The factor has since been named migration i n h i b i t o r y factor (MIF) or macrophage migration i n h i b i t o r y f a c t o r (MMIF) (Pick 1979). MIF can be produced by s e n s i t i z e d B lymphocytes and T lymphocytes i n the presence of the s p e c i f i c antigen (Rosenstreich and Wahl 1979) . Macrophages are required as accessory c e l l s f o r MIF production by both T and B c e l l s (Rosenstreich arid Wahl •1979),. MIF i s not r e s t r i c t e d by the major histocompatability complex (MHC). The stimulation of T lymphocytes to produce MIF might be MHC r e s t r i c t e d i . e . i t i s possible that T lymphocytes could only be stimu-lated by antigen on the surface of macrophages with which they share MHC genes. In most LMI t e s t s , a soluble antigen i s used. Even i f t h i s i s an allogeneic tumour extract, the T lymphocytes would encounter the relevant tumour antigen attached to syngeneic macrophages. 37 In t h i s way the MIF test might be more u s e f u l than c y t o t o x i c i t y assays i n demonstrating cell-mediated immunity to antigens common to tumours of a given h i s t o l o g i c type. However, the c y t o t o x i c i t y assays more c l e a r l y demonstrate the a b i l i t y of lymphocytes from tumour bearers to a c t u a l l y destroy tumour c e l l s . 3. Leukocyte Adherence I n h i b i t i o n Tests The various leukocyte adherence i n h i b i t i o n (LAI) t e s t s are a l l based on the f i n d i n g that the adherence of leukocytes from immune donors to s o l i d surfaces i s i n h i b i t e d i n the presence of the s p e c i f i c antigen. This phenomenon was f i r s t described by H a l l i d a y and M i l l e r (1972) using pe r i t o n e a l leukocytes from mice bearing various chemically-induced tumours with tumour extract as antigens. Their reserach was prompted by the desire to f i n d a more rapid a l t e r n a t i v e to the LMI t e s t . Preliminary studies had suggested that the adherence of macrophages to a s o l i d surface during a b r i e f incubation period might be analogous to macrophage migra-f. t i o n during a longer incubation period. Since c e l l types other than macrophages showed adherence i n h i b i t i o n , the t e s t was c a l l e d the leuko-cyte adherence i n h i b i t i o n (LAI) t e s t . The p e r i t o n e a l c e l l s and':tumour extract were incubated together for 30 minutes and placed i n a haemocyto-meter.- chamber for 60 minutes. The t o t a l number of c e l l s and the number of c e l l s adhering to the haemocytometer s l i d e were then counted and the percentage of adherent c e l l s -.calculated. This type of LAI t e s t i s known as the s l i d e LAI t e s t . The percentage of adherent c e l l s was s i g n i f i c a n t l y reduced when c e l l s from mice with growing tumours, or mice rendered tumour-free by surgery, were incubated with s p e c i f i c tumour 38 extract, compared with'^controls containing no extract or the extract of a d i f f e r e n t tumour. Foeta l c a l f serum (FCS) was present throughout the pro-cedure. Serum from tumour-bearing mice abrogated the LAI reacti o n . Two major modifications of the o r i g i n a l s l i d e LAI test have been developed, the plate or microtest method and the tube method (Halliday 1979). In the microtest or plate method, leukocytes are incubated for 1 to 2 hours with and without antigen i n wells of m i c r o t i t e r p l a t e s , and a f t e r gentle washing the adherent c e l l s are stained and counted micro-s c o p i c a l l y (Holt et a l , 1975; Goldrosen et a l . 1979). Serum i s included i n the incubation mixtures. This method has the advantage of requiring fewer lymphocytes than the other methods (Holt et_ a l . 1975). In the tube method, leukocytes, with and without antigen, are incubated for 2 hours i n large glass test tubes slanted f o r maximization of the glass surface i n contact with the mixture (Grosser and Thomson 1975). A f t e r gentle a g i t a t i o n , nonadherent c e l l s are counted. The tube LAI d i f f e r s from the s l i d e and microtest methods i n that no serum i s present i n the incubation mixtures (Grosser and Thomson 1975; Thomson and Grosser 1979). When the e f f e c t s of serum on the assay are to be determined the leukocytes are always preincubated with serum and then washed before being used i n the LAI assay (Grosser and Thomson 1975; Thomson and Grosser 1979). I n h i b i t i o n of adherence of leukocytes from cancer patients when i n -cubated with extracts of tumours of the same h i s t o l o g i c type as the patient's cancer has been shown using the s l i d e (Maluish and Ha l l i d a y 39 1974; H a l l i d a y and Maluish 1974; H a l l i d a y et ' a l . 1975, 1977; Fujisawa et a l . 1977; Maluish 1979), tube. (Grosser and Thomson:\1975; M a r t i and Thomson 1976; Floras'i et a l . 1977; Tataryn et a l . 1978; Tataryn et a l . 1979; Holan et a l . 1979; Thomson et a l . 1979) and m i c r o t e s t (Russo et a l . 1978; Goldrosen et a l , 1979) v e r s i o n s of the LAI t e s t . A l l of the above s t u d i e s showed r e a c t i v i t y of p a t i e n t s ' leukocytes w i t h e x t r a c t s of a l l o g e n e i c tumours, and s p e c i f i c i t y w i t h regard to h i s t o l o g i c type of tumour, At an i n t e r n a t i o n a l workshop on LAI he l d i n May, 1978, p u b l i c demonstrations of the three LAI methods were he l d using coded leukocyte samples from three cancer p a t i e n t s and appropriate tumour e x t r a c t s . The s l i d e (Maluish and H a l l i d a y 1979) and the tube (Thomson "1979) methods both c o r r e c t l y i d e n t i f i e d the types of tumours a f f l i c t i n g the. leukocyte donors, w h i l e d i f f i c u l t i e s were experienced w i t h the m i c r o t e s t method (Goldrosen et a l . 1979). B l o c k i n g f a c t o r s have been found i n p a t i e n t s ' sera by both the s l i d e (Maluish and H a l l i d a y 1974; H a l l i d a y et a l . 1974, 1975, 1977) and tube (F l o r e s ejt a l , 1977; Thomson and Grosser 1979) LAI assays. The b l o c k i n g f a c t o r s had the same s p e c i f i c i t y as the leukocyte r e a c t i v i t y . The r e l a t i o n s h i p of LAI and b l o c k i n g serum a c t i v i t y to extent of disease depended on the LAI assay used. Studies using the s l i d e LAI t e s t showed a high p r o p o r t i o n of p o s i t i v e LAI t e s t s i n p a t i e n t s w i t h malignant melanoma ( H a l l i d a y et a l . 1975), c o l o r e c t a l cancer ( H a l l i d a y et a l . 1977), and breast cancer (Fujisawa et a l . 1977) regardless of the degree of spread of the disease. In c o n t r a s t , s t u d i e s using the tube 40 LAI method showed a s i g n i f i c a n t decrease i n the p r o p o r t i o n of p a t i e n t s w i t h leukocytes reactive, i n LAI as the disease become more widespread. This was seen i n melanoma ( M a r t i and Thomson 1976), i n c o l o n , p a n c r e a t i c , and stomach cancer ( M a r t i and Thomson 1976; Tataryn. et a l , 1979) and i n breast cancer ( F l o r e s e_t a l . 1977). Serum b l o c k i n g a c t i v i t y was found i n almost a l l p a t i e n t s w i t h a growing tumour using the s l i d e LAT t e s t ( H a l l i d a y e t a l . 1975, 1979), but only i n p a t i e n t s w i t h advanced tumours and negative LAI t e s t s using the tube LAI assay (Fl o r e s et^ a l . 1977; Thomson and Grosser 1979) . The tube LAI r e a c t i v i t y disappeared 3 to 4 months a f t e r surgery i n p a t i e n t s rendered c l i n i c a l l y tumour f r e e and returned some months before c l i n i c a l recurrence (Thomson et a l . 1979). However, s l i d e LAI r e a c t i v i t y p e r s i s t e d 8 months to s e v e r a l years a f t e r surgery even i n p a t i e n t s w i t h no evidence of recurrence (Maluish 1979). With the s l i d e LAI t e s t , monitoring the serum b l o c k i n g a c t i v i t y which disappears a f t e r s u c c e s s f u l surgery and reappears p r i o r to a c l i n i c a l l y d e tectable recurrence may be u s e f u l (Maluish 1979). The mechanisms of LAI i n the s l i d e and tube v e r s i o n s of the t e s t are d i f f e r e n t . In the s l i d e LAI t e s t i n both animals and humans, i t has been shown that s t i m u l a t e d lymphocytes i n contact w i t h s p e c i f i c antigen produce a s o l u b l e mediator c a l l e d leukocyte adherence i n h i b i t i o n f a c t o r (LAT.F) which i n h i b i t s ; t h e adherence of normal leukocytes to glass and p l a s t i c surfaces ( H a l l i d a y 1979; Powell et a l . 1979). H a l l i d a y (1979) reported that LAIF could be produced by both B c e l l s and T c e l l s w i t h accessory adherent c e l l s r e q u i r e d . On the other hand, Powell e_t a_l. (1979) 41 found that only T c e l l s produced LAIF and that T c e l l s were also the c e l l s responding to LAIF. In the tube LAI t e s t , a soluble mediator i s apparently not involved (Grosser and Thomson 1975; Thomson and Grosser 1979). The reactive c e l l , i . e . the c e l l which adhered and which l o s t adherence i n the presence of s p e c i f i c antigen,was found to be a monocyte with Fc receptors (Thomson and Grosser 1979). Normal monocytes could be "armed" by incubation with serum from LAI p o s i t i v e breast cancer patients to give a p o s i t i v e LAI te s t with breast cancer extract. The "arming" factor was IgG. On the other hand, leukocytes from patients with metastatic breast cancer were not r e a c t i v e i n the LAI test and t h e i r serum blocked p o s i t i v e LAI tests with breast cancer antigen. The serum of patients with advanced cancer contained excess tumour antigen. Leukocytes from patients with advanced cancer adhered poorly to glass surfaces, but the addition of antigen did not further decrease the adherence. Thomson and Grosser (1979) proposed that monocytes from patients with early cancer are coated with c y t o p h i l i c anti-tumour antibody which does not decrease t h e i r adherence. In the presence of tumour-specific antigen, the antigen then coats the monocytes, decreasing t h e i r adherence. Serum from such patients contains free anti-tumour antibody which can "arm" normal monocytes. In advanced cancer, monocytes are already coated with antibody and antigen and thus are poorly adherent,.but addition of antigen can have no further e f f e c t on t h e i r adherence. Leukocytes reactive i n LAI would lose t h i s r e a c t i v i t y if.„preincubated with serum from patients with advanced cancer because they would become coated with tumour antigen. 4 2 Another study of the mechanism of the tube LAI: t e s t suggested that s e v e r a l c e l l types i n c l u d i n g lymphocytes, monocytes and polymorpho-nuclear leukocytes could act as r e a c t i v e c e l l s (Tang et a l . 1979). Otherwise, t h e i r proposed mechanism agreed w i t h that of Thomson and Grosser (1979). One study of the mechanism of LAI i n the mic r o t e s t v e r s i o n showed that the monocyte was the r e a c t i v e c e l l and that normal monocytes could be armed by the product of s p e c i f i c a l l y s e n s i t i z e d B c e l l s (Goldrosen et a l , 1979). Another study showed that a s o l u b l e mediator produced by T lymphocytes could mediate LAI (Holt et a l . 1975; Holt e t a l . 1979) i n a mouse tumour system, but serum from ^tumour-bearing mice could arm leukocyte populations depleted of T c e l l s to react i n the LAI t e s t (Holt et a l . 1979) . H a l l i d a y (1979) suggested that the c r i t i c a l d i f f e r e n c e between the ve r s i o n s of the LAI t e s t was the presence of serum i n the s l i d e LAI t e s t but not i n the tube LAI t e s t . He presented evidence that LAIF may be destroyed by proteases present i n tumour e x t r a c t s and n e u t r a l i z e d by serum. He a l s o suggested that the coa t i n g of monocytes by antibody might be i n h i b i t e d by serum p r o t e i n s which might d i s p l a c e Ig from the monocytes. The LAI t e s t s are f a i r l y r a p i d t e s t s f o r tumour immunity. They can detect common tumour antigens on a l l o g e n e i c tumours as can the LMI t e s t . One study using two mouse tumour systems showed a good c o r r e l a t i o n between LAI r e a c t i v i t y and t r a n s p l a n t a t i o n r e s i s t a n c e (Leveson et a l . 1979).„ 43 4. Others Other tests used to detect c e l l mediated anti-tumour immunity include the lymphocyte transformation or stimulation assay i n which the incorporation of t r i t i a t e d thymidine (3H-TdR) into s e n s i t i z e d lymphocytes i s increased i n the presence of tumour antigen (Mavligit et a l , 1974) and skin t e s t i n g with tumour extracts (Black and Leis 1973; Hollinshead et a l . 1974). However, these assays have been used much less than those discussed above and w i l l not be discussed i n d e t a i l here. B. The Role of Humoral Antibody i n Tumour Immunity Although i t has been demonstrated that transplantation immunity to tumour can be transferred with c e l l s but not with serum (Klein and Sjogren 1960; Kleirt et a l . 1960), anti-tumour antibodies can have a major i n - . : fluence on immunity to tumours. Winn (1959) demonstrated that the growth of lymphomas i n mice could be i n h i b i t e d by admixing lymphoma c e l l s with immune serum p r i o r to in; 1 j e c t i o n i n t o mice or by passive immunization with serum before tumour i n j e c t i o n . I n j e c t i o n of guinea pig complement into the r e c i p i e n t mice further increased the i n h i b i t o r y e f f e c t of the serum. However, serum f a i l e d to protect against non-lymphoid tumours which a c t u a l l y grew better i n passively immunized hosts. Early studies with tumour a l l o g r a f t s showed that with some sarcomas (Gorer and K a l i s s 1959)and lymphatic leukaemias (Moller 1963), i n h i b i t i o n of growth took place with i n j e c t i o n of large doses of antiserum and en-hancement of growth with smaller doses. 44 In the above studies, antisera were raised i n allogeneic mice and would have included ahti-H-2 antibodies. Old ejt al_. (1967) reported increased s u r v i v a l i n mice with a syngeneic Gross virus-induced leukaemia upon treatment with rat anti-G (Gross) antiserum. Here the antiserum was s p e c i f i c for a tumour-associated antigen. An antiserum r a i s e d i n r a b b i t s , s p e c i f i c f or a murine mastocytoma, P815, k i l l e d cultured P815 c e l l s i n the presence of complement, protected r e c i p i e n t mice when mixed with P815 c e l l s before i n t r a p e r i t o n e a l i n -j e c t i o n and prevented the recurrence of a proportion--of resected sub-cutaneous P815 tumours (Al-Rammahy and Levy 1979). The mechanisms by which anti-tumour antibodies might i n h i b i t tumour growth i n vivo could include complement-mediated c y t o t o x i c i t y , coating of tumour c e l l s to act as targets i n ADCC, and arming of macrophages and lymphoid c e l l s to become cytotoxic as discussed above. Early studies on the e f f e c t of antibody on cell-mediated anti-tumour immunity have been reviewed by Hellstrom and Hellstrom (1974). In a number of animal and human tumour systems, sera from i n d i v i d u a l s with progressive tumours blocked the cytotoxics and other e f f e c t s of immune lymphocytes from the tumour-bearers. Such blocking factors appeared to be tumour s p e c i f i c antigen-antibody complexes i n some cases and free tumour antigen i n others. Sera from animals and humans rendered tumour-free by surgery or spontaneous tumour regression; often counteract the e f f e c t of blocking sera from i n d i v i d u a l s with the same tumour type. Such sera are said to 45 be unblocking. The unblocking f a c t o r may be an excess of anti-tumour antibody (Hellstrom and Hellstrom 1974) . More recentl y , as discussed above, serum blocking factors have been demonstrated using LMI and LAI assays. Monoclonal antibodies produced by hybrids of myeloma c e l l s and immune spleen c e l l s are currently being used to study tumour-associated antigens. This topic has been reviewed recently by Hellstrom'et a l . (1980). IgG2, but not IgGl, monoclonal antibodies to human melanoma c e l l s were cytotoxic to cultured melanoma c e l l s i n the presence of complement (Yeh et a l . 1979). Two IgG2 monoclonal antibodies against two d i f f e r e n t determinants on human melanoma antigen p97 acted s y n e r g i s t i c a l l y i n complement-mediated l y s i s (Hellstrom e_t al_. 1981) . Koprowski et^ al_. (1978) showed that a monoclonal antibody to a human melanoma could i n h i b i t the growth i n nude mice of:-.a tumour which was a hybrid of human melanoma and mouse f i b r o b l a s t c e l l s . Monoclonal antibodies to human melanoma and c o l o r e c t a l carcinoma could coat the appropriate target c e l l s to act as targets i n ADCC (Herlyn et a l . 1979b). Monoclonal antibodies bound to tumoricidal agents may have p o t e n t i a l i n immunotherapy. Recently, i n a mouse tumour system, a photosensitive cytotoxic agent, bound to a tumour s p e c i f i c monoclonal antibody, was found to decrease tumour s i z e (Mew and Levy 1982). In summary, i t appears that large doses of anti-tumour antibody may be e f f e c t i v e i n i n h i b i t i n g tumour growth by various mechanisms. The 46 e f f e c t of p h y s i o l o g i c a l l e v e l s of anti-tumour antibody produced i n the tumour-bearing host i s less- c l e a r , and i f bound to an excess of tumour antigen may b l o c k cell-mediated anti-tumour r e a c t i o n s . IV. LYMPOCYTE STIMULATION BY PHYTOHAEMAGGLUTININ Phytohaemagglutinin..(PHA) i s derived from the red kidney bean, Phaseolus v u l g a r i s (Yachnin and Svenson 1972). I t i s a mitogen, i . e . a compound which can s t i m u l a t e a l a r g e f r a c t i o n of the lymphocyte p o p u l a t i o n to p r o l i f e r a t e and d i f f e r e n t i a t e (Sharon 1977; Hood et a l . 1978). PHA was o r i g i n a l l y used to a g g l u t i n a t e and thus remove er y t h r o c y t e s when the s e p a r a t i o n of leukocytes from other blood elements was r e q u i r e d (Nowell 1960). Nowell (1960) discovered that PHA causes the p r o l i f e r a t i o n of leukocytes. The subpopulation of leukocytes responding to PHA was l a t e r shown to be T lymphocytes (Geha and Merler 1974; Lohrmann et a l . 1974; Nowell et a l . 1975). However, the a d d i t i o n of B lymphocytes and monocytes to pure populations of T lymphocytes enhances the response to PHA. (Lohrmanri et a l . 1974; P o t t e r and Moore 1975). PHA i s a l e c t i n , i . e . a p l a n t p r o t e i n or g l y c o p r o t e i n which binds s p e c i f i c a l l y to various carbohydrate groups (Sharon 1977; Hood et a l . 1978). The form used i n t h i s study, PHAP, i s a p r o t e i n derived from a g l y c o p r o t e i n p r e c u r s o r , PHAM, by removal of polysaccharide groups (Difco package i n s e r t ) . Both i t s a b i l i t y to a g g l u t i n a t e e r y t h r o c y t e s and i t s mitogenic a c t i v i t y depend upon i t s a b i l i t y to bind to carbohydrate groups on c e l l s u rface g l y c o p r o t e i n s (Sharon 1977). PHA appears to bind s p e c i f i c a l l y to galactose i n the t e r m i n a l or penultimate p o s i t i o n i n the 47 oligosaccharide moiety of a glycopeptide (Kornfeld and Korafeld 1969). It has been suggested that the binding of mitogens to receptors leads to a c l u s t e r i n g of receptor molecules on the lymphocyte surface, which i n turn i n i t i a t e s metabolic changes ins i d e the lymphocyte culminating i n b l a s t transformation and mitosis (Sharon 1977). Lymphocyte a c t i v a t i o n by various mitogens has been widely used as a model for lymphocyte a c t i v a t i o n by antigen. This appears to be justified..-, because lymphocytes stimulated by mitogens can carry out many of the same functions as lymphocytes stimulated by antigen, e.g. immuno-glob u l i n synthesis by B c e l l s and c e l l k i l l i n g by T c e l l s (Sharon 1977; Hood et a l . 1978). The response of peripheral blood lymphocytes to T c e l l mitogens, most commonly PHA, has been used to assess the functional i n t e g r i t y of the cell-mediated immune system i n various diseases in c l u d i n g cancer. The j u s t i f i c a t i o n f o r t h i s i s the concordance between i n vivo tests of c e l l mediated immunity, i . e . delayed type h y p e r s e n s i t i v i t y to serum test antigens, and the response of lymphocytes to PHA. Such concordance has been reported by a number of inves t i g a t o r s (Her.sh and Oppenheim 1965; Sharma et a l . 1971; ''Gatalona et a l . 1975). However, Han and Sokal (1970) found no r e l a t i o n between PHA response of lymphocytes and skin r e a c t i v i t y to mumps and tuberculin antigens i n patients with Hodgkin's d i s e a s e i i n remission, but there was a c o r r e l a t i o n between response to mumps antigen and lymphocyte response to PHA among patients with ac t i v e Hodgkin's disease. Golub et a l . (1974) found no c o r r e l a t i o n between the a b i l i t y to become s e n s i t i z e d to dimitrochlorobenzene (DNCB) and the response of lymphocytes to PHA and other mitogens i n patients with various cancers. 48 However, there was a c o r r e l a t i o n between s e n s i t i z a t i o n to DNCB and the a b i l i t y of lymphocytes to respond i n a mixed lymphocyte reaction- (MLR), and between skin t e s t r e a c t i v i t y to a panel of r e c a l l antigens and responseato mitogens and allogeneic c e l l s . The authors postulated impaired responses to DNCB and i n MLR indicated antigen recognition defects whereas impaired responses to r e c a l l antigens and mitogens indicated p r o l i f e r a t i o n defects which would be a more severe T c e l l de-f i c i e n c y . Thus the MLR would be a more s e n s i t i v e test of T c e l l function than PHA stimulation. The response of lymphocytes to PHA has been assessed bv microscopic observation of the percentage of lymphocytes that have undergone b l a s t transformation a f t e r a sui t a b l e period of incubation. Blast c e l l s are larger than ordinary small lymphocytes. More often, the degree of lympho-cyte "response to PHA i s assessed by measuring the incorporation of 3H-TdR into lymphocyte DNA a f t e r a period of incubation of lymphocytes and PHA. In summary, most studies suggest that lymphocyte response to PHA i s a f a i r l y r e l i a b l e i n d i c a t o r of the function of the cell-mediated immune system. The study of Golub et a l . X1974) suggested that a low response to PHA indicates a f a i r l y severe deficiency of cell-mediated immunity while minor degrees of impairment might be undetected by t h i s assay. V. THE IMPORTANCE OF THE IMMUNE RESPONSE TO BREAST CANCER The i n t e r e s t i n studying: immunosuppression i n cancer depends upon the fi n d i n g , i n many tumour systems, that cancer-bearing i n d i v i d u a l s have an immune response to t h e i r tumours, which may have a bearing on prognosis. 49 In vivo evidence of a cell-mediated anti-tumour immune response i n the form of tumour i n f i l t r a t i o n by lymphocytes and sinus h i s t i o c y t o s i s i n the regional lymph nodes has been associated with a good prognosis i n breast cancer (Black 1973;Humphrey et a l . 1974; Tsakralides 1974 a,b; d i Paola et a l . 1974). However,one study (Flores et al..1974) showed no re l a t i o n s h i p between sinus h i s t i o c y t o s i s and prognosis i n medullary breast cancer, a p a r t i c u l a r p a t h o l o g i c a l v a r i a n t . A recent study of breast cancer patients (Syrjanen and H j e l t 1978) found sinus h i s t i o c y t o s i s and p a r a c o r t i c a l a c t i v i t y , both i n d i c a t i v e of T c e l l a c t i v i t y , i n tumour-free lymph nodes, but degenerative sinus h i s t i o c y t o s i s i n lymph nodes invaded by tumour. Hunter et a l . (1975) examined both a x i l l a r y and internal.mammary, lymph nodes i n patients treated by extended r a d i c a l mastectomy i . e . r a d i c a l mastectomy plus removal of h a l f of the sternum and i n t e r n a l mammary nodes. They put forward the hypothesis that an early tumour releases l i v e c e l l s which stimulate a cell-mediated immune response which helps prevent metastases. Later, the primary tumour becomes p a r t i a l l y n e c r o t i c . M a t e r i a l released from n e c r o t i c c e l l s stimulates an antibody response i n the regional nodes. The antibody blocks the cell-mediated immune response and f a c i l i t a t e s metastases. By the time the tumour i s advanced enough to metastasize to i n t e r n a l mammary nodes, c i r c u l a t i n g immunosuppressive factors are present which decrease the reaction of lymph nodes . This would explain t h e i r f i n d i n g that i n t e r n a l mammary nodes usually show l e s s evidence of immune r e a c t i v i t y than do a x i l l a r y nodes from the same patient. 50 In a s e r i e s of papers (Black et a l . 1974 a, b, 1975, 1976; Zachrau et a l . 1976), Black and h i s co-workers presented evidence f o r a breast cancer-associated glycoprotein antigen of MW approximately 55,000 daltons and r e l a t e d to an antigen of murine mammary tumour v i r u s . Evidence suggested that t h i s antigen i s present on many early breast cancers and often provokes sinus h i s t i o c y t o s i s and l o c a l lymphocytic i n f i l t r a t e s . However, as the tumour progresses the antigen i s often l o s t and the sinus h i s t i o c y t o s i s disappears. This antigen i s associated with patho-l o g i c a l f a c t o r s suggesting a good prognosis. S u r p r i s i n g l y , Stewart and Orizaga (1971) found that a p o s i t i v e delayed-type h y p e r s e n s i t i v i t y s k i n r e a c t i o n to autologous breast tumour extract was associated with a poor prognosis. A number of i n v i t r o studies have also demonstrated cell-mediated immune reactions to human breast cancer. Lymphocytes from a higher proportion of breast cancer patients than of controls have been found to react against autologous and allogeneic breast cancer c e l l s i n colony i n -h i b i t i o n and c y t o t o x i c i t y assays (Hellstrom e_t a l . 1971a; Baldwin e_t a l . 1973; Cummings et a l . 1973; D e l i a Porta et a l . 1973; Warnatz et a l . 1976) and i n lymphocyte blastogenesis assays as measured by the uptake of 3H-TdR (Mavligit et a l . 1974). Patients' lymphocytes also showed greater r e a c t i v i t y than c o n t r o l lymphocytes toward breast tumour extracts i n LMI tests (Jones and Turnbull 1974, 1975; McCoy et a l . 1974, 1976; Cochran et a l . 1973, 1974, 1976; Black et a l . 1976; Rieche et a l . 1976) and i n LAI tests (Maluish and H a l l i d a y , 1974; H a l l i d a y ejt a l . 1974; Flores et a l , 1977; Fujisawa et a l . 1977; Lopez et. a l . 1977; Lopez et a l . 51 1978). In most i n v i t r o studies, cell-mediated immune reactions to breast cancer antigens were found to be stronger i n patients with early breast cancer than i n those with advanced disease (Cochran et^ a l . 1974, 1977; Warnatz et a l . 1976; Flores et al_, 1977 ). However, as mentioned i n I - I I I , Fujisawa et a l (1977) found no s i g n i f i c a n t d i f f e r e n c e i n the percentage of p o s i t i v e s l i d e LAI tests among breast cancer patients with Stage I, I I , and I I I disease. In c o n t r a d i c t i o n to the above r e s u l t s , other studies showed that lymphocytes from healthy women and from breast cancer patients did not d i f f e r i n t h e i r a b i l i t y to i n a c t i v a t e breast tumour c e l l s i n v i t r o (Sinkovics et a l . 1972; Takasugi et a l . 1974; Jeejeebhoy 1975; Canevari et a l . 1976), and that reactive lymphocytes showed a lack of s p e c i f i c i t y f or tumuor type (Sinkovics et a l . 1972; Takasugi et al_.1974; Canevari et a l . 1976). Blocking a c t i v i t y was found i n the sera of many patients with advanced breast cancer (Hellstrom et a l . 1971 b; Cummings et a l , 1973; Warnatz et a l . 1976; Flores et a l . 1977). Sera from breastzcancer patients rendered tumour-free by surgery could often abrogate the e f f e c t of blocking serum (Hellstrom e t a i L . 1971?.c; Maluish and H a l l i d a y 1974), or arm normal lymphocytes to give a p o s i t i v e LAI test (Flores et a l . '1.1'.". 1977) . In summary, although r e s u l t s are contradictory, the majority of studies show evidence that breast cancer patients have cell-mediated immunity toward breast cancers, and that the strength of t h i s immunity 52 decreases with tumour progression. Evidence of cell-mediated a n t i -tumour immunity i n early stage breast cancer i s associated with a good prognosis. VI. IMMUNE COMPETENCE IN BREAST CANCER PATIENTS Patients with a v a r i e t y of tumours frequently show a general depression of cell-mediated immune responses, as shown by.decreased a b i l i t y to become s e n s i t i z e d to dinitrochlorobenzene (DNCB) (Brooks e_t a l . 1972; Golub e_t a l . 1974) or to other newly encountered antigens ( M i l l e r et a l . 1975), by decreased response to skin t e s t i n g with common r e c a l l antigens (Brooks et a l . 1972; Golub et a l . 1974), and by decreased i n v i t r o lymphocyte responses to PHA (Golub et a l . 1974; Buda et a l . 1.1975) i n homologous normal as w e l l as i n autologous serum. Some studies have shown a c o r r e l a t i o n between depresssion of c e r t a i n immune functions and poor prognosis or advanced stage of disease (Buda et a l . 1975; E i l b e r et  a l . 1975; Lee et a l . 1975). Studies of general immune competence i n human breast cancer have been contradictory. Decreased a b i l i t y to be s e n s i t i z e d to new antigens ( M i l l e r et a l . 1975; Bolton et a l . 1975; Adler et_ a l . 1980a) and to .. respond to r e c a l l antigens (Adler et a l . 1980; Bolton et a l . 1975; Nemoto et a l . 1974) has been found. Lymphocyte response to PHA has also been found to be reduced i n patients with breast cancer, even i n early disease (Buda et_ a l . 1975; Knight and Davidson 1975; Adler et a l . 1980). Immuno-suppression has been reported to be more profound i n more advanced breast cancer (Roberts and Jones-Williams 1974; Nemoto at a l . 1974; Knight and 53 Davidson 1975; Adler et al. 1980a). However, other studies have not found evidence of decreased skin reactivity to antigens (Daban et al. 1975)or of impaired lymphocyte responses to PHA (Roberts 1970; Roberts and Jones-Williams 1974). Adler et al. (1980b) found an association between immunocompetence as measured by in vivo and in vitro tests and a favourable prognosis. There is some evidence in human cancer, including breast cancer, that macrophage function may be impaired. Thus lymph nodes draining breasts containing cancer, but not those draining breasts affected by benign disease, showed impaired macrophage function as demonstrated by decreased uptake of a radioactive colloid (Agwanobi and Boak 1978). Decreased peripheral blood monocyte chemotaxis was also demonstrated in patients with breast cancer (Snyderman et al . 1978). Richters (1980) showed that the clustering of lymphocytes about macrophages was significantly greater in tumour-free axillary lymph nodes from breast cancer patients than in tumour-containing nodes. Baum et al. (1973) showed that the phagocytic activity of macrophages from the liver and spleen of patients with localized breast cancer was significantly greater than in those with disseminated disease. Thus the majority of studies indicate a general depression of cell-mediated immune responses in patients with breast cancer, especially in those with advanced disease. The reduction in specific anti-tumour immunity seen in patients with advanced breast cancer,;.as discussed in I-V may be part of the general immunosuppression. The general suppression of immune response may be caused by nonspecific serum inhibitory factors 54 to be d i s c u s s e d i n I-VII," by n o n s p e c i f i c suppressor c e l l s , or by some i n t r i n s i c lymphocyte d e f e c t , or by some combinat ion of t h e s e . The suppress ion of a n t i - t u m o u r immunity cou ld be caused by the above n o n s p e c i f i c mechanisms, but a l s o by s p e c i f i c b l o c k i n g f a c t o r s as men-t i o n e d i n I - I I I and I -V , or by s p e c i f i c suppressor c e l l s . N o n s p e c i f i c suppressor c e l l s have been d e s c r i b e d i n a number of human tumours i n -c l u d i n g b r e a s t (Vose and Moore 1979; Braun et a l . 1980) . C e l l s capable of s u p p r e s s i n g s p e c i f i c a n t i - t u m o u r immunity have been found i n f i l t r a t i n g lung and b r e a s t cancers (Vose and Moore 1979) and i n the p e r i p h e r a l b lood of p a t i e n t s w i t h os teogen ic sarcoma (Yu et a l . 1977) . However, i t was not determined whether or not these same c e l l s cou ld suppress n o n -s p e c i f i c immune r e a c t i o n s . Thus s p e c i f i c suppressor c e l l s have apparent l y not been c o n c l u s i v e l y demonstrated i n humans, but have been found i n murine tumour systems (Takei e_t a l . 1976, 1977) . V I I . IMMUNOSUPPRESSIVE SUBSTANCES IN SERUM AND PLASMA A. Ev idence For and A g a i n s t Serum and Plasma Immunosuppressive  F a c t o r s i n Cancer 1 . Immunosuppression by Serum or Plasma i n Cancers Other Than Breast  Cancer The numerous s t u d i e s in tended to a s c e r t a i n whether or not serum or plasma from cancer p a t i e n t s i s i n h i b i t o r y to the immune responses of :.. normal lymphocytes have g i ven c o n t r a d i c t o r y r e s u l t s . As e a r l y as 1967 some ev idence was presented i n d i c a t i n g t h a t the PHA- induced t r a n s f o r m a t i o n of normal human lymphocytes might be reduced i n the presence of s e r a from p a t i e n t s w i t h cancer of v a r i o u s types ( S i l k 55 1967). S i m i l a r f i n d i n g s were published by Sample et a l . (1971). How-ever, these workers found that v a r i a b i l i t y i n p a t i e n t sera was so great t h a t , w h i l e the o v e r a l l e f f e c t s appeared to be i n h i b i t o r y , the d i f n ferences were not s i g n i f i c a n t . I n h i b i t o r y sera was a l s o found i n p a t i e n t s w i t h symptomatic Hodgkin's disease, but not p a t i e n t s i n remission or w i t h asymptomatic disease (Trubowitz et a l . 1966). In some l a t e r s t u d i e s , plasma or serum from p a t i e n t s w i t h v a r i o u s s o l i d tumours were found to s i g n i f i c a n t l y i n h i b i t the responses of normal lymphocytes to PHA ( G a t t i e t a l . 1970; Brooks et a l . 1972; Menzoian et a l . 1973; Suciu-Foca ej: a l . 1974; Ueda et a l . 1978), to a l l o g e n e i c c e l l s i n MLR (Brooks et a l . 1972; Copeland et a l . 1974; Suciu-Foca et a l . 1974), to t h e i r autologous tumour c e l l s (Brooks et a l . 1972), and to v a r i o u s antigens i n c l u d i n g PPD ( F i e l d and Caspary 1972). Washed lymphocytes from some cancer p a t i e n t s showed subnormal PHA responses when c u l t u r e d i n normal human plasma or serum, apparently i n d i c a t i n g i n t r i n s i c lymphocyte d e f e c t s as w e l l as i n h i b i t i o n by serum ( G a t t i e_t a l . 1970; F i e l d and Caspary 1972; Suciu-Foca et a l . 1974). I n h i b i t o r y sera and i n t r i n s i c lymphocyte defects u s u a l l y occurred together ( G a t t i e_t a l . 1970; F i e l d and Caspary 1972). Menzoian e_t a l . (1973) reported a cor-r e l a t i o n between the presence of i n h i b i t o r y serum and f a i l u r e of the p a t i e n t to react to common r e c a l l antigens and to be s e n s i t i z e d to DNCB. Copeland et_ a l . (1974) showed an increased frequency of i n h i b i t o r y sera i n p a t i e n t s w i t h advanced cancer compared w i t h those w i t h l o c a l i z e d ... disease. Ueda e_t a l . (1978) found that the degree of serum i n h i b i t i o n 56 increased with increasing tumour burden i n ovarian cancer, although i n h i b i t o r y sera were found even i n patients with very small tumours. Similar findings were obtained by F i e l d and Caspary (1972) i n patients with a variety of cancers. In ovarian cancer, the degree of i n h i b i t i o n declined after surgery, and increased with the development of metastases (Ueda et a l . 1979). Leukaemic sera were also found to i n h i b i t the response of normal lymphocytes to PHA (Humphrey et a l . 1974). The i n h i b i t i o n could be overcome by increasing the PHA concentration, and thus may have been due to binding of PHA by components of leukaemic-sera. On the other hand, several studies have f a i l e d to show i n h i b i t i o n of the lymphocyte response to PHA by the serum or plasma of cancer patients (Golub et a l . 1973). Mclllmurray et a l . (1973) actually showed enhancement' of the PHA response of normal lymphocytes i n plasma from patients with advanced colon cancer. Al-Sarraf et a l . (1971) found that lymphocytes from patients with cancer, from patients with non-malignant diseases, and from healthy controls usually gave a higher response to PHA i n the presence of autologous plasma than i n plasma from a member of one of the other two groups. ..They did not study the effect of culturing lymphocytes from a healthy control with PHA i n autologous and allogeneic normal plasma. Some of the discrepancies among such studies may be due to d i f -ferences i n the types of cancer patients studied, i n serum or plasma concentration used, i n PHA concentration used and i n the assay system used 57 to detect PHA stimulation. I t i s also clear that methods of serum storage and the age of serum samples at the time of te s t i n g may have a marked e f f e c t on most i n v i t r o assays for immunologic a c t i v i t y . 2. Immunosuppression by Serum or Plasma i n Human Breast Cancer In studies l a r g e l y or e n t i r e l y r e s t r i c t e d to patients with breast cancer, contradictory r e s u l t s have also been found. Apparently the f i r s t report of the immunosuppressive e f f e c t of serum from breast cancer patients was that of Whittaker et a l . (1971). This group compared the response to PHA of lymphocytes i n autologous .. serum from breast cancer patients with those from women with benign breast disease. ...The percent transformation of lymphocytes i n response to PHA was assessed by autoradiography, and was found to be s i g n i f i c a n t l y lower i n cancer patients. A lower percentage transformation was found i n patients with advanced cancer than i n those with early disease. In s i x patients with advanced cancer, lymphocytes were cultured with PHA both i n autologous serum and i n serum from a normal i n d i v i d u a l . Lymphocytes from the normal person were cultured i n autologous serum and cancer patients' sera. The patients' sera i n h i b i t e d the response of normal lymphocytes to PHA, whereas the response of patients' lymphocytes to PHA i n normal serum was almost equal to that of normal lymphocytes i n normal serum. Shortly a f t e r , G a t t i (1971) reported s i m i l a r findings i n a va r i e t y of cancers inc l u d i n g breast. 58 Matthews and Whitehead (1976) reported the inhibi t ion of l y s i s of antibody-coated.target ce l l s by leukocytes (ADCC) i n the presence of serum from 39% of breast cancer patients compared with 8% of controls. Buda et al. (1975) , in an extension of their previous work (Suciu-Foca et a l . 1974), found that lymphocytes from patients with breast and colon cancer responded to PHA and to allogeneic cel ls in an MLR less well in autologous than in pooled normal serum. There was no significant difference in the responses of lymphocytes from 50 normal controls in autologous compared with pooled serum. Serum from cancer patients inhibited the response of normal lymphocytes to PHA and allogeneic c e l l s . Lymphocytes from cancer patients in pooled normal serum responded i n both assays less well than did lymphocytes from controls. Both the serum inhibi t ion and the apparent i n t r i n s i c lymphocyte d e f i c i t were more marked in patients with more advanced disease. A contradictory report (Blomgren ej: a l . 1975) indicated no s i g n i f i -ficant difference in the response of normal lymphocytes to PHA in breast cancer patients compared with controls with benign breast disease. Another study of breast cancer patients (Whitehead et a l . 1974) supported the previously described results of Al-Sarraf et a l . (1971). Lymphocytes from 19 patients with benign breast disease, 8 with early breast cancer and 14 with advanced breast cancer were stimulated with PHA i n the presence of autologous serum, AB serum from a healthy blood bank donor, pooled serum from patients with advanced breast cancer (none from 59 the lymphocyte donors), pooled serum from patients, with advanced colon cancer, and f o e t a l ca l f serum. In each group of lymphocyte donors, the response to PHA was greatest i n autologous serum. Thus, lymphocytes from patients with advanced breast cancer responded te PHA better i n auto-logous serum than i n normal AB serum. In f a c t , pooled advanced breast cancer serum supported lymphocyte stimulation better than the normal AB serum. The pooled advanced colon cancer serum supported lymphocyte stimulation least w e l l , contrary to the results of Mclllmurray et a l . (1973). With a given serum or serum pool, lymphocytes from benign breast disease patients gave the highest response to PHA and those from advanced breast cancer patients the lowest. The conclusion drawn was that the depression of lymphocyte response to PHA i n the presence of cancer patients' serum compared with autologous serum i s a nonspecific effect of nonself serum. Depression of the lymphocyte response to PHA i n cancer must be due to a lymphocyte defect. This study would have been more con-clusive i f serum from several normal donors or a normal pool had been used. Perhaps the AB serum used i n t h i s study happened to be i n h i b i t o r y . Again, as was the case with studies i n other types of cancer, the differences i n the results of the various studies may be explained i n part by differences i n the details of the assays used, such as differences i n PHA concentration, serum or plasma concentration and incubation times, as well as by differences: i n storage of the sera. B. The Chemical Nature of Immunpregulatory Substances i n Serum arid Plasma Studies designed to determine the chemical nature of nonspecific immunosuppressive substances i n serum or plasma from cancer patients have implicated a number of different substances. Alterations i n the 60 c o n c e n t r a t i o n of many normal serum components commonly occur i n cancer p a t i e n t s . I n a d d i t i o n , some compounds are d e t e c t a b l e i n a p r o p o r t i o n of cancer p a t i e n t s ' s e r a but not i n ^ c o n t r o l s e r a . I n a few s t u d i e s , :;the i n h i b i t i o n of immune f u n c t i o n s of normal lymphocytes by serum from cancer p a t i e n t s o r plasma has been c o r r e l a t e d w i t h the l e v e l s of one or more serum p r o t e i n s or g l y c o p r o t e i n s . V i r t u a l l y every substance known to be i n c r e a s e d i n the s e r a of a p r o p o r t i o n of cancer p a t i e n t s has been c o n s i d e r e d a p o s s i b l e cause of immunosuppressive e f f e c t s commonly seen i n such s e r a . A number of these substances have been p u r i f i e d and t e s t e d f o r i m m u n o s u p p r e s s i v e ; . a c t i v i t y . Few s t u d i e s have been concerned s o l e l y w i t h b r e a s t c a n c e r . Few s t u d i e s have d e a l t w i t h the enhancement of immune r e s p o n s e s . 1. Normal Serum Components w i t h A l t e r e d C o n c e n t r a t i o n s i n Cancer: (1.1) A l t e r a t i o n s i n Normal Plasma P r o t e i n s i n Cancer: The t o t a l plasma p r o t e i n c o n c e n t r a t i o n i s u s u a l l y decreased i n p a t i e n t s w i t h cancer because of a decrease i n albumin c o n c e n t r a t i o n (Schwartz and Young 1979). a i - a n d 012 — g l o b u l i n s and f i b r i n o g e n , are u s u a l l y i n c r e a s e d , 3 - g l o b u l i n s and • T - g l o b u l i n s may be i n c r e a s e d s l i g h t l y i n advanced cancer but l e s s so than the ."..a - g l o b u l i n s . The e l e v a t i o n of a2 - g l o b u l i n s i n cancer i s not due to an e l e v a t i o n of a2 -macroglobul in (Schwartz and Young 1979). Most of the a i - and 012-g l o b u l i n s are acute phase r e a c t a n t s , p r o t e i n s and g l y c o p r o t e i n s which i n c r e a s e i n many d i s e a s e s i n c l u d i n g c a n c e r , major s u r g e r y , i n f e c t i o n s , b u r n s , and many inf lammatory and degenerat ive d iseases ( B r a d l e y et a l . 1977; Schwartz and Young 1979). The acute phase r e a c t a n t s which are 61 often elevated i n cancer are (Xi-antitrypsin, haptoglobin, haemopexin, caeruloplasmin and c t i-acid glycoprotein. T r a n s f e r r i n and pre-albumin are ot-globulins which are usually decreased i n cancer patients (Schwartz and Young 1979). Several studies have shown a r e l a t i o n s h i p between elevation of serum o t-globulins and immunosuppression by serum i n cancer p a t i e n t s . Hsu and Lo Gerfo (1972) found that within a group of 15 patients with colon cancer, most having early disease, high plasma carcino-embryonic antigen (CEA) l e v e l s were associated with high serum ot-globulins and with serum which was i n h i b i t o r y toward the PHA response of normal lymphocytes. F i e l d and Caspary (1972) reported that t h e i r lymphocyte depressive factor was an ot2-globulin. Minton and Bianco (1974) found elevated o t 2-globulins i n 32 of 39 patients with metastatic breast cancer , and i n 1 of 11 with disease confined to the breast and regional nodes. Serum albumin was decreased i n 30 of 39 patients with metastatic disease and i n 2 of 11 with l o c a l i z e d disease. The authors discussed the possible immunosuppressive e f f e c t of Ota-globulins but apparently d i d not tes t the sera for immunosuppression. Vetto e_t a l . (1974) found that sera from 13 of 26 patients with a var i e t y of cancers, (not including breast cancer), i n h i b i t e d the response of normal lymphocytes to PHA and pokeweed mitogen (PWM). Immuno-suppression was correlated with elevated 0t2-globulins detected by electrophoresis. Immunosuppresive sera and elevated serum Ota-globulins occurred more frequently i n patients with progressive disease. 62 This group of workers suggested that the elevation of a-globulins seen i n cancer patients might be the r e s u l t of a per s i s t e n t immune response to a per s i s t e n t antigen (Burger et a l . 1974). They found that serum a-globulins were elevated and secondary immune responses to other antigens were decreased i n guinea pigs during development of a c e l l u l a r immune response to DNCB. On the other hand, G a t t i ej: a l . (1970) found no c o r r e l a t i o n between serum ag-globulins and plasma i n h i b i t i o n of PHA responses. Bradley e_t a l . (1977) measured t o t a l protein-bound carbohydrate, CEA, and 18 serum proteins i n 43 patients with a va r i e t y of non-lymphoid malignancies. They also measured the response of patients' and control lymphocytes to PHA i n 20% normal AB serum. The response of lymphocytes to PHA i n the presence of patients' sera was not studied. The response of patients' lymphocytes to PHA was s i g n i f i c a n t l y lower than that of controls. Low response to PHA was s i g n i f i c a n t l y correlated with low l e v e l s ofo^-HS glycoprotein and albumin and with high l e v e l s of a\-a n t i t r y p s i n and haptoglobin. The response of lymphocytes to PHA may r e f l e c t i n part influences of the donor serum unless the lymphocytes have been washed very thoroughly (Mannick et a l . 1977). A l t e r n a t i v e l y , decreased lymphocyte r e a c t i v i t y and elevation of acute phase reactants with decreased a2~HS glycoprotein and albumin are both found i n advanced disease and may not be causally r e l a t e d to each other. One report described an attempt to remove nonspecific i n h i b i t o r y a c t i v i t y from patients' sera by plasmapheresis(Israel et a l . 1977). 8 of 27 patients showed a p a r t i a l response to t h i s treatment. Af t e r plasmapheresis, responders showed a more marked decrease i n serum 63 c i i - and a2-globulins, a i - a n t i t r y p s i n , haptoglobin, a i - a c i d glycoprotein, IgA, and IgM than did nonresponders. The differences were not s t a t i s t i c -a l l y s i g n i f i c a n t . (1.2) Immunosuppressive E f f e c t s of Some Normal Substances Which are Elevated i n the Serum or Plasma of Cancer Patients: (i ) Normal Immunoregulatory a-Globulins: A number of research groups have demonstrated an immunosuppressive a-globulin f r a c t i o n i n plasma from normal rats (Kamrin 1959; Mowbray 1963a), rabbits (Mowbray 1963a), oxen (Mowbray 1963a), and humans (Mowbray 1963a; Mannick and Schmid 1967; Hanna e_t a l . 1975). The material of Mowbray and h i s colleagues was c a l l e d Fraction C and was usually i s o l a t e d from bovine plasma (Mowbray 1963b). The immunosuppressive f r a c t i o n of human plasma studied by Mannick and h i s co-workers was c a l l e d immunoregulatory a-globulin (IRA) (Cooperband e_t a l . 1968) , while that of Hanna and h i s colleagues was c a l l e d normal immunosuppressive protein (NIP) (Hanna e_t a l . 1975). Lack of species s p e c i f i c i t y i n the immunosuppressive e f f e c t s was found for Fraction C (Mowbray 1963a,b; Mowbray and Scholand 1966; Milton 1971), for IRA (Mannick and Schmid 1967), and for NIP (Hanna et a l . 1975). The immunosuppressive e f f e c t s included prolongation of skin a l l o g r a f t s u r v i v a l by Kamrin's preparation (Kamrin 1959), Fraction C (Mowbray 1963a), and IRA (Mannick and Schmid 1967); i n h i b i t i o n of the antibody response to T cell-dependent antigens by Frac t i o n C (Mowbray 1963b; Mowbray and Scholand 1966); i n h i b i t i o n of lymphocyte p r o l i f e r a t i o n i n response to 64 mitogens and antigens by Fract i o n C (Milton 1971) and IRA (Cooperband et a l . 1968); i n h i b i t i o n of the formation of human lymphocyte E-rosettes by IRA (Menzoian et a l . 1974a) but not by NIP (Hanna et a l . 1975); the enhancement of tumour growth by NIP (Ovadia et a l . 1975); and the i n -h i b i t i o n by NIP of human T - c e l l c y t o t o x i c i t y to chicken erythrocytes and non-T c e l l ADCC to chicken erythrocytes (Hanna et a l . 1975). Lympho-cyte stimulation by antigens including allogeneic c e l l s was more sen-s i t i v e to IRA than was stimulation by mitogens (Cooperband ej; a l . 1972). Although there were many s i m i l a r i t i e s , the various a-globulin pre-parations were not i d e n t i c a l . F r a c t i o n C contained only oi2-globulins (Mowbray 1963a), whereas Kamrin's preparation (Kamrin 1959) and IRA (Mannick and Schmid 1967) contained oti-and 012-globulins. Mowbray and Hargrave (1966) found that F r a c t i o n C contained RNase a c t i v i t y . The lower molecular weight bovine pancreatic RNase, not immunosupppressive i t s e l f , acquired the a b i l i t y to i n h i b i t antibody responses i n mice upon coupling to a v a r i e t y of protein c a r r i e r s (Mowbray and Scholand 1966). IRA did not show RNase a c t i v i t y (Cooperband et^ a l . 1969). The mechanism of ac t i o n of F r a c t i o n C and IRA appeared to be d i f -ferent. Milton and Mowbray (1972) presented evidence that F r a c t i o n C exerted i t s e f f e c t by causing a r e v e r s i b l e decrease i n synthesis of lymphocyte receptors secondary to a decrease i n ribosomal mRNA synthesis. Thus a preincubation of lymphocytes with Frac t i o n C was necessary for the f u l l i n h i b i t o r y e f f e c t to be seen. In contrast, preincubation with IRA followed by washing did not a f f e c t the response of lymphocytes to mitogens and antigens (Cooperband et a l . 1972). IRA was i n e f f e c t i v e i f added a f t e r the p r o l i f e r a t i v e response had begun. It appeared to act as a noncompetitive i n h i b i t o r of mitogen and antigen stimulation. This would imply the presence of separate lymphocyte receptors for IRA and for mitogens and antigens. Occhino e_t a l . (1973) reported that an immunosuppressive peptide (IRA-peptide) could be dissociated from IRA by d i a l y s i s i n buffers of low pH or high i o n i c strength. IRA-peptide, l i k e IRA, i n h i b i t e d T c e l l but. not B c e l l functions (Menzoian at a_l. 1974a) , including prolongation of renal a l l o g r a f t s u r v i v a l i n rats (Menzoian et a l . 1974b). Subsequently, immunosuppressive sera from cancer patients were studied to determine whether the suppression was due to a peptide s i m i l a r to IRA-pep tilde (Glasgow et a l . 1974; Nimberg et a l . 1975). A peptide, chemically s i m i l a r to IRA-peptide, was diss o c i a t e d from albumin and/or 8-globulins of cancer patients'-sera. The albumin and 8-globulin-containing f r a c t i o n of cancer patients' sera was immunosuppressive whereas the corresponding f r a c t i o n of normal sera was not. The authors postulated that IRA-peptide, which i s normally c a r r i e d by a-globulins, increases i n amount i n cancer pa t i e n t s , saturating the a-globulins and binding to other plasma proteins. NIP was measured i n serum by a haemagglutination test (Nelken e_t a l . 1975) . Elevated NIP l e v e l s were found i n the sera of some cancer patients ( i i ) Serum Protease I n h i b i t o r s : The normal plasma protease i n h i b i t o r , i n t e r - a t r y p s i n i n h i b i t o r 66 (IATI), as well as the a n t i g e n i c a l l y related abnormal protease i n h i b i t o r , EDC1, found i n the urine of some cancer patients (Chawla et a l . 1978), were found to i n h i b i t the response of normal lymphocytes to PHA (Chawla et a l . 1980). There was evidence that t h e i r immunosuppressive a c t i v i t y might be re l a t e d to t h e i r a c t i v i t y as protease i n h i b i t o r s , i n that IATI was about 3 times as potent as EDC1 i n both a c t i v i t i e s . Both a c t i v i t i e s of IATI were heat l a b i l e , whereas both functions of KDC1 were heat stable (Chawla et a l , 1980). IATI was immunosuppressive at physiologic con-centrations, whereas EpCl was suppressive only at concentrations ex-ceeding those found i n cancer patients' plasma (Chawla et a l . 1980). e-amino caproic acid (e-ACA) and other low molecular weight protease i n h i b i t o r s had previously been shown to i n h i b i t the lymphocyte response (Hirshhorri et a l . 1971). Other possible mechanisms of action of C-ACA were ruled out, suggesting that i n h i b i t i o n of pr o t e o l y s i s might be re-late d toithe i n h i b i t i o n of lymphocyte stimulation. PHA stimulation of lymphocytes i s associated with an increase i n i n t r a c e l l u l a r protease a c t i v i t y (Grayzel e_t al_. 1975). N-o^-tosyl-L-lysyl chloromethyl ketone (TLCK) i n h i b i t e d both the increase i n protease a c t i v i t y and lymphocyte H-TdR uptake. The major protease i n h i b i t o r s of serum are oti-antitrypsin and ot2-macroglobulin, not IATI ( L a u r e l l and Jeppsson 1975). Chase (1972) p u r i f i e d 0(2-macrogl6bulin from human plasma and serum, and found that i t i n h i b i t e d the response of normal human lymphocytes to ) PHA. The 0t2-macroglobulin contained e s t e r o l y t i c a c t i v i t y , but did not i n h i b i t es-terolysis by t r y p s i n . However, i t s a b i l i t y to i n h i b i t p r o t e o l y s i s by tr y p s i n was apparently not tested. a 2-macroglobulin i s not regula r l y elevated i n cancer patients' sera (Schwartz and Young 1979). Apprently a i - a n t i t r y p s i n , which i s one of the acute phase react-ants often elevated i n the sera of cancer patients (Bradley et a l . 1977), has not been p u r i f i e d and tested f o r immunosuppressive a c t i v i t y . Stachurska et a l . (1975) studied the composition of each of the three f r a c t i o n s obtained by chromatography of normal human plasma on a DEAE-c e l l u l o s e column according to the method of Mowbray (1963 a,b). Each f r a c t i o n was analyzed by immunoelectrophoresis, and the e f f e c t on contact h y p e r s e n s i t i v i t y i n mice was measured, a j - a c i d glycoprotein and o t i -a n t i t r y p s i n were present i n the highest concentration i n the most immuno-suppressive f r a c t i o n C, and i n lower concentrations i n the less immuno-suppressive f r a c t i o n B. The nonimmunosuppressive f r a c t i o n A contained no a i - a c i d glycoprotein and a small amount of a i - a n t i t r y p s i n compared with B and C. Therefore, these two glycoproteins appeared l i k e l y immuno-suppressive substances, but they were not separated from each other. Therefore, i t was not determined whether one or both was immunosuppressive, ( i i i ) C i i - Acid Glycoprotein;" a i - a c i d glycoprotein (a i^AG) i s one of the acute phase reactants elevated i n the sera of some cancer patients (Schwartz and Young 1979). As mentioned above, Stachurska et a l . (1975) found that a i-AG might be one of the immunosuppressive substances i n normal human plasma. More recently, Chiu et a l , (1977) reported that p u r i f i e d Oti-AG,* at physiologic concentrations of 550 to 1400 Jig/ml (Putman 1975), 68 i n h i b i t e d the response, of normal lymphocytes to PHA and other mitogens and to allogeneic c e l l s . a,i-AG interacted with PHA., but i t also i n t e r -acted d i r e c t l y with, lymphocytes as i t decreased the lymphocyte response i n the MLR, and as preincubation of lymphocytes with oti-AG decreased t h e i r response to optimal concentrations of PHA. One study suggested that a i-AG might be elevated i n response to excessive immune stimulation (Jenkins et a l . 1973). In a study of patients with toxaemia of pregnancy, the serum cti-AG was elevated. The most elevated l e v e l s of a i-AG were associated with the greatest in-.:', h i b i t ion by serum of the MLR between the patients' lymphocytes and those of her husband. High l e v e l s of a i-AG were also associated with high responses i n the MLR i n the absence of the -patient:'s serum i . e . with a greater d i s p a r i t y between spouses at the HL-A. locus. - I t was suggested that a i-AG i n pregnancy was elevated i n response to an excess immune reaction of the mother against the foetus, and served to decrease i t . A recent study (Gahmberg and Anderson 1978) showed that ai-AG i s synthesized by lymphocytes, e s p e c i a l l y activated lymphocytes, as w e l l as by the l i v e r . This may explain i t s elevation i n conditions, such as inflammation, major surgery, and cancer, which are associated with p r o l i -f e r a t i o n of leukocytes. (iv) Transcortin: Transcortin, the C o r t i s o l binding protein of serum, was found to be elevated i n patients, with breast cancer, Sej:a;;f;tPW..preas-t'.--.c;a.nceE'patients i n h i b i t e d PHA blastogenesis both before and a f t e r d i a l y s i s , Transcortin was synthesized by malignant breast t i s s u e . Transcortin with t i g h t l y bound Cortisol, at a concentration of 0.1 mg/ml , i n h i b i t e d normal lymphocyte responses to PHA by 35% (Werthamer ejt a l . 1976). This i s j u s t above the normal concentration of t r a n s c o r t i n , and within the range seen in breast cancer. Serum t r a n s c o r t i n i s elevated up to 0.12 mg/ml. during administration of oestrogens because of increased hepatic synthesis (Werthamer et a l . 1976). (v) Polyamines : Polyamines are small c a t i o n i c molecules which are widespread in nature. Increased synthesis and accumulation of polyamines are seen in s i t u a t i o n s involving rapid c e l l growth. Increased 24-hour urinary ex-c r e t i o n of one or more of the polyamines, putrescine, spermidine, and spermine, was found in a high proportion of cancer patients (Russell 1971). Increased serum polyamines are also common in cancer (Schwartz and Young 1979). Recently, Gaugas and Curzen (1978) found that serum from pregnant women a f t e r 15 weeks gestation combined with the polya-mines spermine and putrescine to markedly i n h i b i t the response to PHA of normal rat lymphocytes. Apparently,similar studies in cancer patients have not been reported, but perhaps polyamines might i n t e r a c t with sera of cancer patients with r e s u l t i n g immunosuppression. (vi) Lipoproteins: Immunosuppressive substances have been found in the very low density l i p o p r o t e i n and chylomicron f r a c t i o n (Waddell et a l . 1976; C h i s a r i 1977). and in the low density l i p o p r o t e i n f r a c t i o n s ( C u r t i s s and Edgington 1976) of normal human serum, and i n the high density l i p o p r o t e i n f r a c t i o n (Stimson and Blackstock 1976) of sera from pregnant women. However, serum li p o p r o t e i n s are usually decreased rather than increased i n cancer (Schwartz and Young 1979) and immunosuppressive l i p o p r o t e i n s have apparently not been implicated as a cause of the immunosuppressive e f f e c t of cancer patients' sera. ( v i i ) Albumin and T r a n s f e r r i n : Very l i t t l e has been written about serum substances which enhance immune responses. One study a t t r i b u t e d a l l or almost a l l the a b i l i t y of serum to support lymphocyte stimulation by mitogens to albumin (Polet and Spieker-Polet 1975). Serum albumin i s sometimes reduced i n cancer patients (Schwartz and Young 1979). Another study (Tormey and Mueller 1972) showed that t r a n s f e r r i n was necessary f or normal lymphocyte DNA synthesis i n response to PHA. In these experiments, t r a n s f e r r i n was used at a concentration of 10 yg/ml which was s u f f i c i e n t to support normal stimulation. However, since the normal t r a n s f e r r i n concentration i n serum i s 2 to 4 mg/ml, (Putman 1975) i t seems u n l i k e l y that any modest reduction i n serum t r a n s f e r r i n seen i n cancer would have much e f f e c t on the serum immunosuppression. 2. Abnormal Substances i n Serum or Plasma of Cancer Patients (i ) Carcinoembryonic Antigen (CEA): CEA i s a protein-polysaccharide complex of MW 200,000, which i s found i n f o e t a l i n t e s t i n e , l i v e r and pancreas, and i n many human cancers (Schwartz and Young 1979). It appears to be synthesized by the tumour c e l l s themsleves, and i s elevated i n the plasma of many cancer patients. A l e v e l below about 2.5 ng/ml i s considered normal. About 18% of breast cancer patients have plasma CEA l e v e l s of 2.5 to 5 ng/ml and 27% greater than 5.0 ng/ml (Schwartz and Young 1979). Two studies have shown a p o s i t i v e c o r r e l a t i o n between elevated CEA and i n h i b i t i o n of PHA blastogenesis by cancer patients' sera (Hsu and Lo Gerfo 1971; Suciu-Foca et a l . 1974), while one has shown a negative c o r r e l a t i o n (Steward et a l . 1974). P u r i f i e d CEA did not i n h i b i t the response of normal lymphocytes to PHA (Steward et a l . 1974; Suciu-Foca et a l . 1974). Therefore, the association between i n h i b i t o r y serum and high CEA l e v e l s was probably due to t h e i r common association with advanced disease (Hsu and Lo Gerfo 1971; Schwartz and Young 1979). ( i i ) a-foetoprotein: a-foetoprotein, a f o e t a l a i - g l o b u l i n , i s often detectable i n the serum of patients with l i v e r cancer and embryonal malignant te r a t o -blastomas of the testes and ovaries (Abelev 1971; Schwartz arid."Young 1979). It has been reported to i n h i b i t various immune responses including PHA blastogenesis (Murgita and Tomasi 1971 a, b; Yachnin 1976). However, since a-foetoprotein i s detectable i n sera of le s s than 1% of patients with cancers other than l i v e r and teratoblastomas (Abelev 1971), i t would not contribute to immunosuppression by sera of breast cancer patients. ( i i i ) Pregnancy-Associated a2 _Macroglobulin: Pregnancy-associated macroglobulin (PAM) i s a glycoprotein of MW 506,000 found i n the serum of women"in l a t e pregnancy, but usually un-detectable i n normal serum (Stimson 1972). PAM i s frequently elevated 72 i n i n d i v i d u a l s treated with oestrogens, ei t h e r as a component of o r a l contraceptives or as treatment for p r o s t a t i c or breast cancer (Berne 1973; Home et^ a l . 1973) . PAM i s also elevated i n some cancer patients not on oestrogens'(Stimson 1972; Berne 1973; Horne e_t al. 1973). PAM l e v e l s i n pregnancy are much higher than those seen under other circumstances (Home et a l . 1973). One study indicated that f l u c t u a t i o n s i n PAM i n patients with breast cancer treated with chemotherapy might r e f l e c t changes i n disease burden (Stimson 1975). Plasma from pregnant women suppresses the response of normal lympho-cytes i n the MLR (Kasakura 1971). P u r i f i e d PAM i n h i b i t e d the response of normal lymphocytes to PHA (Stimson 1-972; Stimson 1976), and to Con A, PPD, and allogeneic c e l l s (Stimson 1972). The i n h i b i t i o n was concentra-t i o n dependent. However, the amount of PAM present i n sera of pregnant women was i n s u f f i c i e n t to account for a l l the immunosuppressive e f f e c t of such sera (Stimson 1976), and removal of PAM from pregnancy serum by a f f i n i t y chromatography removed only part of the i n h i b i t o r y a c t i v i t y (Stimson 1980). As PAM i s les s elevated i n cancer patients than i n pregnancy i t seems .u n l i k e l y that PAM makes a major contribution to immunosuppression by cancer p a t i e n t s ' sera, except perhaps.'in patients treated with oestrogens. Indeed, the degree of immunosuppression by serum was found not to co r r e l a t e with PAM_levels i n pregnancy (Stimson 1976; Stimson 1980; Okamura e_t a l . 1980) and i n patients with p r o s t a t i c cancer on oestrogens,.. and patients with primary b i l i a r y c i r r h o s i s (Horne e_t a l . 1973). (iv) C-reative P r o t e i n : C-reactive protein (CRP) (MW 110,000 to 140,00 0)is an acute phase 73 reactant which i s elevated i n a number of diseases including cancer (Claus et a l . 1976). It has usually been measured by a r a d i a l immunodiffusion procedure by which the l e v e l s i n most normal sera are undetectable. How-ever, by a s e n s i t i v e radioimmunoassay the normal range was found to be 68-8200 ng/ml, with a median of 580 ng/ml (Claus et a l . 1976). \ There i s some heterogeneity of CRP, and forms of both 8- ..(Hornung and F r i t c h i i 1971; Hokama et a l . 1972) and ..(Hokama et a l . 1972; O i s h i et --al.,..1973;'..Mortensen. et .al. 1.1975) el e c t r o p h o r e t i c m o b i l i t y have been described. However, the Y form i s apparently the one that has been more in t e n s i v e l y studied (Hokama et a l . 1972; O i s h i et_ a l . 1975; Mortensen and Gewurz 1976). C-reactive protein i s so .named because i t p r e c i p i t a t e s with the C-polysaccharide of pneumococcal c e l l walls. P u r i f i e d Y-CRP has been shown i n some studies to decrease the response of normal lymphocytes to PHA at concentrations ranging from 16 to 40 Ug/ml. (Hokama et a l . 1973; O i s h i et a l . 1973). Other studies found that Y ~ C R P bound to T c e l l s but not to B c e l l s and i n h i b i t e d E-rosette formation and MLR but not PHA or Con A blastogenesis at 10 ug/ml. (Mortensen ej; a l . 1975; Mortensen and Gewurz 1976). Phosporyl choline binds to CRP and could counteract i t s immunosuppressive e f f e c t s (Oishi et a l . 1973). With the upper l i m i t of normal CRP considered v a r i o u s l y 8.2 ug/ml (Claus et a l . 1976) or 12 ug/ml (Putman 1975), i t i s possible that the elevated l e v e l s seen i n some cancer patients might contribute to the serum immunosuppresive e f f e c t . 74 O i s h i et a l . (1973) found that leukocytes from patients with various cancers i n 40% autologous plasma and 4% PCS responded to PHA s i g n i f i c a n t l y l e s s w e l l than d i d c o n t r o l leukocytes i n autologous plasma and FCS. The PHA response was s i g n i f i c a n t l y lower for leukocytes and plasma from CRP+ patients compared with CRP- patients. However, i t i s doubtful that t h i s d i f f e r e n c e was due to CRP, as the addition of phosphoryl choline improved the PHA response l i t t l e i n CRP+ pa t i e n t s , and more i n controls and CRP-patients. CRP elev a t i o n and serum immunosuppression may both r e f l e c t advanced disease. (v) Immune Complexes: Ag-Ab complexes involving tumour antigen have been implicated as the cause of s p e c i f i c blocking of cell-mediated anti-tumour immunity (Sjogren et a l . 1971). In a mouse tumour system, Gorczynski at a l . (1975) found that such complexes could s p e c i f i c a l l y block cell-mediated immunity to tumour-associated antigens at low concentrations, but could block T c e l l func-tion s , eg. PHA blastogenesis, n o n s p e c i f i c a l l y at higher concentrations. A l l lymphocyte cultures contained 10% FCS. Kilburn et a l . (1976) showed that immune complexes alone were not n o n s p e c i f i c a l l y immunosuppressive, but synergized with normal and espe-c i a l l y tumour-bearer mouse serum to i n h i b i t Con A blastogenesis of normal mouse lymphocytes. A recent study of breast cancer (Tanaka e_t a l . 1979) has shown that immune complexes, but not antigen or antibody alone, can mediate s p e c i f i c blocking i n the LAI assay, and also nonspecific blocking of the response of normal lymphocytes to PHA. Immune complexes were p r e c i p i t a t e d from 75 serum by polyethylene g l y c o l . The amount of immune, complexes p r e c i p i t a t e d was lowest i n normal controls, highest i n breast cancer patients with blocking sera, and s i m i l a r and intermediate i n patients with benign breast disease and breast cancer without blocking sera. To test t h e i r e f f e c t on LAI and PHA blastogenesis, the:' immune complex p r e c i p i t a t e was dissolved i n phosphate buffered s a l i n e (PBS) of the o r i g i n a l serum volume. Most of the sera that were blocking i n LAI and PHA blastogenesis also had " .~.o blocking immune complexes (92% f o r LAI, 100% for PHA). 8 of 11 sera which were negative f o r blocking of LAI yie l d e d non-blocking immune complexes. However, of 4 breast cancer sera which were not blocking to PHA b l a s t o -genesis, 3 yielded blocking immune complexes. Of ten patients whose sera and complexes were studied i n both LAI and PHA blastogenesis assays, 6 of 10 had blocking sera and complexes i n both assays, 1 of 10 had blocking serum and complexes i n LAI only, and 3 of 10 had blocking serum and complexes i n the PHA blastogenesis assay only. The greater frequency of blocking sera i n the PHA blastogenesis assay may simply r e f l e c t the fact that a serum sample was c a l l e d blocking i f i t i n h i b i t e d PHA blastogenesis by 35%, but i f i t i n h i b i t e d LAI by 70%. Blocking i n the LAI assay was s p e c i f i c , i . e . blocking sera from breast cancer patients usually did not block the LAI of - lymphocytes from melanoma patients with melanoma extracts. The lymphocyte cultures containing immune complexes and PHA did not contain added serum, but perhaps a small amount of a s y n e r g i s t i c serum molecule, as described by Kilburn et a l . (1976) might be present i n the immune complexes that were p r e c i p i t a t e d from serum. 76 ( v i ) Prostaglandins,-: Some tumours produce prostaglandins (PG), e s p e c i a l l y p r o s t a g l a n d i n E 2(PGE 2) ( P l e s c i a e_t a l . 1975). There i s considerable recent evidence f o r a r o l e f o r PG i n immune r e g u l a t i o n . Several s t u d i e s have shown that v a rious PG can i n h i b i t the response of mouse (Webb and Nowowiejski 1978; Fu l t o n and Levy 1980) and human (Mihas e t a l . 1975; Goodwin. e_t a l . 1977; Novogrodsky et a l . 1979)<lymphocytes to PHA. Human p e r i p h e r a l blood monocytes produce PG i n q u a n t i t i e s s u f f i c i e n t to reduce PHA bl a s t o g e n e s i s (Goodwin et a l . 1977; Novogrodsky et a l . 1979). The d i f f e r e n t i a t i o n of n o n s p e c i f i c T suppressor c e l l s i n mice may be induced by PGE 2 (Webb and Nowowiejski 1978) and by PGEj (Fulton and Levy 1981). The T suppressor c e l l exerts i t s immunosuppressive e f f e c t by the r e l e a s e of a suppressive substance. F u l t o n and Levy (1981) found that the T suppressor c e l l s produced PG, whereas Webb and Nowowiejski (1978) found that the suppressive substance was not PG. No s i g n i f i c a n t c o r r e l a t i o n was found between plasma l e v e l s of p r o s t a -glandins and immunosuppression i n cancer p a t i e n t s (Harvey e_t al^. 1977) . These authors suggested that immunosuppression by PG might be important i n the neighbourhood of the tumour. F u l t o n and Levy (1980, 1981) found that the type of T suppressor c e l l induced b y PG was.'•present i n the spleens of tumour-bearing mice. ( v i i ) Miscellaneous Tumour-Related Substances: U n i d e n t i f i e d immunosuppressive f a c t o r s have been found i n e x t r a c t s of tumour c e l l s from animals (Motoki e_t a l . 1974; Wong e_t a l . 1974; Kamo et a l . 1975). 77 F i b r i n o g e n degradation products are o f t e n found i n p a t i e n t s w i t h advanced cancer. One study (Girmann et a l . 1976) showed that serum i n -h i b i t i o n of PHA b l a s t o g e n e s i s p a r a l l e l e d evidence of f i b r i n o l y t i c a c t i v i t y i n most cancer patients-. Low- molecular weight fibrinogen, degradation products were shown to be immunosuppressive. 3. I n t e r a c t i o n of PHA w i t h Serum P r o t e i n s : I t had been suggested that the suppressive e f f e c t of a - g l o b u l i n s on PHA b l a s t o g e n e s i s might be caused by i n t e r a c t i o n s between PHA and a-g l o b u l i n s l e a d i n g to the removal of PHA by p r e c i p i t a t i o n . PHA was known to p r e c i p i t a t e w i t h plasma p r o t e i n s . Yachnin (1972) f r a c t i o n a t e d PHA i n t o two f r a c t i o n s , both of which were mitogenic, w h i l e only one p r e c i p i t a t e d serum p r o t e i n s , a - g l o b u l i n s depressed the response to both f r a c t i o n s e q u a l l y . Therefore, p r e c i p i t a t i o n w i t h PHA was not necessary f o r the immunosuppressive e f f e c t of a - g l o b u l i n s . A l s o , most s t u d i e s of immune i n h i b i t o r s have found that the i n h i b i t i o n of MLR was at l e a s t as great as the i n h i b i t i o n of the PHA response (see above). 4. Summary I t i s apparent that a number of substances which are o f t e n elevated i n cancer p a t i e n t s may be immunosuppressive. The a b i l i t y of a p a r t i c u l a r serum to support the s t i m u l a t i o n of lymphocytes by PHA depends": on the i n t e r p l a y of the e f f e c t s of many substances which probably d i f f e r some-what even between p a t i e n t s w i t h the same type of cancer. Therefore, any e l u c i d a t i o n of c a u s a l subtances must account f o r a l l the v a r i a b l e s which may a f f e c t the s e r a tested such as: age of serum, storage c o n d i t i o n s , age of p a t i e n t s and r e s p e c t i v e c o n t r o l s , disease s t a g i n g , c o n d i t i o n at the time 78 serum and treatment p r o t o c o l s , V I I I . THE EFFECT OF SEX HORMONES ON THE IMMUNE SYSTEM As the s t u d i e s reported i n t h i s t h e s i s have shown a p o s s i b l e r e l a -t i o n s h i p between immunosuppressive sera and treatment w i t h v a r i o u s hor-monal manipulations, i t was of i n t e r e s t to review the l i t e r a t u r e on the e f f e c t of sex hormones on the immune system. Many s t u d i e s have been done but w i t h c o n t r a d i c t o r y r e s u l t s . Of greater relevance to the problem of immunosuppressive sera i n cancer p a t i e n t s are s t u d i e s of the e f f e c t of oestrogens and androgens on the l e v e l s of various serum proteins,"some of which may i n t u r n i n f l u e n c e immune responses as reviewed i n I - V I I . The i n h i b i t o r y e f f e c t of a d r e n o c p r t i c o s t e r o i d s on the immune system i s w e l l known (Heilman et a l . 1973; G i l l i s "et a l . 1979) and w i l l not be reviewed here. S u r p r i s i n g l y , the e f f e c t s of c o r t i c o s t e r o i d s on the l e v e l s of i n d i v i d u a l serum p r o t e i n s have apparently not been s t u d i e d . A. Normal Plasma Le v e l s and Transport of Oestrogens arid Androgens The v a r i o u s androgens and oestrogens are a l l s t e r o i d hormones derived from c h o l e s t e r o l (Ganong 1977b). The most potent androgen, or m a s c u l i n i z i n g hormone, i s t e s t o s t e r o n e . Testosterone i s the p r i n c i p a l hormone of the t e s t e s , but i s a l s o produced i n s m a ll amounts by the adrenals. Other l e s s potent androgens, such as aridrostenedione, are produced by the adrenals and o v a r i e s . The plasma l e v e l of t e s t o s t e r o n e i s 0.65 ug/100 ml (2.3 x 10 8M ) i n normal males and 0.03 g/100 ml (1.0 x 10 9M) i n females (Ganong 1977b). In the c i r -c u l a t i o n , t e s t o s t e r o n e i s bound to albumin and to.',a '8-globulin (gonadal 79 steroid-binding globulin) which also binds o e s t r a d i o l (Paulsen 1974; Ganong 1977b). Estimates of the percent<• of' binding:-;to prbteins'.vary• Ganong (1977b) reported 66% binding, whereas Paulsen (1974) stated that 97-99% of the testosterone was bound, but that the binding was of - low a f f i n i t y . Oestrogens are secreted mainly by the ovaries, but also i n smaller amounts by the adrenals and testes. Adrenal androgens are converted to oestrogens i n the c i r c u l a t i o n (Ganong 1977a). The major secreted oestrogen i s 173-oestradiol. This i s i n equilibrium i n the c i r c u l a t i o n with oestrone. Oestrone i s matabolized to o e s t r i o l , mainly i n the l i v e r . Oestradiol i s the most potent oestrogen and o e s t r i o l the least potent (Ganong 1977b). About 70% of the c i r c u l a t i n g oestrogens are bound to pr o t e i n , mainly to the gonodal steroid-binding g l o b u l i n (Ganong 197bb). The normal plasma l e v e l s of o e s t r a d i o l and oestrone i n human females of reproductive age are shown i n Table V (reproduced from Ross and Vande Wiele 1974). The secretion of androgens by the testes i s under the con t r o l of l u -teinizing.ihormone (IH) and the secretion of oestrogens by the ovaries i s c o n t r o l l e d by LH and f o l l i c l e stimulating hormone (FSH) (Ganong 1977a). Various synthetic androgens and oestrogens are i n c l i n i c a l use. B. Sex Differences irt Immune Responses A number of studies have shown an increased s u s c e p t i b i l i t y to i n f e c t i o n i n human males compared with females. This d i f f e r e n c e i s seen i n b a c t e r i a l (Thompson et a l . 1963; Washburn e_t a l . 1965; Goble and 80 TABLE V NORMAL PLASMA LEVELS OF 178-OESTRADIOL AND OESTRONE IN VARIOUS PHASES OF THE MENSTRUAL CYCLE IN WOMEN Oestrogen Phase of Menstrual Cycle Concentration i n Plasma (ug/100 ml) 17 8-oestradiol E a r l y F o l l i c u l a r 0.006 (2.2 x 10" 1 0M) Late F o l l i c u l a r 0.033-0.070 (1.2 x 10" 9 2.6 x 10"9M) Midluteal 0.020 Oestrone Ea r l y F o l l i c u l a r 0.005 Late F o l l i c u l a r 0.015-0.030 Midluteal 0.011 * Reproduced from Ross and Vande Wiele (1974). 81 Konopka 1973) , v i r a l (Goble and Konopka 1973) and fungal and p a r a s i t i c (Goble and Konopka 1973) i n f e c t i o n s . One exception appears to be malaria, which has a greater incidence and se v e r i t y i n females (Goble and Konopka 1973). The increased s u s c e p t i b i l i t y to i n f e c t i o n has also been described i n mice (Willoughby and Watson 1964; Friedman e_t a l . 1972; Anderson and Hanson 1974). The difference i n s u s c e p t i b i l i t y to i n f e c t i o n begins early i n l i f e when hormonal differences between the sexes are not great. Therefore, i t has been postulated that the double X-chromosome i n the female i s important for resistance to disease (Washburn et a l . 1965; Goble and Konopka 1973). Autoimmune diseases occur much more frequently i n females. This i s true i n humans for rheumatoid a r t h r i t i s (Lawrence 1969), systemic lupus erythematosis (Dubois 1966), and Hashimoto's t h y r o i d i t i s (Ingbar and Woeber 1977) and also i n mice (Weinstein and Berkovich 1981). The l e v e l of immunoglobulins i s higher i n women than i n men. This diff e r e n c e i s due to higher IgM l e v e l s i n women, while most studies found no consistent differences i n IgG and IgA l e v e l s between the sexes (Butterworth et a l . 1967; Rhodes et a l . 1969; Wood et a l . 1969, A d i n o l f i et a l . 1978). Several studies have shown a c o r r e l a t i o n between IgM l e v e l s and the number of X-chromosomes i n humans (Rhodes et a l . 1969; Wood et a l . 1969; A d i n o l f i et a l . 1978). The higher immunoglobulin l e v e l s among females are also found i n c e r t a i n stains of mice (Cohn and Hamilton 1976). 82 C. E f f e c t s of Castration arid Sex Hormone Administration on Immune Responses 1. E f f e c t s of Sex Hormones on the Development or Recovery of Immune  Competence Ei t h e r androgens or oestrogens can cause f a i l u r e of development of the bursa of F a b r i c i u s i n chick embryos, or i n v o l u t i o n of the bursa i n chicks (Kappas and Palmer 1973). In a mammalian system, R e i i l y et a l . (1967) found that the adminis-t r a t i o n of pharmacological doses of oestrogen to neonatal mice led to a runting syndrome characterized by thymic hypoplasia, lymphoid hypoplasia, impaired immune responses, and increased mo r t a l i t y . Oestrogens (Thompson et a l . 1967) and androgens (Kotani et a l . 1974) impaired the recovery of c e l l u l a r and humoralMmmune responses and reduced the s u r v i v a l of mice following sublethal i r r a d i a t i o n or l e t h a l i r r a d i a t i o n and bone marrow r e c o n s t i t u t i o n . This occurred at doses having l i t t l e e f f e c t upon the immune responses of i n t a c t mice. Metcalf (1956) described a factor i n an extract of human thymus that stimulated lymphocyte production i n mice. I t s e f f e c t s were blocked by ©estradiol. I t has been suggested that oestrogens have an adverse e f f e c t on the development and recovery of the immune system secondary to an e f f e c t on the thymus or a blocking of a thymic fa c t o r ( R e i i l y et_ a l . 1967). In mice, and p o s s i b l y also i n humans, a c o r r e l a t i o n has been found between high, s e n s i t i v i t y to androgens and low immunoglobulin l e v e l s i n adult males (Cohn and Hamilton 1976; Cohn 1980). The l e v e l s of immuno-globulins were not rel a t e d to adult testosterone l e v e l s , and were not a f f e c t e d by c a s t r a t i o n i n mice. I t was po s t u l a t e d that androgens i n  utero i n s e n s i t i v e mice might program the immune system to produce low l e v e l s of immunoglobulin. In comparing females of two s t r a i n s of mice, the s t r a i n most s e n s i t i v e to oestrogens gave a b e t t e r antibody response to SRBC and an increased response a f t e r a d m i n i s t r a t i o n of exogenous oestrogens (Stern and Pavidsohn 1955). On the other hand, the s t r a i n l e s s s e n s i t i v e to oestrogens had a lower immune response to SRBC and t h i s was decreased by exogenous oestrogens. 2. E f f e c t s of Oestrogen A d m i n i s t r a t i o n on the Mature Immune System Oestrogens have been shown to enhance the humoral immune response to Type I pneumococcus i n r a b b i t s (von Haam and Rosenfeld 1942) , to a l l o g e n e i c tumour c e l l s i n mice (Peter et a l . 1970) and to E s c h e r i c h i a  c o l i ( E . c o l i ) i n mice (Kenny e t a l . 1976). Hulka et^ al_. (1965) showed that o e s t r a d i o l i n high doses had no s i g n i f i c a n t e f f e c t on antibody production to human serum albumin i n complete Freund's adjuvant i n r a b b i t s . Other s t u d i e s have shown an i n h i b i t o r y e f f e c t of oestrogens on the antibody response to E . c o l i antigens i n guinea pigs (Toivanen 1967), to p e n i c i l l o y l ^ - c o u p l e d guinea p i g Y - g l o b u l i n i n guinea pigs (Feigen et a l . 1978), and to SRBC (Warr and S l j i v i c 1972) and to type I I I pneumococcal polysaccharide ( S l j i v i c and Warr 1974) i n mice. Pharmacological doses of oestrogens increased s u s c e p t i b i l i t y to encephalomyocarditis v i r u s i n mice (Friedman et a l . 1972) and to 84 staphylococci i n mice (Toivanen 1967). OestradioL-administration during immunization slowed the decline i n anti-hapten t i t e r caused by repeated i n j e c t i o n s of p e n i c i l l o y l - c o u p l e d guinea pig Y~gl°hulin, probably due to an i n h i b i t o r y e f f e c t of oestrogens on the production of suppressor c e l l s (Feigen e_t a_l. 1978) . Some studies a t t r i b u t e d the decreased antibody response to the reduced a v a i l a b i l i t y of antigen because of increased phagocytosis i n response to oestrogen (Warr and S l j i v i c 1972; S l j i v i c and Warr 1974). On the other hand, Nichol e_t a l . (1964) a t t r i b u t e d increased resistance to various i n f e c t i o n s i n mice and increased Y~globulin l e v e l s i n guinea pigs to stimulation of phagocytosis by oestrogens. Most studies have shown an i n h i b i t o r y e f f e c t of oestrogens on c e l l -mediated immune responses. Oestrogens i n h i b i t e d the development of delayed h y p e r s e n s i t i v i t y reactions to tuberculin and thyroglobulin i n guinea pigs immunized with thyroglobulin and complete Freund's adjuvant, but had a v a r i a b l e e f f e c t on the development of autoimmune t h y r o i d i t i s (Kappas et a l . 1963). In r a t s , oestrogens also had a va r i a b l e e f f e c t on the development of autoimmune t h y r o i d i t i s , reduced the incidence of ad-juvant-induced a r t h r i t i s , but had no e f f e c t on the incidence of experi-mental a l l e r g i c e n cephalitis or on skin a l l o g r a f t r e j e c t i o n (Kappas ejt a l . 1963). Oestrogen administration has been found to prevent the r e j e c t i o n of grafts of trophoblast t i s s u e i n patients with advanced c e r v i c a l cancer (Lajos and Dorcs 1962), to delay the r e j e c t i o n of skin a l l o g r a f t s i n mice (Simmons e_t a l . 1968) , to prevent the r e j e c t i o n of corneal transplants i n some rabbits which had received a skin g r a f t from 85 the same donor (Whitman et al, 1976), to decrease the a l l e r g i c r e a c t i o n to p i c r y l c.h.Qloride. i n mice, (Ca.rter 1976), and to decrease the production of n a t u r a l k i l l e r c e l l s i n the bone marrow of mice (Seaman and Gindhart 1979? Seaman et a l . 1979), Bilde.r (1976) found that i n j e c t i o n of crude preparations of p i t u i -t a r y gonadotrophins or c a s t r a t i o n , but not hypophyseetomy, a c c e l e r a t e d the r e j e c t i o n of g r a f t e d mouse s k i n by male and female r a t s . A d m i n i s t r a -t i o n of .17 ^ - o e s t r a d i o l p l u s progesterone to ca s t r a t e d r a t s r e s t o r e d g r a f t r e j e c t i o n to normal, but was without e f f e c t i n hypophysectomized r a t s . Therefore, i t was suggested that p i t u i t a r y hormones s t i m u l a t e d the immune system, and the, e f f e c t s of c a s t r a t i o n and oestrogen a d m i n i s t r a t i o n were brought about by the increase and decrease i n p i t u i t a r y gonadotrophins which they caused, A b l i n e_t a l . (1974c) showed a decrease i n the response to PHA of lymphocytes from p a t i e n t s w i t h p r o s t a t i c cancer c u l t u r e d i n autologous serum a f t e r the i n i t i a t i o n of oestrogen therapy. Guinari et a l . (1975) confirmed that the lymphocyte response to PHA was decreased i n p r o s t a t i c cancer p a t i e n t s on oestrogens compared w i t h untreated p a t i e n t s , but s k i n t e s t responses to PPD were increased. The i n h i b i t o r y e f f e c t of autologous serum from p a t i e n t s r e c e i v i n g oestrogens- on the response of lymphocytes to PHA was not confined to p a t i e n t s w i t h p r o s t a t i c cancer, but was. a l s o seen i n transsexuals r e c e i v i n g oestrogen therapy (Ablin e t a l , 1976). 86 Stimson and Hunter (1976) gave 173-oestradiol d a i l y f o r 14 days to normal and thymectomized male r a t s . Serum was taken from the rats before and a f t e r treatment and incubated with lymphocytes from healthy human males. Incubation with serum from oestrogen-treated rats caused a de-crease i n E-rosette formation, and an increase i n lymphocytes lacking T and B c e l l markers. The t o t a l number of c e l l s forming EAC-rosettes and/ or bearing surface Ig did not change, but the number with both markers increased. Serum from thymectomized oestrogen-treated rats did not have these e f f e c t s . Apparently oestrogen caused release from the thymus of a factor a f f e c t i n g lymphocytes. Fr a c t i o n a t i o n of serum from oestrogen-treated rats revealed the active agent to be an ether-soluble compound with MW less than 1000, s i m i l a r to a substance previously described i n the serum of women i n l a t e pregnancy. Hulka et a l . (1965) found that high doses of o e s t r a d i o l had no e f f e c t on a l l o g r a f t r e j e c t i o n i n ra b b i t s . 3. E f f e c t s of Androgen Administration on the Mature Immune System As with oestrogens, the reports of the e f f e c t s of androgens on the immune system have been contradictory. Most studies have shown that androgens i n h i b i t the humoral:.immune response. Testosterone was found to reduce the incidence of autoimmune t h r y o i d i t i s and adjuvant-induced a t h r i t i s i n rats i n j e c t e d with thyroglobulin and complete Freund's adjuvant (Kappas et a l . 1963), to decrease the resistance of female mice to Group A str e p t o c o c c i (Willoughby and Watson 1964), to reduce the antibody response to human serum albumin i n hens (Oberdorfer and Herrmann 1971), and to decrease the number of 87 antibody-producing c e l l s i n the spleens- of rats immunized with SRBC without reducing the t i t e r of antibodies (Aschkenasy 1976). Castro (1974) showed that orchidectomy i n mice increased the a n t i -body response to SRBC which i s known to be l i m i t e d by the a v a i l a b i l i t y of helper T c e l l s . Antibody responses to the T - c e l l independent antigen^ type I II pneumococcal polysaccharide, and to H-2 antigens, i n which T c e l l s are not limiting,&were,>not: affected'. Nichol et_ a l , (1965) found that testosterone alone had no e f f e c t on the r e t i c u l o e n d o t h e l i a l system i n mice, but potentiated the stimulant e f f e c t of 17-8 o e s t r a d i o l while i n h i b i t i n g the stimulant e f f e c t of o e s t r i o l and oestrone. Variable e f f e c t s of androgens on cell-mediated immune responses have been reported. At least one study has shown a stimulatory e f f e c t . Weinstein and Berkovich (1981) studied the e f f e c t s of testosterone on various aspects of the immune system i n an attempt to c l a r i f y the reasons f o r the higher incidence of autoimmune diseases i n females both i n humans and i n ce r t a i n mouse s t r a i n s . Mice of the B/W s t r a i n i . e . (NZB x NZW) Fj develop e a r l i e r incidence (by 6 mo of age) of autoimmune disease than do normal males, and long-term administration of testosterone i n h i b i t s the develop-ment of autoimmune disease i n females and castrated males. The enzyme 20a-hydroxysteroid dehydrogenase (20aSDH) i s an enzyme found i n some lymphocytes of T - c e l l lineage. At 6 months of age, female and castrated male B/W mice show a 50% decrease i n t h e i r marrow 20aSDH a c t i v i t y 88 compared w i t h normal male mice. C a s t r a t i o n of B/W, BALB/c and BALB/c nude mice caused a decrease i n marrow 20aSDH, a decrease i n l a r g e marrow c e l l s (those which have 20aSDH a c t i v i t y ) , h y p e r t r o p h y of the thymus but low thymus 20aSDH a c t i v i t y and a poor response of thymic lymphocytes to PHA, and a decrease i n s p l e n i c suppressor T c e l l s (Weinstein and Berkovich 1981). Testosterone implant-a t i o n i n t o c a s t r a t e d males caused the reverse e f f e c t s . Serum te s t o s t e r o n e l e v e l s were 30 ng /ml f o r normal male mice, 1 n g / m l f o r c a s t r a t e d males, and 15 ng./ml f o r males w i t h . t e s t o s t e r o n e implants. In v i t r o , 2800 ng /ml of testosterone caused a 30%-40% decrease i n the response of thymocytes to PHA. The authors suggested that i f was u n l i k e l y that serum testost e r o n e l e v e l s as low as 15 ng ;/ml would have a primary e f f e c t on the responsiveness of thymic c e l l s when such high i n v i t r o concentrations were r e q u i r e d . They p o s t u l a t e d that 20aSDH p o s i t i v e bone marrow c e l l s were precursors of medullary.thymic lymphocytes which thus increased w i t h t e s t o s t e r o n e whereas the precursors of c o r t i c a l thymic lymphocytes which decreased w i t h testosterone were 20aSDH negative. Suppressor T c e l l s , r e s p o n s i b l e f o r preventing the development of autoimmune dis e a s e , and the T c e l l s responding to PHA apparently were of the 20aSDH p o s i t i v e subpopulation. Other s t u d i e s have shown that t e s t o s t e r o n e decreases c e l l - m e d i a t e d immune responses. In experiments i n v o l v i n g two congenic mouse s t r a i n s d i f f e r i n g at the minor h i s t o c o m p a t i b i l i t y l o c u s , H-12, females of both s t r a i n s r e j e c t e d . s k i n g r a f t s from the other more f r e q u e n t l y than d i d males (Graff et_ a l . 1969). 89 Castration of male mice led to increased r e j e c t i o n while administration of testosterone to females decreased r e j e c t i o n . Castro (1974) showed that prepurbertal orchidectomized mice showed accelerated skin graft r e j e c t i o n . Postpubertal orchidectomy had the same e f f e c t eventually, but i t took 12 weeks to develop. The e f f e c t s of orchidectomy were counteracted by testosterone. Thymectomy plus or c h i d -ectomy was no d i f f e r e n t i n e f f e c t from thymectomy alone. Therefore, apparently the e f f e c t of orchidectomy was mediated through the thynus. In one study, testosterone did not a l t e r the s u r v i v a l of mice a f t e r i n j e c t i o n of allogeneic tumour c e l l s although castrated male mice sur-vived a s i g n i f i c a n t l y shorter time than normal male mice (Peter et^ a l . 1970). Oestradiol treatment further reduced the s u r v i v a l of the castrated mice. The author postulated that physiologic doses of t e s t o s t -erone are stimulatory to the c e l l mediated immune system whereas larger doses are not. A l t e r n a t i v e l y , low l e v e l s of testosterone are necessary to antagonize the e f f e c t s of adrenal oestrogens. D. In V i t r o E f f e c t s of Sex Hormones 1. E f f e c t s of Oestrogens i n V i t r o A b l i n et a l . (1974a,b) studied the e f f e c t of oestrogen added to cultures on the response of normal human lymphocytes to PHA. The syn-t h e t i c oestrogen d i e t h y l s t i l b o e s t r o l diphosphate added to lymphocyte cultures with PHA caused a 57% reduction i n 3H-TdR incorporation at a concentration of 2000 yg/ml, the highest tested (Ablin et a l . 1974b), and a 50% reduction at a concentration of 500 Ug/ml (Ablin et a l . 1974a,b) which i s about 10 6 times physiologic plasma l e v e l s . As therapeutic doses of oestrogen i n breast cancer are only about 6 times replacement doses (Canadian/.Eharmaceutical Association 1980, p. 213) , one might assume that plasma l e v e l s of oestrogens i n cancer patients on 90 oestrogen treatment might be about 6 times p h y s i o l o g i c l e v e l s , and thus w e l l below 500 yg/ml. Kenny et a l . (1976) found that moderate doses of o e s t r a d i o l (50-5000 pg/ml) i n v i t r o increased the number of plaque forming c e l l s to E_. c o l i antigens i n the spleens of mice i n j e c t e d w i t h E_. c b l i . This i s w i t h i n to 1 0 - f o l d above the p h y s i o l o g i c range (see Table I V ) . Higher concentrations of o e s t r a d i o l (20,000 pg/ml) i n v i t r o decreased the production of plaque forming c e l l s . Oestrogens may act on thymic lymphocytes (at l e a s t i n the r a t ) at the l e v e l of the plasma membrane by preventing calcium ions from inducing h y p e r p o l a r i z a t i o n which may t r i g g e r m i t o s i s (Morgan et a l . 1976) . K i t z m i l l e r and R o c k l i n (1980) found that doses of o e s t r a d i o l , progesterone, and human c h o r i o n i c gonadotrophin much higher than phys-i o l o g i c d i d not i n h i b i t MIF production by preimmunized guinea pig, leuko-cytes exposed to antigen. The response of normal macrophages to MIF was l i k e w i s e not a l t e r e d by the hormones. Progesterone and o e s t r a d i o l at sup r a p h y s i o l o g i c l e v e l s , but not te s t o s t e r o n e , i n h i b i t e d DNA sy n t h e s i s i n the mouse MLR and a l s o i n h i b i t e d the generation of c y t o t o x i c lymphocytes (Pavia et^ a l . 1979). Hormones d i d not :affeet the c y t o t o x i c i t y of p r e v i o u s l y generated c y t o t o x i c lymphocytes. Soweine et a l . (1980) presented evidence that subpopulations of human p e r i p h e r a l blood lymphocytes may have receptors f o r 1 7 8 - o e s t r a d i o l and dihydrotestosterone, the form to which testos t e r o n e i s converted i n tar g e t organs (Paulsen 1974). Incubation of normal human lymphocytes w i t h p h y s i o l o g i c a l amounts of 1 7 8 - o e s t r a d i o l caused an increased form-a t i o n of e a r l y E-rosettes which are formed by lymphocytes having a high a f f i n i t y f o r SRBC. 91 2. E f f e c t s of Androgens In V i t r o Testosterone Inhibited the response of human peripheral blood lymph-ocytes to PHA (Mendelsohn et a l . 1977). A concentration of 20 mM, which i s about 101* times normal male plasma l e v e l s (see I-VIII A) , caused about 50% i n h i b i t i o n of incorporation of 3H-TdR. A higher concentration was required to i n h i b i t stimulation of human lymphocytes by Con A. Androgen doses f o r treatment of breast cancer i n females are 2 to 3 times the replacement dose for males with androgen d e f i c i e n c y (Murad and Gilman 1975). :,. : ::Similaf findings were .reported by ;Wyle and Kent (1977). They found that low concentrations of testosterone, 0.01 to 1.0 ug/ml, augmented the response of human lymphocytes i n v i t r o to PPD but not to PHA. About 40 yg/ml testosterone, 10^ times normal male plasma l e v e l s , were required to cause a 50% i n h i b i t i o n of the response of human lymphocytes to PHA. E. E f f e c t of Sex Hormones on Serum Protein Levels 1. E f f e c t of Oestrogens on Serum Protein Levels Pregnancy and ingestion of o r a l contraceptives and oestrogens alone are associated with changes i n the l e v e l s of many serum proteins. The serum proteins generally found i n increased concentration i n these s i t u a t i o n s are a i - a n t i t r y p s i n , ( L a u r r e l l e_t al.-1968; Song et a l . 1970; Stimson 1972; Peters 1979), t r a n s f e r r i n ( L a u r r e l l et a l . 1968; Home et a l . 1970,1971; Song et_ a l . 1970; Stimson 1972; Peters 1979), caeruloplasmin (Musa et a l . 1967; L a u r r e l l et a l . 1968; Song et a l . 1970; Stimson 1972; Peters 1979), Gc glo b u l i n (Musa et a l . 1967; Stimson 1972), 012-macroglobulin ( L a u r r e l l et a l . 1968; Horne et_ a l . 1970, 1971: Stimson 1972), c o r t i c o s t e r o i d binding g l o b u l i n (Musa et a l . 1967; L a u r r e l l et a l . 1968), and thyroxine binding g l o b u l i n (Musa et a l . 1967; L a u r r e l l et a l . 1968). On the other hand, Musa et_ al_. (1967) reported ho change i n serum 92 t r a n s f e r r i n and i n serum a 2-macroglobulin a f t e r treatment with the syn-t h e t i c oestrogen, e t h i n y l o e s t r a d i o l and Song et al_. (1970) reported no change i n a2-macroglobulin a f t e r at le a s t 3 months of treatment with a combined oestrogen-progestogen oral_contraceptive. The serum proteins usually found i n decreased concentrations i n pregnancy and a f t e r treatment with oestrogens or combined o r a l contra-ceptives are albumin (Musa et a l . 1967; L a u r r e l l et a l . 1968; Stimson 1972), a i - a c i d glycoprotein ( L a u r r e l l et a l . 1968; Song et a l . 1970; Stimson 1 9 7 2 ; Peters 1979), and haptoglobin (Musa et a l . 1967; L a u r r e l l et a l . 1968; Song et a l . 1970; Peters 1979). Home et a l . (1971) .. reported no change i n serum albumin a f t e r treatment with mestranol, a synthetic oestrogen, while Song et a l . (1970) reported only a non-s i g n i f i c a n t decrease with a. combined.oral conctraceptive. Stimson (1972) reported no change i n serum haptoglobin i n pregnancy. Prealbumin i s unusual i n that i t i s increased i n women taking o r a l contraceptives (Peters 1979) but i s decreased i n pregnancy (Stimson 1972; Peters 1979). Stimson (1972) found that IgG and IgA were decreased i n pregnancy whereas IgM l e v e l s were unchanged. Horne e_t a l . (1971) reported no . change i n serum IgG with mestranol treatment although they had previously reported an increase i n IgG i n women on combined o r a l contraceptives (Horne et a l . 1970). No changes i n immunoglobulins occurred with . combined o r a l contraceptives ( L a u r r e l l et a l . 1968; Song et a l . 1970). At l e a s t i n preganancy, a i - a c i d glycoprotein was the only serum protein decreased i n t o t a l amount i n the body. A l l other decreases were 93 secondary to an i n c r e a s e i n plasma volume (Stimson 1 9 7 . 2 ) . I t i s b e l i e v e d that oestrogen i s the a c t i v e agent i n combined o r a l c o n t r a c e p t i v e s w i t h regard to a l t e r a t i o n s i n serum p r o t e i n s , s i n c e i t . has been shown that oestrogens alone have a s i g n i f i c a n t e f f e c t on p r o t e i n s y n t h e s i s (Doe at a l . 1967). The a l t e r a t i o n s i n serum p r o t e i n s brought about by oestrogen treatment i n c l u d e increases i n a j - a n t i t r y p s i n , a2 -macroglobulin and c o r t i -c o s t e r o i d b i n d i n g g l o b u l i n ( t r a n s c o r t i n ) a l l of which may be immuno-suppressive as o u t l i n e d i n Sect i o n I-VIL Oestrogens may a l s o cause a decrease, i n serum albumin which may be one of the p r i n c i p l e serum subtstances supporting lymphocyte s t i m u l a t i o n by PHA. However, oestrogens a l s o decrease the con c e n t r a t i o n of a i - a c i d g l y c o p r o t e i n which may be immunosuppressive and increase the concentration of t r a n s f e r r i n which may be a f a c t o r supporting lymphocyte s t i m u l a t i o n . Therefore, i t would be d i f f i c u l t to p r e d i c t whether these changes should have a net " i n h i b i t o r y or enhancing e f f e c t on the lymphocyte, response to PHA. S i m i l a r l y , i f the oestrogen antagonist Tamoxifen causes serum p r o t e i n changes opposite to those produced by oestrogens, the net e f f e c t of these changes on lymphocyte s t i m u l a t i o n by PHA would be unclear. One study ( K e l l e r e_t a l . 1976) showed that a - f e t o p r o t e i n (a-FP) i s only immunosuppressive when complexed w i t h o e s t r a d i o l . a-FP binds .. ;.. : oestrone,but the r e s u l t i n g complex i s not immunosuppressive. The authors p o s t u l a t e d that the d i s c r e p a n c i e s between v a r i o u s a-FP preparations w i t h respect to immunosuppression might be due to d i f f e r e n c e s i n the oestrogens a v a i l a b l e f o r b i n d i n g . A l t e r n a t i v e l y there might e x i s t two forms of a-FP, one of which binds oestrogen and i s suppressive>. and one which does not 94 bind oestrogen and is- not suppressive,. Oestrogen alone, i n the concen-t r a t i o n i n which, i t was found i n the AFP-oestrogen complex, was not suppressive. 2. E f f e c t s of Androgens on Serum Protein Levels Dickinson et a l . (1969) studied a l t e r a t i o n i n plasma proteins i n 10 normal adult males who had received 150 mg of testosterone c y c l o -pentyl propoionate on two occasions 7 days apart. This has been shown to cause an increase of plasma testosterone by 1.0 to 1.5 ug/100 ml f o r at l e a s t 7 to 10 days. Plasma proteins were measured before the f i r s t dose, before the second dose, and 7 days a f t e r the second doses of testosterone. Testosterone administration produced a small but s i g n i f i c a n t increase i n a i - g l o b u l i n s but no s i g n i f i c a n t change i n serum albumin ,a-, •, B-, or y-globulins or t o t a l p r o t e i n , A s i g n i f i c a n t decrease i n t r a n s f e r r i n , fibrinogen, thyroxine binding g l o b u l i n , c o r t i c o s t e r o i d binding g l o b u l i n and a borderline s i g n i f i c a n t decrease i n a i - a n t i t r y p s i n followed testosterone treatment. However, d i f f e r e n t e f f e c t s were noted with some synthetic androgen de r i v a t i v e s , Barbosa et a l . (1971) studied the e f f e c t s of d i f f e r e n t anabolic steroids i n c l i n i c a l use upon the l e v e l s of a number of serum proteins i n 80 patients and 65 healthy volunteers. Fluoxymesterone, the androgen used to tr e a t some of the breast cancer patients i n t h i s study, and other a - a l k y l anabolic steroids caused an increase i n serum, haptoglobin with no s i g n i f i c a n t change i n s i a l i c a c i d , a s i g n i f i c a n t increase i n 95 plasminogen, a n o n s i g n i f i c a n t decrease i n f i b r i n o g e n , and no s i g n i f i c a n t change i n t o t a l plasma p r o t e i n s . Oxymetholone, another 17a-alkyl s t e r o i d , caused an increase i n a i - a n t i t r y p s i n and d i - a c i d g l y c o p r o t e i n , but no- s i g n i f i c a n t change i n t r a n s f e r r i n or caerirloplasmin. These plasma p r o t e i n s were not measured i n fluoxymesterone-treated i n d i v i d u a l s but the r e s u l t s might be expected to be s i m i l a r to those w i t h - • oxymetholone because of t h e i r s t r u c t u r a l s i m i l a r i t y . In view of the p o s s i b l e immunosuppressive e f f e c t of protease i n h i b i t o r s , a paper on the e f f e c t s of a n a b o l i c s t e r o i d s on l e v e l s of plasma protease i n h i b i t o r s i s of i n t e r e s t (Walker et a l . 1975) . 5 of 6 17a-alkylated a n a b o l i c s t e r o i d s produced elevated l e v e l s of antithrombin I I I , 5 produced decreased l e v e l s of a2-macroglobulin and 3 produced e l e -vated l e v e l s of C i i - a n t i t r y p s i n . Fluoxymesterone was not among the s t e r o i d s s t u d i e d . The study i n v o l v e d 20 males. w n o had s u f f e r e d at l e a s t one myocardial i n f r a c t i o n . They were given a l t e r n a t e l y 4 weeks of placebo and 4 weeks of a n a b o l i c s t e r o i d therapy. The one non-17a-a l y k y l a t e d s t e r o i d s t u d i e d d i d not produce s i g n i f i c a n t changes i n any of the three protease i n h i b i t o r s . Thus 17op ;alkylated s t e r o i d s , a c l a s s to which fluoxymesterone belongs, cause an inc r e a s e i r i ; : a i - a c i d g l y c o p r o t e i n and C i i - a n t r i t r y p s i n , both p o s s i b l y immunosuppressive. On the other hand, 17a-aklyated s t e r o i d s may cause a decrease i n Ci2-macroglobulin which may be immuno-suppressive. Therefore, as w i t h oestrogens, i t would be d i f f i c u l t to p r e d i c t the net e f f e c t of these serum p r o t e i n changes on the response of normal lymphocytes to PHA. 96 F. E f f e c t s of Hormonal Treatement of Breast Cancer on Immune Responses Webster et a l . (1979) studied 37 patients with advanced breast cancer r e c e i v i n g hormonal therapy i n the form of oophorectomy or Tamo-x i f e n or androgens. Hormonal treatment did not a l t e r the various indices of immune status from pretreatement values. Response to DNCB and PPD skin t e s t i n g , lymphocyte blastogenesis with PHA, t o t a l p e r i -pheral lymphocyte count, and serum l e v e l s of immunoglobulin were used to assess immune competence. No attempt was made to study the e f f e c t s of the three d i f f e r e n t hormonal treatments separately. Franks and Williams (1978) studied patients with advanced breast cancer treated with androgens or oestrogens. Among both oestrogen and androgen-treated patients, the successful responders had a higher mean pretreatment lymphocyte count than did patients with a p a r t i a l or with no response. The high lymphocyte counts of the responders remained high during the 6 month study period, whereas the intermediate responders and f a i l u r e s showed a r i s e i n lymphocyte count with treatment, always re-maining below that of the successful responders. The authors concluded that oestrogens and androgens might have a s l i g h t stimulatory e f f e c t on the immune system. In summary, although i t i s apparent that androgens, oestrogens and agents which a l t e r t h e i r production or e f f e c t may have e f f e c t s on the immune system, the contradictory r e s u l t s obtained from various experi-mental systems make a cl e a r p r e d i c t i o n of the r e s u l t s of a given t r e a t -ment impossible. 97 CHAPTER JI THE EFFECT OF VARYING EXPERIMENTAL CONDITIONS ON THE RESPONSE OF NORMAL LYMPHOCYTES TO PHA IN CONTROL AND BREAST CANCER PATIENTS' SERA I. INTRODUCTION As discussed i n Section I-VII , studies designed to determine whether sera from patients with breast cancer support the stimulation of normal lymphocytes by PHA s i g n i f i c a n t l y less well than c o n t r o l sera have given contradictory r e s u l t s . The discrepancies among studies may be due i n part to differences i n such t e c h n i c a l d e t a i l s of the experiments as the concentrations of serum and of PHA used, and the duration of i n -cubation of the lymphocyte cultures. Accordingly, a number of experiments were done to define the most su i t a b l e conditions, including reagent concentrations and incubation times-to use i n assessing the degree to which sera support lymphocyte stimula-t i o n , to observe the e f f e c t s of varying these parameters, and to ascertain the r e l i a b i l i t y of the assay system. I I , MATERIALS AND METHODS The methods used d i f f e r e d i n some d e t a i l s between the early experi-ments, designated phase 1, and the l a t e r or phase 2 experiments, as noted below. 1. Patients Patients, whose sera and/or lymphocytes were studied, were women re-ceiving treatment at the Cancer Control Agency of B r i t i s h Columbia for metastatic breast cancer. Patients with a recent h i s t o r y of systemic or l o c a l i n f e c t i o n were excluded. In phase 1 experiments, most patients were, being treated with chemo-therapy or with one of the conventional hormonal agents (see I - I I ) while a few-were r e c e i v i n g no treatment. In phase 2 experiments, patients receiving chemotherapy were not included, and the patients studied were receiving no treatment or one of the newer hormonal treatments (see I - I I ) . In both phases, some patients had recently been treated by r a d i a t i o n with or without some other treatment. Most of the patients studied had developed c l i n i c a l l y apparent me-t a s t a t i c disease a v a r i a b l e length of time a f t e r t h e i r primary cancer had been removed by mastectomy with or without radiotherapy. In other patients the cancer was inoperable at the time of diagnosis and was treated with r a d i a t i o n alone, often without any period of apparent com-pl e t e absence of disease. 2. Controls The controls, whose sera and/or lymphocytes were studied f o r com-parison with those of the patients, were women over 30 years of age, with most over 40 years i . e . i n the same age group as most of the patients. None had ever had any malignant disease diagnosed. A l l were i n good general health, although some were taking regular medications for chronic conditions such as hypothyroidism, mild hypertension, and o s t e o a r t h r i t i s , as were some of the patients. None was taking any hormone treatment, including o r a l contraceptives. 99 3. Normal Lymphocyte Donors The donors of normal lymphocytes, on which the e f f e c t s of various sera were studied,were vhealtliy adults of / e i t h e r sex, None had had any i n f e c t i o n i n the preceding 2 weeks. 4. Preparation of Sera Blood was allowed to c l o t f o r up to 36 hours at 4°C before serum was removed. Where dialysed serum was required, an aliquot of each serum sample was dialysed against at least 30 volumes of RPMT-1640 tiss u e c u l -ture medium (Gibco,Grand Island, N. Y.) at 4°C f o r 24 hours with one change of medium during d i a l y s i s f o r the early experiments (Phase 1) and for 48 hours with two changes of medium for the l a t e r experiments (Phase 2. Ordinary d i a l y s i s membranes were used i n Phase 1 and small pore mem-branes which excluded molecules of MW 3500 (A.H. Thomas, Phi l a d e l p h i a , Pa.) were used i n Phase 2. The protein concentration of each dialysed serum sample was estimated spectrophotometrically using the following formula: protein concentration (mg/ml) = 1.56 x OD280-0.76 x OD2 6o(Layne 1957). The concentration was adjusted to 35 mg protein/ml. A l l sera, dialysed and undialysed, were heated at 56°C f o r 30 min. to i n a c t i v a t e complement, s t e r i l i z e d by f i l t r a t i o n (through 0.45u M i l l i p o r e f i l t e r s ( M i l l i p o r e Corp., Bedford, Mass.), and stored frozen at -20°C. In Phase 2 experiments, repeated freezing and thawing were e l i -minated by using dialysed sera immediately a f t e r d i a l y s i s without r e -freezing. 100 5. P r e p a r a t i o n of Leukocytes and Lymphocytes Normal leukocytes or lymphocytes to be used f o r t e s t i n g sera were separated from 20 to 50 ml of venous blood c o l l e c t e d i n a s t e r i l e h e p a r i n i z e d s y r i n g e or i n s t e r i l e vacuum tubes (Becton-Dickinson, Mississauga, Ont.) and t r a n s f e r r e d i n t o a s t e r i l e heparinized: f l a s k (Sodium Heparin, 1000 USP u n i t s / m l , w i t h 0.9% b e n z y l alcohol,,Or ganon,, Toronto, Ont. i n phase 1 or A l l e n and Hanburys, Toronto, Ont. i n phase 2). In a few p r e l i m i n a r y experiments, as noted i n I T - I I I , u n f r a c t i o n a t e d leukocytes were used. The blood was mixed i n the h e p a r i n i z e d syringe w i t h 3 ml s t e r i l e ' . Plasmagel ( L a b o r a t o i r e Roger B e l l o n , N e u i l l y , France) per 10 ml blood, and incubated at 37°C-, 5% CQ 2 f o r 45 to 90 min to a l l o w erythrocytes to sediment. The l e u k o c y t e - r i c h supernatant was removed and c e n t r i f u g e d . The leukocytes were washed three times i n 10 ml of phosphate buff e r e d s a l i n e (PBS) and resuspended i n RPMI-1640 c o n t a i n i n g a n t i b i o t i c s . The number of v i a b l e c e l l s per m i l l i l i t r e was determined by Trypan blue e x c l u s i o n and adjusted as r e q u i r e d . Lymphocytes were separated i n e i t h e r of two ways: (1) Leukocytes obtained by the Plasmagel method described above were resuspended i n 10 ml. of PBS and layered onto 13 ml of s t e r i l e Ficoll-Hypaque, s p e c i f i c g r a v i t y T.0:8" ( F i c o l l , MW 400,000, Pharmacia Fine Chemicals, Uppsala, Sweden;sodium d i a t r i z o a t e (Hypaque sodium), Winthrop, Aurora, Ont.). Lymphocytes were recovered by the method of Perper et al. (1968). (2) Heparinized blood was mixed 2:1 w i t h s t e r i l e PBS and layered onto F i c o l l -Hypaque. About 2 volumes of blopd-PBS mixture to 1 volume of F i c o l l -Hypaque was used. Lymphocytes were obtained as i n (1). In e a r l y 101 phase 1 experiments method (1) was. used, L a t e r phase 1 and a l l phase 2 experiments used method, ( 2 ) , Neither of these methods removes p l a t e l e t s or monocytes from lymphocytes. P a t i e n t s ' and control, lymphocytes used f o r t e s t i n g lymphocyte f u n c t i o n were prepared by t a k i n g 14 ml of venous blood from each donor i n t o h e p a r i n i z e d tubes (Becton-Dickinson, Mississauga, Ont.). The blood was d i l u t e d 1:2 w i t h PBS and layered onto 10 ml - of Ficoll-Hypaque. Lymphocytes were recovered by the method of Perper et_ ' a l . (1968) as above. In a l l i n s t a n c e s , lymphocytes were washed three times w i t h PBS and resuspended i n RPMI-1640 c o n t a i n i n g a n t i b i o t i c s and sera as s p e c i f i e d below. The number of v i a b l e c e l l s per m i l l i l i t r e was determined by Trypan blue e x c l u s i o n and adjusted as r e q u i r e d . F i f t e e n of the lymphocyte preparations to be used f o r t e s t i n g lymphocyte f u n c t i o n were s t a i n e d w i t h W r i g h t ' s u s t a i n and the percentage of lymphocytes determined. The preparations contained 82% to 100% lymphocytes, w i t h an average of 95%. 6. Lymphocyte S t i m u l a t i o n w i t h PHA . A l l experiments were c a r r i e d out i n m i c r o t i t e r p l a t e s (Linbro Chemical Co. Inc., New Haven, Conn.) co n t a i n i n g a n : . ' i n i t i a l volume of 0.25 ml , per w e l l . Leukocyte and lymphocyte c u l t u r e s contained a n t i b i o t i c s (Potassium P e n i c i l l i n G, 100 u n i t s / m l ; ; Streptomycin, 0.1 mg /ml ; and, i n phase 1 only, Amphotericin B,0.025 yg /ml ; Gibco). I n i t i a l l y , i n -d i v i d u a l s e r a or lymphcoyte preparations were t e s t e d w i t h and without PHA i n t r i p l i c a t e or q u a d r u p l i c a t e . Subsequently, c o n t r o l s without PHA 102 were omitted as t h i s was found not to a l t e r the conclusions (see I I - I I I ) . C u ltures were incubated at 37^G i n a moist 5% CO2 atmosphere f o r 72±1 hours unless otherwise i n d i c a t e d . At that time, 0.05 ml of methyl 3H-TdR (Amersham-Searle, Don M i l l s , Ont. i n Phase 1 and New England Nuclear Canada, Lachine, P.Q. i n Phase 2 ) , 20 uCi/ml i n RPMI-1640 w i t h a n t i b i o t i c s was added to each w e l l to give a f i n a l volume of 0.30 ml. A f t e r an a d d i t -i o n a l 16 to 18 hours of i n c u b a t i o n , h a r v e s t i n g was performed by a s p i r a t i n g the contents of each w e l l onto a g l a s s . f i b e r f i l t e r u s i n g a m u l t i p l e sample harvestor (Hartzman et a l . 1972; Thurman et_ al_. 1973). Each f i l t e r was washed w i t h methanol, d r i e d , immersed i n 3 ml of s c i n t i l l a t i o n f l u i d c o n s i s t i n g of toluene c o n t a i n i n g 4 gm/1 Omnifluor (New England Nuclear Canada) i n Phase 1, or 4 gm/1 PPO/Bis IV (Snydel L a b o r a t o r i e s , Vancouver, B.C.) i n Phase 2, and counted i n a Nuclear Chicago U n i l u x s c i n t i l l a t i o n counter. (a) T e s t i n g the E f f e c t of Sera on Lymphocyte S t i m u l a t i o n w i t h PHA: The PHA used to t e s t the e f f e c t of sera on normal lymphocyte respons-iveness to PHA was Bacto-Phytohaemagglutinin P (Difco L a b o r a t o r i e s , D e t r o i t , Mich.). This was supplied as a powder to be r e c o n s t i t u t e d w i t h 5 ml s t e r i l e d i s t i l l e d water. PHA concentrations are expressed as d i l u t i o n s of t h i s stock s o l u t i o n . Unless otherwise i n d i c a t e d i n I I - I I I , each w e l l contained 10 5 lymphocytes. The usual c o n d i t i o n s f o r t e s t i n g a.serum f o r immune i n h i b i t o r y a c t i v i t y c o n s i s t e d of usi n g serum d i a l y s e d against RPMI-1640 at a serum 103 protein concentration of 14 mg/ml, a PHA concentration, of 1:1500, and an incubation time of 72 hours before the addition of 3H-TdR. Detai l s of i n d i v i d u a l experiments, e s p e c i a l l y departures from the usual expe-rimental conditions, are given with the r e s u l t s . The e f f e c t of a serum sample on the response of normal lymphocytes to PHA i s expressed as the serum support of lymphocyte stimulation (SSLS) or the serum support of lymphocyte stimulation index (SSLS-T), calculated as follows: SSLS = mean counts/min (c.p.m.) i n r e p l i c a t e wells containing the serum plus PHA. In early experiments, SSLSs == '(mean c.p.m. i n r e p l i c a t e wells ) - (mean c.p.m, i n r e p l i c a t e ) (with serum + PHA ) (wells with serum only ) SSLS-I = SSLS of an i n d i v i d u a l serum x 100% mean SSLS of control sera i n the same experiment : 1/ . Dialysed sera with SSLS-I<30% under the standard conditions of 14 mg serum protein/ml, PHA 1:1500, and 72 hours incubation were designated i n h i b i t o r y sera. 30% was chosen a r b i t r a r i l y before analysis of the r e s u l t s . (b) Testing the Response of Control and Patients Lymphocytes to PHA: In experiments assessing PHA responsiveness of patients' lymphocytes, each w e l l contained 1.5 x ID5 lymphocytes. For lymphocytes from each donor eight wells were used, four with and four without PHA. Lymphocytes were cultured i n 8% heat-inactivated f o e t a l c a l f serum (FCS) (Gibco). Two experiments were c a r r i e d out to determine whether peri p h e r a l lymphocytes from breast cancer patients responded to PHA l e s s w e l l than co n t r o l s . In experiment 1, the PHA used was Bacto-Phytohaemagglutinin P (Difco) as above, at 1:750 f i n a l concentration. In experiment 2, PHA, reagent grade (Burroughs-Wellcome, Beckenham, England) was used at a f i n a l concentration of 1:125 of the reconstituted powder. These were the optimal PHA concentrations f o r the two types of PHA. A l l lymphocyte preparations comprising one experiment were not tested on the same day, but i n groups of two or three over several weeks. For each i n d i v i d u a l ' s lymphocytes,the'.lymphocyte stimulation i s calculated as : (mean c.p.m. i n wells with PHA) - (mean c.p.m. i n wells without PHA). The percent lymphocyte•stimulation. .is;caleulated as: (stimulation with Individual's lymphocytes )_ x 100%. (mean stimulation with co n t r o l lymphocytes i n the same experiment) 7. Testing Dialysed Sera for Cytotoxicity;-toward Lymphocytes Cultures of normal lymphocytes with sera to be tested were set up i n test tubes. Each culture contained 106- lymphocytes and 14 mg dialysed serum protein i n RPMI-1640 plus a n t i b i o t i c s i n a t o t a l volume of 1 ml. The cultures were incubated at 37°C.,."5% CO2 f o r four days. Samples of each culture were removed d a i l y f o r four days, d i l u t e d appropriately, and the number of v i a b l e lymphocytes determined. by'.JTrypan...bluef.exclusion. 8. Mixed Lymphocyte Reaction The e f f e c t of some patients' sera on a two-way mixed lymphocyte reaction was tested i n order to determine whether sera which i n h i b i t e d the response of normal lymph.ocy/t5es---.to.'-PHA',.couldr.also::-intilb.±t".'the' 'MLR. Two-way mixed lymphocyte reactions were set up i n m i c r o t i t e r p l a t e s . Each w e l l contained 10 5 lymphocytes i n RPMI-1640 from eachjof..two un-r e l a t e d normal donors or 2 x 10 5 lymphocytes from one donor i n c o n t r o l 105 wells, and 20% whole patient's serum i n a t o t a l volume of 0,25 ml. Each patient's serum was tested i n t r i p l i c a t e wells, Plates were incubated for 5 days at 37°C and 5% C0 2. Then 0.05 ml of 3H-TdR, 20 UCi/ml i n RPMI-1640 was added, and the plates incubated an a d d i t i o n a l 16 to 18 hours before being harvested and counted as above. The serum support of MLR for an i n d i v i d u a l ' s serum was calculated as the mean c.p.m. i n t r i -p l i c a t e wells containing 20% serum from that i n d i v i d u a l and lymphocytes from both donors. 9. S t a t i s t i c a l Methods The hypotheses that patients' sera supported lymphocyte stimulation by PHA les s w e l l than control sera (Figure 1), that 40% undialysed sera from patients with i n h i b i t o r y dialysed sera were i n h i b i t o r y (Table IX), and that patients' lymphocytes responded. tooPHA'-signifleantly..less.'.well than co n t r o l lymphocytes (Figure 7) were tested by an analysis of variance with r e p l i c a t i o n s (Snedecor and Cochran 1967). This was performed by computer. Because the standard deviation of r e p l i c a t e measurements was approximately l i n e a r l y r e l a t e d to the mean, a l l analyses of variance were performed on the logarithms of the SSLS readings. The r e p r o d u c i b i l i t y of the SSLS on d i f f e r e n t days with d i f f e r e n t lymphocyte donors (Tables XI and XII) was analysed by a components of variance analysis(Kempthorne 1952; Snedecor and Cochran 1967). This analysis was performed by computer. Again the analysis was performed on the logarithms of the SSLS readings. 106 The r e l a t i o n between SSLS-I and serum support of MLR (Table XIII), was studied by c o r r e l a t i o n analysis and the r e l a t i o n between SSLS-I and the percent lymphocyte stimulation of patients' lymphocytes (Table XIV) was studied by regression analysis (Snedecor and Cochran 1967). The regression analysis was performed by computer. I I I . RESULTS 1. Determination of the Duration of Incubation to be Employed Routinely  i n Lymphocyte Stimulation Experiments Several early experiments were set up i n duplicate, and 3H-TdR was added to one plate a f t e r 48 hours of incubation and to the duplicate p l a t e a f t e r 72 hours of incubation. In each case, the plate was incu-bated an a d d i t i o n a l 16 to 18 hours before being harvested. In these experiments each w e l l contained 1.5 x 10 5 white blood c e l l s (not p u r i f i e d lymphocytes) from controls or patients,. 10% FCS and PHA 1:750 or PHA 1:375. The r e s u l t s are shown i n Table VI. In a l l but CI, PHA 1:750 and C7, PHA 1:750, 3H-TdR incorporation . was greater when 3H-TdR was added a f t e r 72 hours. Thereafter 72 hours was the routine incubation time used. The majority of studies reported i n the l i t e r a t u r e have used a 72 hour incubation time f o r the stimulation of-human lymphocytes with PHA. TABLE VI EFFECT OF DURATION OF INCUBATION ON PHA STIMULATION OF NORMAL LYMPHOCYTES IN 10% FCS ' • (A PRELIMINARY EXPERIMENT) Mean Lymphocyte S t i m u l a t i o n (c.p.m.) Lymphocyte 4 8 H o u r I n c u b a t i o n 72 Hour Incubation Donor PHA 1:750 PHA 1:375 . PHA 1:750 PHA 1:375 C 1* 44,102 + 269** 43,725 + 2658 C 2 56,955 + 5557 44,482 + .37.77. , 132,904 + 7244 122,612 + 5635 C 3 50,279 + 3215 38,104 + 1920 108,260 + 9710 89,146 + 6168 C 4 55,308 + 1774 51,803 + 1314 104,316 + 4571 111,996 + 2019 C 5 55,342 + 1752 60,510 + 1756 100,906 + 3292 111,678 + 365 C6 74,807 + 2069 69,277 + 1173 131,942 + 2607 155,690 + 4188 C 7 118,280 + 8282 94,598 + 2938 109,772 + 4225 142,302 + 3019 P 1 55,008 + 756 44,918 + 1149 64,298 + 1883 56,842 + 584 P 2 35,588 + 966 29,658 + 1428 54,160 + 913 47,322 + 2747 *C 1-7 designate i n d i v i d u a l c o n t r o l s ; P 1-2 designate i n d i v i d u a l p a t i e n t s . **Each r e s u l t i s the mean ± standard e r r o r of the mean (S.E.) of 4 w e l l s . o 108 2. P r e l i m i n a r y Experiments Suggesting the Value of Using D i a l y s e d Sera In a very e a r l y experiment, f r a c t i o n a t i o n of serum by p r e c i p i t a t i o n w i t h 50% saturated ammonium sulphate (SAS) was c a r r i e d out i n order to t e s t the f r a c t i o n s f o r i n h i b i t i o n of the PHA response of normal lympho-cytes. The g l o b u l i n f r a c t i o n of sera from breast cancer p a t i e n t s i n h i b -i t e d the response of normal lymphocytes to PHA compared w i t h the co r r e s p -onding f r a c t i o n of normal serum ( r e s u l t s not shown). However, i n a l a t e r p r e l i m i n a r y experiment, 20% sera from 14 p a t i e n t s w i t h m e t a s t a t i c breast cancer d i d not i n h i b i t the response of normal lymphocytes to PHA, 1:750 compared w i t h c o n t r o l sera ( r e s u l t s not shown). I t was pos t u l a t e d that the removal of small molecules from the g l o b u l i n f r a c t i o n by t h e . . d i a l y s i s r e q u i r e d to remove the ammonium sulphate may have caused the discrepancy i n the r e s u l t s of these two experiments. Therefore, these 14 sera were d i a l y s e d against PBS and the experiment was repeated u s i n g d i a l y s e d sera at a p r o t e i n c o n c e n t r a t i o n of 14 mg/ml which would be equivalent to the p r o t e i n i n 20% whole serum (Ravel 1969). The d i a l y s e d sera from the cancer p a t i e n t s supported lymphocyte s t i m u l a t i o n l e s s w e l l than d i d c o n t r o l sera ( r e s u l t s not shown). However, i t was found that the sub-s t i t u t i o n of PBS f o r some of the t i s s u e c u l t u r e medium RPMI-1640 i n the w e l l s caused a considerable decrease i n 3H-TdR i n c o r p o r a t i o n . A c c o r d i n g l y , a l l l a t e r experiments used sera d i a l y s e d against RPMI-1640 where d i a l y s e d sera were r e q u i r e d . 3. Determination of the PHA Concentration and the Number of Lymphocytes  per Well to be Employed R o u t i n e l y i n Lymphocyte S t i m u l a t i o n E x p e r i - ments (Phase 1)' An experiment was set up i n which each w e l l contained pooled normal serum, d i a l y s e d against RPMI-1640, at a p r o t e i n c o n c e n t r a t i o n of 14 mg/ ml, w i t h v a r y i n g numbers of lymphocytes from one of two normal donors, 109 and varying amounts of PHA. The experiment was incubated for 72-hours before 3H-TdR was added. The r e s u l t s are shown i n Table VII. Depending upon the lymphocyte donor, and the number of lymphocytes per w e l l , the optimum PHA concentration was 1: 500 to 1:1000, most often 1:750. There i s evidence that suboptimal PHA concentrations may reveal lymphocyte defects not apparent at the optimal concentration (Ducos et a l . 1970). Therefore, 1:1500 was chosen as the usual f i n a l PHA concentration to be used i n t e s t i n g for serum i n h i b i t i o n . The usual number of lymphocytes per w e l l was chosen to be 10 5. From the r e s u l t s of Table VII, i t appeared that t h i s number of c e l l s would give a good but probably suboptimal response to PHA. It was thought that i n h i b i t i o n of lymphocyte stimulation by serum might be more r e a d i l y demonstrated at s l i g h t l y suboptimal c e l l numbers, since at optimal or supraoptimal c e l l numbers the i n a c t i v a t i o n of a proportion of lymphocytes might not r e s u l t i n lower observed 3H-TdR incorporation. 4. Background 3H-TdR Incorporation into Lymphocytes i n the Absence of  PHA (Phase 1) In the i n i t i a l experiments to test the e f f e c t of patient and c o n t r o l dialysed sera on the response of normal lymphocytes to PHA, t r i p l i c a t e wells containing dialysed sera, lymphocytes, but no PHA were set up. The mean lymphocyte stimulation i n the wells without PHA could be subtracted from the stimulation i n wells with PHA to give the SSLS. Two t y p i c a l sets of r e s u l t s are shown i n Table VIII. Because the background stimu-l a t i o n s for both controls and patients were so low r e l a t i v e to the stimu-lations'-with PHA for a l l but the most i n h i b i t o r y sera, f a i l u r e to subtract the background stimulations would not a l t e r any conclusions to be drawn from these experiments. Accordingly, i n the i n t e r e s t of conserving serum, wells without PHA were omitted. TABLE V I I EFFECT OF PHA CONCENTRATION AND NUMBER OF LYMPHOCYTES PER WELL ON PHA STIMULATION OF NORMAL LYMPHOCYTES IN POOLED DIALYSED NORMAL SERUM (PHASE 1) Mean Lymphocyte S t i m u l a t i o n (c.p.m.) Normal Number of Lymphocyte Lymphocytes Donor Per Well PHA Concentration 0 1:1250 1:1000 1:750 1:500 1:250 1 0.75 x 10 5 4320 ± 165* 101,042 ± 3298 102,352 ± 818 110,872 ± 4032 108,960 ± 8530 97,715 ± 8485 1.5 x 10 5 2498 ± 428 123,962 ± 1762 130,592 ± 9732 134,325 ± 4050 147,238 ± 3852 145,025 ± 14,790 3.0 x 10 5 • 1815 ± 140 67,910 ± 2061 63,865 ± 20,135 87,975 ± 12,070 84,238 ± 12,048 75,805 ± 9685 2 0.75 x 10 5 1260° ± 120 41,735 45,275 41,015 ±:.1660 26,118 ±..1322 14,695 ± 6405 1.5 x 10 5 1078 ± 412 96,052 ± 5582 97,090 ± 2545 107,283 ± 312 86,678 ± 15,068 73,738 ± 3618 3.0 x 10 5 1655 106,910 107,215 119,350 94,885 81,550 *Results are the mean ± S.E. of 2 w e l l s i n most cases. Where the S.E. i s omitted, r e s u l t s are f o r 1 w e l l only. TABLE VIII SSLS WITH AND WITHOUT PHA (PHASE 1) Experiment (a) Experiment (c) SSLS (c.p m.) SSLS (c.p.m.) Serum Donor PHA Concentration Serum Donor PHA Concentration 0 1:1500 .0 1:1500 C 1* 333 + 62+ 19,499 .1090 C 1 1562 ± 81* 88,780 ± 2904 C 2 268 + 21 47,938 + 2656 C 7 372 ± 68 43,308 ± 3971 C 3 1060 + 755 35,463 + 707 C 8 287 ± 75 29,485 ± 1951 C 8 566 + 123 32,574 + 1428 C 9 437 ± 81 113,640 ± 2707 C 9 .7.21 + 314 16,026 + 3846 C 10 ' 1233 ± 114 65,082 ± 4131 C 10 529 + 182 15,898 + 933 C 12 338 ± 76 80,545 ± 5553 C . l l 385 + 66 21,052 + 4963 C 13 278 ± 56 124,750 ± 4570 P 3 527 + 122 36,742 + 2273 C 14 402 ± 32 117,543 ± 4078 P 4 402 + 101 38,770 + 2391 C 15 200 ± 45 84,448 ± 3125 P 5 565 + 127 31,134 + 665 P 8 1340 103,020 TABLE V I I I (continued) Experiment (a) Experiment (c) SSLS (c.p.m.) SSLS Cc. p.m.) Serum Serum Donor PHA Concentration Donor PHA Concentration 0 1:1500 0 1:1500 P 6 302 ± 57 47,844 ± 2702 P 9 1325 + 250 51,378 ± 4802 P 7 369 ± 56 49,386 ± 2960 P 10 742 + 50 48,525 ± 6745 P 11 398 + 9 73,668 ± 9010 P 12 435 + 46 7298 ± 2097 P 13 463 + 58 1577 ± 151 - P 14 575 + 40 31,922 ± 4923 P 15 385 + 118 69,003 ± 4762 P 16 177 + 16 10,085 ± 848 P 17 358 + 79 88,053 ± 4186 P 18 268 + 52 18,260 ± 1571 P 19 268 + 29 12,888 ± 508 TABLE VIII (continued) Experiment (a). , Experiment (c) SSLS (c.p.m.) SSLS (c.p.m.) Serum Donor PHA Concentration Serum Donor PHA Concentration 0 1:1500 0 1:1500 -P 20 1 7 8 + 7 15,970 ± 1378 P 21 400 ± 67 718 ± 47 P 22 362 ± 82 9585 ± 3755 p 23 300 ± 80 3032 ± 192 *C 1-15 designate i n d i v i d u a l controls; P 3-23 designate i n d i v i d u a l p a t i e n t s , +Each r e s u l t i n Experiment (a) i s the mean ± SE of 4 wells. ^Each r e s u l t i n Experiment (c) i s the mean ± SE of 3 wells, with the exception of P 8 f o r whom only one well was a v a i l a b l e . 114 5, E f f e c t of Patients' Dialysed Sera on the Response of Ngprnal Lympho-cytes to PHA (Phase 1) The e f f e c t of patients' as compared with control dialysed sera on the stimulation of normal lymphocytes by PHA was tested i n four separate experiments on four d i f f e r e n t days, The r e s u l t s are shown i n Figure 1. Experiments (a) and (b) used lymphocytes from the same normal donor c o l l e c t e d on d i f f e r e n t days. Experiments (c) and (d) used lymphocytes from two other normal donors. Most con t r o l sera i n (c) and (d) had been used i n (a) or (b). Some cont r o l sera were used i n both (c) and (d), but only one was used i n both (a) and (b). Altogether, 18 control sera were used. In Experiment (d) 6 patients' sera had been used i n one of the previous experiments. Experiments (a), (b), and (c) used separate patients' serum samples. In (b), ( c ) , and (d) lymphocyte stimulation was lower i n the presence of patients' sera than of control sera. The differ e n c e was s t a t i s t i c a l l y s i g n i f i c a n t only i n ( c ) , f e l l j u s t short of s i g n i f i c a n c e i n (b), and was not s i g n i f i c a n t i n (d). In (b), ( c ) , and (d) a few patients' sera were very i n h i b i t o r y to lymphocyte stimulation. On the other hand, i n (a), the patients' dialysed sera supported lymphocyte stimulation s i g n i f i c a n t l y better than the normal dialysed sera, and there were no individual i n h i b i t o r y sera from the pa t i e n t s . The patients i n these experiments were r e c e i v i n g a v a r i e t y of treatments for metastatic breast cancer inc l u d i n g the conventional hormone treatments and chemo-therapy , 115 4 0 r 20 ^ \20\ 80} 60 \ 20} (a) • p<0.05 (c) p<0.01 (b) p=0.06 (d)| N.S. Controls Patients Controls Patients SERUM DONORS . FIGURE 1: EFFECT OF CONTROL AND PATIENTS' DIALYSED SERA ON THE RESPONSE OF NORMAL LYMPHOCYTES TO PHA, 1:1500 (PHASE 1) (a ) , ( b ) , ( c ) , and (d) represent the r e s u l t s of four separate experiments. Each c i r c l e , and square, • , represents the SSLS of an i n d i v i d u a l c o n t r o l or p a t i e n t ' s serum r e s p e c t i v e l y . Each p o i n t i s the mean of 3 w e l l s . The h o r i z o n t a l bars rep-resent the mean SSLS of each c o n t r o l or p a t i e n t group. 116 6• Effect of Inhibitory Dialysed Sera on the Viability of Normal  Lymphocytes (Phase 1) Three dialysed sera from breast cancer patients, known to inhibit the response of normal lymphocytes to PHA, and three control sera were tested for cytotoxicity to normal lymphocytes. There was no difference in viability of the lymphocytes in patients' and control sera (results not shown). 7. Dose-Response Effect of Dialysed Sera (Phase 1) The response of normal lymphocytes to PHA in the presence of a range of concentrations of dialysed serum protein is shown in Figure 2. In each experiment, (a) and (b), dialysed sera from healthy controls, breast cancer patients with noninhibitory dialysed sera, and breast cancer patients with inhibitory dialysed sera were tested at several concentrations. In general, the lymphocyte stimulation increased to a maximum with increasingxserum protein concentration and then decreased. The one exception was the most inhibitory serum which would probably have reached its optimal concentration at less than 0.7 mg/ml. In most cases, the inhibitory sera were more immunosuppressive than other sera at a l l serum protein concentrations tested, but the degree of inhibition increased markedly with increasing concentration. The inhibitory dialysed sera supported lymphocyte stimulation maximally at 1.4 mg/ml compared with control and noninhibitory patients' dialysed sera which stimulated maximally at 3.5 mg/ml to 7.0 mg/ml in most cases. 118 FIGURE 2: EFFECT OF CONCENTRATION OF DIALYSED SERUM PROTEIN ON NORMAL LYMPHOCYTE STIMULATION BY PHA 1:1500 (PHASE 1) (a) and (b) represent the r e s u l t s of two d i f f e r e n t experiments each u s i n g lymphocytes from a d i f f e r e n t normal donor. D i a l -ysed s e r a are from ( i ) h e a l t h y c o n t r o l s , ( i i ) p a t i e n t s w i t h n o n i n h i b i t o r y d i a l y s e d s e r a , and ( i i i ) p a t i e n t s w i t h i n h i b i t -ory d i a l y s e d s e r a . Each curve rep resents an i n d i v i d u a l serum sample. Each p o i n t i s the mean of 3 w e l l s . 119 8, Dose-Response Effect of Undialyse.d. Sera (Phase, 1) As lymphocytes i n vivo are i n contact with undialysed rather than with dialysed serum, i t was of interest to compare the effect of un-dialysed and;.dialysed sera on normal lymphocyte stimulation. The PHA-induced stimulation of lymphocytes from a normal donor i n the presence of control and patients' sera at various concentrations i s shown i n Figure 3. Up to and including." 20% serum (approximately equivalent to a r p r o t e i n con-centration of 14 mg/ml), lymphocyte stimulation remained almost constant with changing serum concentration and did not d i f f e r greatly between control sera, ( i ) , and patients' s e r a , ( i i ) and ( i i i ) , although there was perhaps a s l i g h t decline i n stimulation i n 20% undialysed serum from patients with i n h i b i t o r y dialysed sera, ( i i i ) . Lymphocyte stimulation was greater and v a r i a b i l i t y less i n 20% whole serum than i n 14 mg/ml dialysed serum protein. At serum concentrations greater than 20%, lympho-cyte stimulation declined and the v a r i a b i l i t y within each group increased. Although the sample sizes were too small for s t a t i s t i c a l t e s t i n g , i t appeared that the patients' sera might be more in h i b i t o r y than control sera at 40% and 60% serum.. In this experiment, the data on the effect of the serum when dialysed had been obtained from previous experiments using lymphocytes from different donors. Therefore, to make the comparison between dialysed and undialysed serum more meaningful, undialysed sera at 40% and the same sera dialysed at 14 mg' protein/ml were tested with lymphocytes from a single donor i n the experiments with PHA 1:1500. The results are shown i n Table IX. 40% undialysed sera of patients whose dialysed sera were i n h i b i t o r y : 120 FIGURE 3: EFFECT OF CONCENTRATION OF UNDIALYSED SERUM ON NORMAL LYMPHO-CYTE STIMULATION BY PHA,'1:1500 (PHASE 1) Sera are from (i) healthy controls, ( i i ) patients with non-inhibitory dialysed sera, and ( i i i ) patients with inhibitory dialysed sera. Each curve represents an individual serum sample. Each point i s the mean of 3 wells . TABLE IX SSLS IN DIALYSED AND UNDIALYSED SERA (PHASE 1) Control or Patient Group Serum Donor SSLS (c.p.m.) Dialysed Serum at 14 mg protein/ml Undialysed Serum at 40% Controls C 3 C 5 C 17s* 65,040 ± 2257* 56,052 ± 1190 56,155 ± 1701 Mean=59,082 62,063 ± 12,256 55,102 ± 5184 66,127 ± 5771 Mean=61,097** Patients with Noninhibitory Dialysed Sera P 7 P 15 P 24 46,005 ± 786 35,518 ± 1040 36,590 ± 1090 Mean=39,371 17,628 ± 830 83,345 ± 6772 73,155 ± 6867 Mean=58,043** Patients with P 16 2095 ± 95 28,073 ± 1264 Inhibi t o r y Dialysed Sera P-21 3137 ± 244 17,663 ± 390 P 25 7287 ± 330 38,777 ± 3037 TABLE IX (continued) C o n t r o l or P a t i e n t Group SSLS (c.p.m.) Serum Donor Dia l y s e d Serum at 14 mg, protein/ml Undialysed Serum at 40% P a t i e n t s w i t h I n h i b i t o r y D i a l y s e d Sera P 26 7072 + 1372 Mean=4898 48,476 ± 6666 Mean=33,247** + C 3-17 designate i n d i v i d u a l c o n t r o l s ; P 7-26 designate i n d i v i d u a l p a t i e n t s , 4: These were a l s o c o n t r o l s i n Figure 3. * Each r e s u l t i s the mean ± SE of 3 w e l l s . ** There are no s i g n i f i c a n t d i f f e r e n c e s among these 3 means. 123 supported lymphocyte s t i m u l a t i o n by PHA. less, w e l l than d i d 40% undialysed sera from c o n t r o l s or from breast cancer p a t i e n t s whose d i a l y s e d sera were not i n h i b i t o r y . This d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t , perhaps because of the small numbers of i n d i v i d u a l s i n each group. 9, Dose-Response E f f e c t bf PHA (Phase 1) As found by Humphrey et a l . (1974), i t was p o s s i b l e that the inhibit-• t'Oi?y-.:ser.a:;ne.cess:il5afcedr,a'. -.h.£gh.e.r' PHA. con c e n t r a t i o n f o r optimal lymphocyte s t i m u l a t i o n than d i d normal s e r a , and that the i n h i b i t o r y e f f e c t could be overcome by i n c r e a s i n g PHA c o n c e n t r a t i o n . With d i a l y s e d s e r a , t h i s p o s s i b i l i t y was r u l e d out i n an experiment shown i n Figure 4. With d i a l y s e d sera at 14 mg p r o t e i n / m l , the maximum lymphocyte s t i m u l a t i o n was achieved at PHA 1:750 or 1:375 f o r sera from both p a t i e n t s and ::. :\.v c o n t r o l s . The se r a which were i n h i b i t o r y at 1:1500 , (the usual concen-t r a t i o n ) , remained immunosuppressive at the optimal PHA c o n c e n t r a t i o n . However, w i t h 40% undialysed s e r a , the e f f e c t of those se r a that were i n h i b i t o r y at PHA 1:1500 could be overcome by i n c r e a s i n g the PHA co n c e n t r a t i o n . This i s i l l u s t r a t e d i n Figure 5. This f i g u r e shows one very anomalous serum i n ( i i ) w i t h which lymphocyte s t i m u l a t i o n d e c l i n e d w i t h i n c r e a s i n g PHA c o n c e n t r a t i o n . 124 FIGURE 4: RESPONSE OF NORMAL LYMPHOCYTES TO VARIOUS CONCENTRATIONS OF PHA IN THE PRESENCE OF 14 MG/ML DIALYSED SERUM PROTEIN (PHASE 1) Dialysed sera are from ( i ) healthy controls, ( i i ) patients with noninhibitory dialysed sera, and ( i i i ) patients with i n h i b i t o r y dialysed sera. Each curve represents an i n d i v i d u a l serum sample. Each point i s the mean of 3 wells. 125 75 0 375 250 1875 150 125 ( P H A D I L U T I O N ) " ' FIGURE 5: RESPONSE OF NORMAL LYMPHOCYTES TO VARIOUS CONCENTRATIONS OF PHA IN THE PRESENCE OF 40% UNDIALYSED SERUM (PHASE 1) Sera are from ( i ) healthy controls, ( i i ) patients with non-i n h i b i t o r y dialysed sera, and ( i i i ) patients with i n h i b i t o r y dialysed sera. Each curve represents an i n d i v i d u a l serum sample. Each point i s the mean of 3 wells. 126 10. E f f e c t of the Duration of Incubation on SSLS at D i f f e r e n t PHA, Concentrations (Phase 2) Experiments to measure SSLS were rou t i n e l y incubated f o r approxi:-.: mately 72 hours before the addition of 3H<-TdR, and for an a d d i t i o n a l 16 to 18 hours thereafter. It was considered p o s s i b l e that some or a l l sera which appeared to be i n h i b i t o r y under these conditions might support a maximum lymphocyte stimulation equal to that of controls, but might reach that maximum at a d i f f e r e n t time. Therefore, an experiment was set up i n quadruplicate t e s t i n g sera from controls and cancer patients at three d i f f e r e n t PHA concentrations, Due to a shortage of some sera, not a l l were tested at 25 hours or at PHA .1:750 or PHA 1:375. 3H-TdR was added to each of the quadruplicate experiments at a d i f f e r e n t time, i . e . a f t e r 25, 49, 73.5 and 97 hours: of ..incubation. Each experiment was then incubated an a d d i t i o n a l 18.5 hours before harvesting and counting. The r e s u l t s are shown i n Figure 6 and Table X. In summary, at the three PHA concentrations tested, the more stimu-l a t o r y patients' and control sera reached a maximum SSLS at 73.5 hours of incubation whereas theless stimulatory sera from patients and controls, in c l u d i n g the i n h i b i t o r y sera (SSLS-I<30%), reached a maximum SSLS at 49 hours. However, even at 49 hours, the i n h i b i t o r y sera had generally lower values of SSLS than did the noninhibitory and control sera. Some i n h i -b i t o r y sera would not have been c a l l e d i n h i b i t o r y ( i , e , SSLS-I<30%) i f tested only at a 49 hour incubation time and/or at PHA 1:750 or 1:375, but they would s t i l l have been le s s stimulatory than most control and non-i n h i b i t o r y patients sera. Therefore, the i n h i b i t o r y character of sera 1273 128 FIGURE 6: RESPONSE OF NORMAL LYMPHOCYTES TO VARIOUS CONCENTRATIONS OF PHA AFTER DIFFERENT INCUBATION TIMES IN THE PRESENCE OF 14 MG/ML DIALYSED SERUM PROTEIN (PHASE 2) (a), (b), and (c) show the r e s u l t s using d i f f e r e n t PHA con-centrations: (a) PHA 1:1500, (b) PHA 1:750, and (c) PHA 1:375. Dialysed sera are from ( i ) healthy controls, ( i i ) patients with noninhibitory dialysed sera, and ( i i i ) patients with i n h i b i t o r y dialysed sera. Each combination of symbol and type of l i n e , e.g. O — — O , represents the serum from the same one i n d i v i d u a l each time i t i s used. Each point i s the mean of 2 or 3 we l l s . TABLE X EFFECT OF THE DURATION OF INCUBATION ON SSLS AT DIFFERENT PHA CONCENTRATIONS (PHASE 2) PHA Concen-t r a t i o n Duration of Co n t r o l Sera N o n i n h i b i t o r y P a t i e n t s ' Sera I n h i b i t o r y P a t i e n t s ' Sera Incubation (hours be-.' : fore 3H-TdR added) •- Mean SSLS (c.p.m.).. Proportion ; w i t h SSLS-I<30%** Mean SSLS (c.p.m.) Pr o p o r t i o n w i t h SSLS-I<30% Mean SSLS (c.p.m.) Pr o p o r t i o n w i t h SSLS-I<30% 1:1500 25 49 . 7029 ± 1608* 23,108 ± 4234 1/6 0/8 10,088 ± 1230 33,991 ± 3742 0/6 0/8 2141 ± 1068 9639 ± 2854 3/5 3/7 73.5 27,974 ± 11,089 1/8 34,621 ± 6681 0/8 3638 ± 995 7/7 (by d e f i n i t i o r 97 12,443 ± 5583 2/8 11,229 ± 2956 2/8 736 ± 166 7/7 1:750 25 49 8836 ± 4562 37,481 ± 6816 1/3 1/6 16,801 ± 1259 51,434 ± 3244 0/4 0/6 N.A. 24,769 ± 3487 N.A.+ 0/3 73.5 48,464 ± 16,337 1/6 53,923 ± 7178 0/6 20,075 ± 5078 1/3 97 24,520 ± 9562 1/6 21,246 ± 3907 0/6 5333 ± 2021 2/3 TABLE X (continued) PHA Concen-t r a t i o n Duration of Incubation (hours be-fore 3H-TdR added) Control Sera Mean SSLS Proportion (c.p.m.) with SSLS-I<30% Noninhibitory Patients' Sera Mean SSLS Proportion (c.p.m.) with SSLS-1<30% Inh i b i t o r y Patients' Sera Mean SSLS Proportion (c.p.m.) with SSLS-I<30% 25 10,176 1/3 18,900 0/4 N.A. N.A. ± 4658 ± 1092 1:375 49 42,514 1/6 56,393 0/4 N. A. N.A. ± 8092 ± 4524 73.5 56,020 1/6 63,760 0/4 N.A. N.A. ± 15,999 ± 3563 97 31,620 1/6 27,516 0/4 N.A. N.A. ± 10,963 ± 3705 *Each r e s u l t under Mean SSLS i s the mean ± SE of the SSLS for the persons:-. i n the con t r o l of patient group. The SSLS for each person i s , i n turn, the mean of 2 or 3 wells. **The control mean used for the c a l c u l a t i o n of the SSLS-I i n each case i s the c o n t r o l mean for that PHA concentration and duration of incubation. N.A. means not applicable because i n s u f f i c i e n t serum was av a i l a b l e for t e s t i n g that combination of PHA concentration and duration of incubation. i—1 o 131 does not r e f l e c t merely a d i f f e r e n t time of maximum SSLS or a d i f f e r e n t dose-response r e l a t i o n s h i p to PHA compared with noninhibitory sera. In the preliminary experiment shown i n Table VI, i n which i t was determined that the maximum 3H-TdR incorporation occurred a f t e r 72 hours, the 3H-TdR was higher than i n t h i s l a t e r experiment mainly because 10% FCS was used instead of dialysed sera, and PHA was used at 1:750 and 1:375 but not at 1:1500. As seen i n Figure 6, the maximum 3H-TdR incorp-oration more often occurs at 72 hours with the higher concentrations of PHA, but at 48 hours with PHA 1:1500. 11. R e p r o d u c i b i l i t y of Measurements of SSLS To avoid overcrowding of the f i g u r e s , standard errors of the means of r e p l i c a t e measurements have not been shown. . These were les s than 10% i n most cases and were of the same order of magnitude as those shown i n the tables. It was considered possible that a serum sample might i n h i b i t the PHA response of lymphocytes from some donors but not from others. For example, a serum sample might i n h i b i t c e r t a i n lymphocytes because i t contained anti-HL-A antibodies or antibodies against blood group antigens present on those lymphocytes. However, there i s no reason to expect such antibodies with higher frequency i n the sera of cancer patients than of controls. Nevertheless, to r u l e out t h i s p o s s i b i l i t y i n the early phase of the project (Phase 1), several sera were tested with lymphocytes from d i f f e r e n t donors on d i f f e r e n t occasions. In t h i s phase, a l l serum c o l l e c t e d from a p a r t i c u l a r patient or control at any one time was heat i n a c t i v a t e d , dialysed against RPMI-1640, then stored frozen. Aliquots were thawed as needed. Therefore, the serum from a given patient of c o n t r o l tested on lymphocytes from several donors had a l l been heat inactivated, and dialysed i n one batch. The SSLS-I measurements obtained for one serum sample tested on d i f f e r e n t donors' lymphocytes had a range of l e s s than 10% for 15 of 27 sera and a range of greater than 30% for only 2 of 27 sera. However, i n the l a t e r phase of the project (Phase 2), i t seemed that when one serum sample was tested on d i f f e r e n t occasions, using lymphocytes from d i f f e r e n t donors, there was often considerable v a r i a t i o n i n SSLS-I. Therefore, an experiment was set up to study the source of t h i s v a r i a b i l i t y i n more d e t a i l . In t h i s experiment, 3 c o n t r o l sera and 14 p a t i e n t s ' sera (including more than one sample from c e r t a i n patients) were used. The SSLS was measured for each serum sample i n duplicate or i n t r i p l i c a t e as the amount of serum a v a i l a b l e permitted, with 3 d i f f e r e n t lymphocyte donors on each of two d i f f e r e n t days at an i n t e r v a l of 2 weeks. In t h i s exper-iment, a d i f f e r e n t a l i q u o t of each serum sample was thawed, heat i n a c t -ivated, and dialysed f o r each of the d i f f e r e n t days. However, the same ali q u o t , heat i n a c t i v a t e d and dialysed as a whole, was used with lymphocytes from three d i f f e r e n t donors on each day. The r e s u l t s are shown i n Table XI. The r e s u l t s of the s t a t i s t i c a l a n a lysis are shown i n Table XII. Analysis of variance performed on the r e s u l t s from 17 sera showed that the variance due to each of the other possible sources of v a r i a t i o n was very s i g n i f i c a n t l y greater than the error variance. For 9 patients' sera with t r i p l i c a t e r e s u l t s , a components of variance analysis was done. The v a r i a t i o n due to patients ( i . e . d i f f e r e n t serum donors) was much greater than the v a r i a t i o n due to d i f f e r e n t lympho-TABLE XI EFFECT OF DIFFERENT DAYS AND DIFFERENT LYMPHOCYTE DONORS ON SSLS (PHASE 2) Serum Donor Day 1 Lymphocyte Donors Day 2 Lymphocyte Donors a a C 18* 205,212 II*'* ± 15,192* 95^338 H ± 6868 173,470 H ± 7750 134,898 H ± 48,112 45,620 M ± 19,800 48,532 H ± 6042 C 19 220,473 H ± 7142 98,980 H ± 7003 180,878 H ± 2908 208,272 H ± 952 103,868 H ± 3202 55,940 H ± 2420 C 20 41,882 L ± 7852 26,055 M ± 2505 50,928 M ± 368 115,802 H ± 11,132 53,245 H ± 5645 49,355 H ± 7230 P 27 870 L ± 575 172 L ±-62" 175 L ± 90 255 L ± 55 130 L ± 65 165 L ± 25 P 28" P 29 90,548 M ± 6216 131,550 H ± 3086 48,097 M ± 6190 71,272 H ± 4734 92,120 M ± 3927 133,677 H ± 4129 70,550 M ± 6285 117,538 H ± 6116 15,728 L ± 1953 24,432 M ± 4019 15,500 M ± 391 26,893 M ± 444 P 30* Sample 1 178,450 H ± 6325 74,643 H ± 2018 118,837 H ± 3590 130,480 H ± 23,981 47,048 M ± 4496 36,408 H ± 1004 P 31' 61,718 M ± 3182 20,258 L ± 558 48,270 M ± 1323 1995 L ± 572 323 L 167 118 L ± 26 TABLE XI (continued) Day 1 Day 2 Serum Donor Lymphocyte Donors Lymphocyte Donors a B y a B Y P 32, Sample 1 163,952 H ± 2422 77,078 H ± 2138 125,758 H ± 4118 104,147 M ± 11,351 33,460 ± 2254 M 25,343 M ± 540 P 33* 213,925 H ± 11,499 111,338 H . ± 1746 148,503 H ± 1595 140,682 H ± 21,536 79,660 ± 6248 H 65,842 H ± 2350 P 34 186,032 H ± 118 77,330 H ± 8390 138,722 H ± 6262 161,422 H ± 16,142 84,333 ± 9696 H 62,678 H ± 10,896 P 30f Sample 2 113,918 H ± 10,474 59,452 H ± 8471 90,272 M ± 6285 146,068 H ± 40,217 42,785 ± 3345 M 36,045 H ± 3215 P 35* 154,543 H ± 7569 74,238 H ± 1086 131,940 H ± 10,684 116,473 H ± 8229 50,908 ± 6522 H 48,355 H ± 3540 P 36* 151,493 H ± 9837 70,937 H ± 6018 118,858 H ± 1691 152,592 H ± 20,835 72,642 ± 7188 H 51,890 H ± 4267 P 37 82,292 M ± 9948 45,378 M ± 8014 76,093 M ± 10,151 162,792 H ± 16,332 82,345 H ± 21,330 57,562 H ± 5312 P 32, Sample 2 224,552 H ± 9952 97,968 H ± 5578 157,112 H ± 6692 124,035 H ± 36,265 82,190 H ± 8585 66,180 H ± 2630 -P-TABLE XI (continued) Day 1 Day 2 Serum Donor Lymphocyte Donors Lymphocyte Donors a 3 Y a 3 Y P 3 8 + 166,533 H 62,795 H 119,263 H 155,712 H 49,360 H 29,533 M ± 8626 ± 6885 ± 491-3 ± 11,073 ± 3784 ± 3745 * C 18-20 designate i n d i v i d u a l c o n t r o l s ; P 27-38 designate i n d i v i d u a l p a t i e n t s . Where more than one blood sample has been obtained at d i f f e r e n t times from one p a t i e n t , these are c a l l e d Sample 1 and Sample 2. ** H,M, and L stand f o r h i g h , medium, and low as defined i n the t e x t . + Each r e s u l t i s the mean ± SE of 2 or 3 w e l l s . * Sera used i n the components of variance a n a l y s i s shown i n Table X I I . t—• 136 TABLE X I I COMPONENTS OF VARIANCE ANALYSIS ON THE EFFECT OF DIFFERENT PATIENTS' SERA, DAYS, AND LYMPHOCYTE DONORS ON SSLS (PHASE 2) Source of V a r i a t i o n Variance Percent of T o t a l Variance A - P a t i e n t s 0.11634 25.5 (Serum Donors) B - Lymphocyte Donors 0.03441 7.5 C - Days 0.07714 16.9 AB I n t e r a c t i o n 0.00028 0.06 BC I n t e r a c t i o n 0.03355 7.3 AC I n t e r a c t i o n 0.17595 38.5 . ABC I n t e r a c t i o n 0.007839 1.7 E r r o r * 0.010957 2.4 A l l other sources of v a r i a t i o n are h i g h l y s i g n i f i c a n t compared w i t h e r r o r v a r i a n c e . 137 cyte donors. There was also considerable v a r i a t i o n due to d i f f e r e n t days. However, the greatest si n g l e contribution to the t o t a l variance came from the patient-day i n t e r a c t i o n . The i n t e r a c t i o n terms can be understood i n the following way. I f there were no patient-day i n t e r a c t i o n s , then the SSLS for each patient or control serum would d i f f e r from Day 1 to Day 2 by a^constant amount. The patient-day i n t e r a c t i o n i s a measure of the depart-ure from t h i s additive r e l a t i o n s h i p . The other i n t e r a c t i o n s can be under-stood s i m i l a r l y . Considering the actual r e s u l t s i n Table XI, the SSLS values have been designated as high i f the SSLS-I > 70%, medium i f the SSLS-I i s 30%-70%, and low i f the SSLS-I < 30%. Only one serum had both low and high SSLS values with d i f f e r e n t donors and d i f f e r e n t days, but many changed between high and medium, and two between medium and low. Although the o v e r a l l mean SSLS was greater on Day 1 than on Day 2 f o r each lymphocyte donor, some sera had higher values of SSLS on.Day 2. The r e l a t i v e lack of r e p r o d u c i b i l i t y of the SSLS measurements i s apparently due mainly to differences introduced into the serum during d i a l y s i s . P ossibly small v a r i a t i o n s i n the thoroughness with which a serum i s dialysed, perhaps brought about by i t s l o c a t i o n i n the d i a l y s i s f l a s k , may have an e f f e c t on how much dial y s a b l e stimulatory material i s a v a i l a b l e to counteract i n h i b i t o r y large molecules. Because of t h i s imperfect r e p r o d u c i b i l i t y , designations of SSLS as high or low may be more meaningful than attempts to be q u a n t i t a t i v e . 138 12. Relationship Between Serum Support of Lymphocyte Stimulation by PHA and i n MLR (Phase 1) A two-way mixed lymphocyte reaction was set up i n 20% whole serum from several breast cancer patients whose dialysed sera had previously been tested f or i t s e f f e c t on PHA stimulation. The r e s u l t s are shown i n Table XIII. The c o r r e l a t i o n between SSLS-I for PHA stimulation and serum support of MLR was s t a t i s t i c a l l y s i g n i f i c a n t both with and without logarithmic transformation of the data. 13. Response of Patients' Lymphocytes to PHA (Phase 1) Since i n t r i n s i c lymphocyte defects, as well as serum immune i n h i b i t o r s , have been reported i n cancer patients (see Chapter I ) , an:, experiment was undertaken to determine whether washed lymphocytes from our breast cancer patients could respond normally to PHA, and, i f not, whether there was any r e l a t i o n between i n t r i n s i c lymphocyte function and the a b i l i t y of serum to support the function of normal lymphocytes. The r e s u l t s of two small experiments comparing the response of control lymphocytes and patients' lymphocytes to PHA i n 8% FCS are shown i n Figure 7. In both, lymphocytes from patients responded s i g n i f i c a n t l y l e s s well to PHA than did control lymphocytes. The r e l a t i o n between the percent lymphocyte stimulation and SSLS-I i s shown i n Table XIV. Regression analysis revealed no s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between percent lymphocyte stimulation and SSLS-I. TABLE XIII RELATION BETWEEN SSLS-I IN DIALYSED SERUM AND SERUM SUPPORT OF MLR (PHASE 1) Serum Donor SSLS-I i n Serum Support Dialysed Serum of MLR (c.p.m.) p 16* 1 2272 + 328 + p 39 32 15,905 + 574 p 40 6.5 1337 + 132 p 1 14 15,100 + 8235 p 26 13 683 + 92 p 42 46 29,720 + 3266 . p 43 101 34,400 + 150 p 24 62 12,918 + 570 p 44 140 32,747 + 4617 Co r r e l a t i o n c o e f f i c i e n t on logarithms of the data i s r=0.741, p<0.05. *P 1-44 designate i n d i v i d u a l patients. +Each r e s u l t represents the mean ± SE of 3 wells. 140 (b) P<0.05 100! z o 3 u o 50 p< 0.005 Controls Patients Controls Patients LYMPHOCYTE DONORS FIGURE 7: RESPONSE OF LYMPHOCYTES FROM HEALTHY CONTROLS AND BREAST CANCER PATIENTS TO PHA IN THE PRESENCE OF 8% FCS (PHASE 1) (a) and (b) show the r e s u l t s of two separate experiments using two d i f f e r e n t brands of PHA: (a) Difco PHA-P, 1:750 and (b) Wellcome PHA, 1:125. Each c i r c l e , * , and square, I , represents the stimulation of lymphocytes from an i n d i v i d u a l control or patient r e s p e c t i v e l y . Each point i s the mean of 4 wells. The h o r i z o n t a l bars represent the mean lymphocyte stimulation f o r each control or patient group. The lympho-cyte samples comprising each experiment were not a l l tested on the same day. 141 TABLE XIV RELATION BETWEEN % LYMPHOCYTE STIMULATION AND SSLS-I OF SERA FROM THE LYMPHOCYTE DONORS (PHASE 1) Donor of Serum and Lymphocytes % Lymphocyte Stimulation SSLS-I P 59* 85 38 P 43 71 138.5 P 60 63 147 P 1 63 14 P 9 62 70 P 22 60 15 P 40 36 6.5 P 61 20 190 P 62 5 3 * P 1-62 designate individual patients. No s i g n i f i c a n t correlation between % lymphocyte stimulation and SSLS-I 142 IV. DISCUSSION As noted i n the introduction, attempts to demonstrate an immune i n h i b i t o r i n the plasma or serum of patients with breast cancer have given contradictory r e s u l t s . From 5 to 20% undialysed serum or plasma was used i n a l l these studies. We could not demonstrate an i n h i b i t o r i n the undialysed serum of patients with metastatic breast cancer at a concentration of 20% serum and PHA 1:1500. However, some such sera be-came i n h i b i t o r y at an equivalent concentration when dialysed, and many of these were i n h i b i t o r y at a 40% concentration even when undialysed. The discrepancy among the various i n v e s t i g a t i o n s cannot be explained by the extent of the disease since Knight and Davidson (1975) found serum i n h i b i t o r (s) i n early breast cancer and Whittaker et a l . (1971) i n advanced disease, while others (Whitehead et a l . 1974; Blomgren et a l . 1975)failed to demonstrate them i n advanced breast cancer. The disparate r e s u l t s may be explained i n part by the use of d i f f e r e n t PHA brands and concentrations i n d i f f e r e n t studies. In our experiments the i n h i b i t i o n of lymphocyte stimulation produced by 40% undialysed serum from cancer patients could be overcome by increasing the PHA concentration. This i s i n agreement with the finds of Humphrey et a l . (1974), using sera from leukaemia patients. In one small experiment i t appeared that 20% undialysed sera from some patients with i n h i b i t o r y dialysed sera might be immunosuppressive at very low PHA concentrations, i . e . PHA. 1:3750 and PHA 1:5000 (results not shown). However, Blomgren et a l . (1975) f a i l e d to demonstrate such an 143 i n h i b i t o r i n breast cancer patients' sera oyer a range of PHA concent-r a t i o n s . Differences i n culture media and culture duration could also play a part i n the d i f f e r e n t r e s u l t s obtained. A 72-hour incubation time appeared to maximize the, differences between SSLS. From the shape of the curves of lymphocyte stimulation versus con-centration of undialysed or dialysed serum, i t appears that control and patients' sera contain competing stimulatory and i n h i b i t o r y f a c t o r s . The i n h i b i t o r y sera may contain an abnormally high concentration of i n h i b i t -or (s-) or a low concentration of stimulator(s). The immune i n h i b i t o r y e f f e c t of sera i s more r e a d i l y demonstrated a f t e r d i a l y s i s . Stimulatory factors i n the serum d i a l y s a t e may mask the e f f e c t of a nondialysable i n h i b i t o r or may compensate for low concentrations of macromolecular st i m u l a t o r ( s ) . It i s of i n t e r e s t that an u l t a f i l t r a t e of pooled normal human serum passed through a UM10 f i l t e r . (Amieon) supported lymphocyte stimulation better than d i d the unfractionated serum (Table XXXIII). Yachnin and Raymond•(1975) showed that serum d i a l y s a t e added with dialysed serum increased lymphocyte PHA-induced stimulation compared with dialysed serum alone. Adjusting the protein concentration of dialysed sera to a constant value might a r t i f i c i a l l y increase the concentration of a normal i n h i b i t o r i n p atients' sera i f such sera had lower t o t a l protein concentrations than normal sera. S i m i l a r l y , the concentration of a normal immune stimulatory substance would be a r t i f i c i a l l y decreased i f the patients had abnormally high t o t a l serum protein concentrations. However, i n h i b i t o r y sera did not di f f e r from normal sera i n t o t a l protein concentration. Therefore, the use of a standardized protein concentration i n dialysed sera was not the cause of some sera appearing i n h i b i t o r y . 144 As mentioned above, the i n h i b i t o r y e f f e c t of c e r t a i n undialysed sera at 40% concentration could be overcome by increasing the PHA con-centration, Humphrey et a l , (1974) interpreted t h i s e f f e c t as an increased binding of PHA by the patients' sera. I t could equally w e l l represent a competitive i n h i b i t i o n of PHA binding to lymphocytes by lymphocyte-binding serum constituents. The l a t t e r i s more l i k e l y since the serum-preciptitating and lymphocyte-stimulating components of PHA are d i f f e r e n t molecules (Yachnin 19.72), It i s not e n t i r e l y clear why the i n h i b i t o r y e f f e c t of dialysed sera at 14 mg/ml protein cannot also be overcome by increasing PHA concen-t r a t i o n . It may be r e l a t e d to the fact that PHA becomes i n h i b i t o r y at high PHA: serum r a t i o s (Yachnin and Raymond 1975). The good c o r r e l a t i o n between dialysed serum inhibition of PHA stimu-l a t i o n and the i n h i b i t i o n of MLR by whole serum gives support to the possible i n vivo relevance of studying dialysed sera i n a PHA assay. The complete lack of c o r r e l a t i o n between the depression of lymphocyte function and the degree of serum i n h i b i t i o n i n our breast cancer patients i s contradictory to the findings of Whitehead e_t a l . (1976) who found that the proportion of E-rosette-forming T - c e l l s i n breast cancer patients was reduced only because such c e l l s were coated with serum constituents which prevented rosette formation. Others have shown a good c o r r e l a t i o n between PHA-response and E-rosette formation (Nowell et a l . 1975). S i m i l a r l y , Mannick et a l , (1977) found the PHA'responsiveness of cancer patients' lymphocytes was increased a f t e r thorough washing. As lymphocytes 145 were washed more thoroughly i n our experiments (three times compared with twice i n Whitehead e t ,aL, 1976) and were, suspended i n a d i f f e r e n t medium, we may have removed any lymphocyte-bound serum i n h i b i t o r more completely. This could account for the lack of c o r r e l a t i o n between the two measures of immune function. Unlike Whitehead et a l . (1976) we did not f i n d the patients' lymphocyte function restored to normal by the removal of the serum i n h i b i t o r . However, t h e i r study excluded patients who had received radiotherapy or chemotherapy wit h i n the preceeding twelve months. Since radiotherapy (Jenkins et a l . 1975; Raben et a l . 1976) and chemotherapy (Hersh e_t a l . 1975) are known to depress immune functions, i n c l u d i n g lymphocyte response to PHA, these treatments may have been a major contributor to the depressed PHA responses of lympho-cytes from our patients with advanced breast cancer, while the serum i n h i b i t i o n seen i n some patients may be caused by other unrelated f a c t o r s . Tn summary, i t appears that a proportion of patients with metastatic breast cancer have sera which i s i n h i b i t o r y to the PHA response of normal human lymphocytes. Such immunosuppression i s more e a s i l y demonstrated a f t e r d i a l y s i s . A l t e r a t i o n s i n experimental conditions such as serum protein concentration, PHA concentration, and duration of incubation may a f f e c t the degree of immunosuppression seen and hence determine whether or not differences between patients and controls are s i g n i f i c a n t , but do not o b l i t e r a t e the immunosuppressive e f f e c t . 146 CHAPTER III THE EFFECT OF CLINICAL VARIABLES ON THE ABILITY OF SERUM FROM BREAST CANCER PATIENTS TO SUPPORT NORMAL LYMPHOCYTE STIMULATION BY PHA I. INTRODUCTION Having determined that dialysed sera from some breast cancer patients i n h i b i t the response of normal human lymphocytes to PHA, an attempt was made to determine any c l i n i c a l factors d i s t i n g u i s h i n g patients with i n -h i b i t o r y sera from others. I n i t i a l l y , c l i n i c a l information was obtained regarding breast cancer patients on various treatments whose SSLS-I had been measured i n the experiments described i n Chapter I I , and s t a t i s t i c a l analyses were performed to determine which c l i n i c a l v a r i a b l e s might be r e l a t e d to SSLS-I. Subsequently, other patients receiving various hormonal treatments or no treatment were selected, and the SSLS of t h e i r dialysed sera were tested to determine whether there was a diffe r e n c e i n mean SSLS among the treatment groups or between patients and controls. I I . MATERIALS AND METHODS 1. Patients The patients were as described i n I I - I I . The conventional hormonal agents most often used to treat patients studied i n the Phase 1 experiments were the oestrogen, d i e t h y l s t i l b o e s t r o l , usually 5 mg twice d a i l y for 4 of every 6 weeks; the androgen, fluoxymesterone, usually 5 mg four times d a i l y ; and the c o r t i c o s t e r o i d , prednisone,usually 5 mg two to four times d a i l y . The newer hormonal agents used to treat patients studied i n Phase 2 147 experiments were Tamoxifen (ICI Americas Inc., Newark, De.) usually 10 mg two or three times d a i l y and Aminoglutethimide (Ciba-Geigy Canada Ltd., Dorval, P. Q.) usu a l l y 250 mg , four to eight times d a i l y . 2. Controls: As described i n I I - I I . 3. Normal Lymphocyte Donors: As described i n I I - I I . 4. Preparation of Sera: As described i n I I - I I . 5. Preparation of Lymphocytes: Blood;., was d i l u t e d 2:1 with s t e r i l e PBS and layered onto F i c o l l -Hypaque. Lymphocytes were recovered by the. method of Perper et_ a l . as described i n I I - I I . 6. Lymphocyte Stimulation with PHA: The methods used were described i n I I - I I . With the exception of experiments to tes t the e f f e c t of Tamoxifen and Aminoglutethimide on the response of normal lymphocytes to PHA, a l l experiments discussed i n Chapter I I I involved t e s t i n g the e f f e c t of sera on lymphocyte stimulation with PHA. In a l l these experiments each well contained 10 lymphocytes,, serum dialysed against 1640 at a concentration of 14 mg protein/ml. , and Bacto PHA-P, 1:1500. The incubation time before the addition of 3H-TdR was always 72 hours. In Phase 2 experiments to compare the e f f e c t s of dialysed sera from patients on various treatments and controls, each of the t r i p l i c a t e wells 148 f o r each serum sample was assigned at random. The d e t a i l s of the experiments for, t e s t i n g the e f f e c t s of Tamoxifen and Aminoglutethimide on the response of normal lymphocytes„to PHA are given with the r e s u l t s . 7. Estimation of Extent of Disease i n Patients: The...amount of tumour present i n patients at the time the blood was taken was estimated from the patients' charts and was c a l l e d Group I to IV (as opposed to stage of disease which i s the amount of disease present at diagnosis). Group I - no c l i n i c a l evidence of disease. Group II - disease < 5 ..gm., ..confined _ to ..skin Group I I I - metastases»5 ;gm,y, confined to chest wall or a single bone or v i s c e r a l metastatic deposit. Group IV- more widespread metastatic disease. 8. Measurement of Serum C o r t i s o l : C o r t i s o l (hydrocortisone) was measured by a radioimmunoassay using 125 the Gammacoat I - C o r t i s o l radioimmunoassay k i t (Travenol Laboratories ? Inc., Cambridge, Mass.). The unknown samples and standards were incubated with 1 2 5 I - C o r t i s o l tracer i n tubes onto which a n t i - c o r t i s o l antibody had been immobilized. The r a d i o a c t i v i t y bound to the tube was counted. This assay was k i n d l y performed by the s t a f f of the D i v i s i o n of Chemical Chemistry,. Vancouver General H o s p i t a l , Vancouver, B. C. 9. S t a t i s t i c a l Methods; In the phase. 1 study., the r e l a t i o n s h i p between the various c l i n i c a l v a r i a b l e s and SSLS-I and log SSLS-I was studied by multiple regression 149 an a l y s i s , both stepwise multiple regression (Snedecor and Cochran 1967) and a "step up and down" technique ( T h e i l 1971). ..The analysis was per-formed by computer. The s u r v i v a l times f o r the patients i n the Phase 1 study were analysed by the l i f e table method (Gross and Clark 1975). The r e l a t i o n -ship between SSLS-I and s u r v i v a l time was determined by the regression of log s u r v i v a l time on SSLS-I and on log SSLS-I (Snedecor and Cochran 1967). These analyses were performed by computer. The X analysis (Snedecor and Cochran 1967) was used to analyse the proportion of patients with i n h i b i t -ory sera among pateints with d i f f e r e n t amounts of disease i n the Phase 1 and Phase 2 experiments. In the Phase 2 experiment, the X 2 test was also used to analyse the proportion of patients with i n h i b i t o r y sera among patients r e c e i v i n g no treatment or various hormonal treatments, among patients of d i f f e r e n t ages on hormonal treatments, and among patients on hormonal treatments whose sera had been stored d i f f e r e n t lengths of time before use. In the phase 2 experiment, the mean values of SSLS for controls, untreated patients, and patients on various hormonal treatments were compared using a two-way analysis of variance with r e p l i c a t i o n s (Cochran and Cox 1957). In the ana l y s i s , the sum of squares due to days was subtract-ed from the treatments sum of squares i n order to remove the e f f e c t of day to day v a r i a t i o n s i n lymphocyte stimulation from the v a r i a t i o n among patients within each treatment group. In Phase IraridPhase 2 experiments, the mean values of variables measuring the duration of disease f o r patients with i n h i b i t o r y and non-i n h i b i t o r y sera were compared using a t - t e s t (Snedecor and Cochran 1967) . 150 Correlation analysis (Snedecor and Cochran 1967) was used to analyse the r e l a t i o n s h i p between C o r t i s o l l e v e l s i n dialysed sera and SSLS, and between the storage time o f serum and SSLS. This analysis was performed on the logarithms of the SSLS data. I I I . RESULTS 1. Relation Between C l i n i c a l Variables and SSLS-I (Phase 1) C l i n i c a l information was c o l l e c t e d from the charts of 42 breast cancer patients whose SSLS-I had been determined. Sur v i v a l data were obtained a f t e r 611/2 years of follow-up. Where sera had been tested on lymphocytes from more than one donor, the SSLS-I used was the average of the determinations. A l l i n h i b i t o r y sera, i . e . with SSLS-I of 30% or l e s s , :•: :-. were tested with lymphocytes from three d i f f e r e n t donors. Any serum which was not c o n s i s t e n t l y i n h i b i t o r y was excluded. (i ) M u l t i p l e Regression Analysis of SSLS-I on a Number of C l i n i c a l V ariables: The regression of the SSLS-I (the dependent variable,Y ) on a number of c l i n i c a l v a r i a b l e s (independent v a r i a b l e s , Xito; • Xi 7) was determined by m u l t i p l e regression analysis i n order to determine which of these variables might be r e l a t e d to SSLS-I. The variables considered are shown i n Table XV. The multiple regression analysis can be done i n several d i f f e r e n t . ways, . y i e l d i n g somewhat d i f f e r e n t r e s u l t s . In the stepwise multiple regression analysis, . each.independent va r i a b l e was considered separately i n r e l a t i o n to the dependent v a r i a b l e , Y , and only those variables s i g n i f i c a n t l y c orrelated with Y were used to construct TABLE XV RESULTS OF STEP UP AND DOWN MULTIPLE REGRESSION ANALYSIS OF SSLS-I AND LOG SSLS-I ON VARIOUS CLINICAL VARIABLES (PHASE 1) C o e f f i c i e n t s of Regression V a r i a b l e A l l 42 P a t i e n t s 31 Group IV P a t i e n t s Y=SSLS-I Y=log SSLS-I Y=SSLS-I Y=log SSLS-I Treatment w i t h . -0.516 (pO.Ol) -44.57 -0.597(p<0.05) oestrogens (0.05<p<0.10) Treatment w i t h . . 40.92 (p=0.01) androgens Treatment w i t h 64.95 (p<0.01) c o r t i c o s t e r o i d s Treatment w i t h -48.51 (p<0.05) -0.290 chemotherapy (0.05<p<0.10) Radiotherapy Presence of bone -30.89 metastases (0.05<p<0.10) Presence of s o f t -57.16 (pO.Ol) -0.437 (p<0.05) t i s s u e metastases Presence of i n t e r n a l organ metastases -40.27 (p<0.05) Table XV (continued) C o e f f i c i e n t s of Regression Variables A l l 42 Patients 31 Group IV Patients Y=SSLS-I Y=log SSLS-I Y=SSLS-I Y=log SSLS-I Peripheral blood lymphocyte count Serum a l k a l i n e 0.29 (p<0.05) phosphatase Age of Patient -0.019 (p<0.05) -0.017 C l i n i c a l stage (0.05<p<0.10) at diagnosis Pathological stage at diagnosis Degree of d i f f e r e n - -0.183 (p<0.05) -0.215 t i a t i o n of tumour (0.05<p<0.10) Time period a* Time period b* Time period c* •*a- months from detection of breast mass to f i r s t treatment b- disease free i n t e r v a l (months) following treatment c- months from f i r s t recurrence to taking of blood sample 153 the regression equation. When t h i s was done, treatment with oestrogens (p=0.012) and the presence of i n t e r n a l organ metastases (p=0.025) had a s i g n i f i c a n t negative l i n e a r r e l a t i o n s h i p with the SSLS-I. These variables were l a r g e l y independent of each other. The same variables had a s i g n i f -icant negative l i n e a r r e l a t i o n s h i p with log SSLS-I, the p values being 0.002 for oestrogens and 0.007 for i n t e r n a l organ metastases. The p values r e f e r to the p r o b a b i l i t y that the true c o e f f i c i e n t i n the regression equation i s zero. If more c l i n i c a l variables were entered into the regression equation i n order of increasing p values, c o r r e l a t i o n s between pai r s of variables caused the s i g n i f i c a n c e of some of the previously entered variables to decline and the s i g n i f i c a n c e of others to increase. That i s , the e f f e c t s of some apparently s i g n i f i c a n t v a r i a b l e s on SSLS-I could be described i n terms of other v a r i a b l e s . Consequently a procedure, "step up and down" multiple regression analysis, was followed i n which variables were entered into the equation i n order of increasing p values u n t i l a l l but the few lea s t s i g n i f i c a n t had been entered. Then variables were removed i n order of decreasing p values, u n t i l only those with c o e f f i c i e n t s having p<0.10 -remained. These were considered the var i a b l e s s i g n i f i c a n t l y r e l a t e d to SSLS-I. The r e s u l t s are summarized i n Table XV. When a l l 42 patients were considered, treatment with androgens and with c o r t i c o s t e r o i d s had a s i g n i f i c a n t p o s i t i v e l i n e a r r e l a t i o n s h i p with SSLS-I, while treatment with chemotherapy and the presence of i n t e r n a l organ metastases both had a s i g n i f i c a n t negative l i n e a r :relationship. Age, the degree of tumour d i f f e r e n t i a t i o n , and treatment with oestrogens a l l had a s i g n i f i c a n t negative l i n e a r r e l a t i o n s h i p with l og SSLS-I. When only the 31 patients with Group IV disease were considered, the presence of soft t i s s u e metast-ases had a s i g n i f i c a n t negative l i n e a r r e l a t i o n s h i p and the l e v e l of serum a l k a l i n e phosphatase a s i g n i f i c a n t p o s i t i v e l i n e a r r e l a t i o n s h i p with SSLS-I. Among Group IV patients, treatment with oestrogens and the presence of soft t i s s u e metastases had a s i g n i f i c a n t negative l i n e a r r e l a t i o n s h i p with l o g SSLS-I. The c o r r e l a t i o n c o e f f i c i e n t s between pai r s of the independent c l i n i c a l v a r i a b l e s were calculated by computer i n the process of perform-ing the multiple regression analysis. The 1 c o r r e l a t i o n : c o e f f i c i e n t s which were s i g n i f i c a n t at p<0.05 are given below. Although these c o e f f i c i e n t s were s i g n i f i c a n t when pair s of var i a b l e s were considered i n i s o l a t i o n from the others, i f a l l the independent v a r i a b l e s and t h e i r i n t e r r e l a t i o n s h i p s had been considered together, some of these c o r r e l a t i o n c o e f f i c i e n t s might not have remained s i g n i f i c a n t at the 5% l e v e l . When a l l patients were considered, treatment with oestrogens was s i g n i f i c a n t l y p o s i t i v e l y correlated with patient age (r=0.41) and with delay i n r e c e i v i n g the i n i t i a l treatment for breast cancer,(r=0.34), and s i g n i f i c a n t l y negatively c o r r e l a t e d with androgen treatment (r=-0.35) and with corticosteroid, treatment (r=-0.33). Treatment with c o r t i c o -steroids had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with chemotherapy (r=0.46) and n e g a t i v e : c o r r e l a t i o n with disease free i n t e r v a l (r=-0.38). The presence of i n t e r n a l organ metastases had a s i g n i f i c a n t p o s i t i v e correla-r'. t i o n with patient age (r=0.35). Delay i n rec e i v i n g treatment had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with the time from f i r s t recurrence u n t i l the blood sample was taken (r=0.74). Generally s i m i l a r r e s u l t s were obtained when only the Group IV pat-ients were considered. Treatment with oestrogens was s i g n i f i c a n t l y p o s i t -i v e l y correlated with patient age (r=0.39), with delay i n rec e i v i n g t r e a t -ment (r=0.44), and with the time from the f i r s t recurrence u n t i l the blood 155 sample was taken (r=0.35). Oestrogen treatment was negatively correlated with androgen treatment (r=-0.37) and with c o r t i c o s t e r o i d treatment (r=-0.40)'. Treatment with c o r t i c o s t e r o i d s had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with chemotherapy (r=0.42) and a s i g n i f i c a n t negative c o r r e l -ation with patient age (r=-0.48) and with disease free i n t e r v a l (r=-0.42). Chemotherapy had a s i g n i f i c a n t negative c o r r e l a t i o n with patient age (r=-0.38). The presence of i n t e r n a l organ metastases had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with patient age (r=0.40). Delay i n r e c e i v i n g treatment had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with the time from f i r s t recurrence u n t i l the blood sample was taken (r=0.80). ( i i ) Relationship of the Amount of Disease Present to the Presence of I n h i b i t o r y Sera: X 2 t e s t s : According to the, above analysis:, the presence of soft t i s s u e metast-ases i n Group IV patients and of i n t e r n a l organ metastases when a l l patients were considered were s i g n i f i c a n t l y negatively r e l a t e d to SSLS-I or l o g SSLS-I. The presence of bone metastases had a negative l i n e a r r e l a t i o n s h i p of borderline s i g n i f i c a n c e with SSLS-I when a l l patients were considered. The t o t a l number of s i t e s involved with metastases (consider-ing each of bone, soft t i s s u e , and i n t e r n a l organs as one s i t e ) might be considered an i n d i c a t i o n of the amount of disease present. As can be seen from Table XVI, i n h i b i t o r y sera were found at a s i g n i f i c a n t l y higher frequency i n patients with disease at a l l three s i t e s than i n patients with no, one or two s i t e s involved. When the amount of disease present was expressed as the disease group, i n h i b i t o r y sera were found at a higher frequency i n Group IV patients than i n patients of other groups, but the d i f f e r e n c e was not s i g n i f i c a n t at p<0.05. The r e s u l t s are shown i n Table XVI. Group IV i s quite a broad c l a s s i f i c a t i o n i n c l u d i n g patients s t i l l i n good general health with a few TABLE XVI PROPORTION OF PATIENTS WITH INHIBITORY SERA AMONG PATIENTS WITH DIFFERENT AMOUNTS OF.DISEASE (PHASE 1) Pro p o r t i o n of P a t i e n t s w i t h I n h i b i t o r y Sera No. of S i t e s of M e t a s t a t i c Disease P Value, f o r Di f f e r e n c e s Among Pr o p o r t i o n s * 1/6 1/14 1/9 9/12 p<0.005 Pr o p o r t i o n of P a t i e n t s w i t h I n h i b i t o r y Sera Disease Group P Value f o r Di f f e r e n c e s Among pr o p o r t i o n s * I I - I I I IV 1/6 0/5 11/30 N.S.** Pr o p o r t i o n of Group IV P a t i e n t s w i t h I n h i b i t o r y Sera No. of S i t e s of M e t a s t a t i c Disease P Value f o r Di f f e r e n c e s Among Pr o p o r t i o n s * 1/9 1/9 11/19 p<0.005 2 * p values were determined u s i n g the x a n a l y s i s . ** N.S.- not s i g n i f i c a n t 157 bony metastases as w e l l as those terminally i l l . The c l a s s i f i c a t i o n by the number of s i t e s of metastases may further subdivide Group IV patients according to the amount of disease. Considering Group IV patients only, the proportion with i n h i b i t o r y sera was s i g n i f i c a n t l y higher i n those with metastases at three s i t e s than i n those with one or two metastatic s i t e s , as shown i n Table XVI. According to a x t e s t , there was no s i g n i f i c a n t d i f f e r e n c e at p<0.05 i n the proportion of patients with no and one or with two and three s i t e s of metastases i n the d i f f e r e n t treatment groups (data not shown). ( i i i ) Relation of SSLS-I to Prognosis: The s u r v i v a l times of the patients from the time blood was taken for the SSLS measurement were found to follow an exponential d i s t r i b u t i o n . This indicated that an approximately constant proportion of the patients who were a l i v e at the beginning of each time i n t e r v a l died during that i n t e r v a l . The regression of the l o g s u r v i v a l time on the SSLS-I for a l l patients, and for Group IV patients only, revealed no l i n e a r r e l a t i o n s h i p between log s u r v i v a l time and SSLS-I, nor i s there a s i g n i f i c a n t r e l a t i o n -ship between l o g s u r v i v a l time and log SSLS-I. Therefore, SSLS-I does not appear to be a prognostic i n d i c a t o r . (iv) Relationship of SSLS-I to Duration of Disease: It i s perhaps s u r p r i s i n g that, although patients with i n h i b i t o r y sera had a greater disease burden than others, they did not have a s i g n i f i c a n t l y shorter s u r v i v a l time. In f a c t , i f only Group IV patients were considered, the mean s u r v i v a l time was somewhat greater for patients with i n h i b i t o r y sera. This would suggest the hypothesis that patients with I n h i b i t o r y sera might often be those with advanced but slowly progressing disease, whereas patients with noninhibitory sera would 158 include those with l i t t l e disease and those with moderately advanced but r a p i d l y progressing disease. Against the hypothesis of an a s s o c i a t i o n between i n h i b i t o r y sera and advanced but slowly growing tumours, i s the complete lack of any s i g n i -f i c a n t a s s o c i a t i o n i n the regression analysis between SSLS-I and variables, measuring the duration periods of the disease up u n t i l the blood: sample was taken. However, patients with i n h i b i t o r y sera had had on the average a s i g n i f i c a n t l y longer disease-free i n t e r v a l a f t e r f i r s t treatment than patients without i n h i b i t o r y sera. The d i f f e r e n c e f e l l j u s t short of s i g n i f i c a n c e i f only Group IV patients were included. Considering only Group IV p a t i e n t s , the average time during which c l i n i c a l l y evident disease was continously present was greater f o r patients with i n h i b i t o r y sera but the d i f f e r e n c e was j u s t short of s i g n i f i c a n c e . On the other hand, the average t o t a l time the disease was present i n c l u d i n g time before i n i t i a l treatment, disease free i n t e r v a l , and duration of metastases was highly s i g n i f i c a n t l y greater (p<0.01) for patients with i n h i b i t o r y sera. This data i s presented i n Table XVII. The f a c t that these differences were not r e f l e c t e d i n the regression analysis .is probably due to a lack of c o r r e l a t i o n between values of SSLS-I and the disease duration within the categories of i n h i b i t o r y and non-i n h i b i t o r y .sera, or to the c o r r e l a t i o n of duration with oestrogen treatment. 2. SSLS for Patients on Current Hormonal Therapies for Breast Cancer Compared with SSLS for Controls and Patients on No Treatment (Phase 2) Experiments were c a r r i e d out i n Phase 2 to further c l a r i f y the e f f e c t s of various c l i n i c a l v a r i a b l e s , e s p e c i a l l y hormonal treatments and amount TABLE XVII: COMPARISON OF MEAN DURATION OF PERIODS OF DISEASE FOR PATIENTS WITH INHIBITORY AND NONINHIBITORY SERA (PHASE 1) Mean Duration of Period (Months)  Period and Patients Patients with ' Patients with P Value :for difference • Included Noninhibitory Sera I n h i b i t o r y Sera between means** Mean disease free i n t e r v a l (months), a l l patients 21.6 + 3.4* 54.0 + 17.3 P<0.025 Mean disease free i n t e r v a l (months), Group IV only 21.3 + 4.8 52.5 + 18.8 0,05<P<0.10 Mean duration of c l i n i c a l l y apparent disease (months), Group IV only 22.0 + 3.50 44.4 + 13.9 0.05<P<0.10 Mean t o t a l duration of disease (months), Group IV only 48.2 + 7.1 99.4 + 21.3 P<0.01 *Each r e s u l t i s mean±§tandard error of the mean (S E.) of durations. **P values were determined by a t - t e s t . Ln 160 of disease, on SSLS, In t h i s phase, the hormonal treatments i n use were Tamoxifen, Aminoglutethimide, and a combination of these. Patients r e c e i v i n g chemotherapy were excluded from these experiments because d i v e r s i t y of chemotherapeutic agents and dosage schedules used would have made the i n t e r p r e t a t i o n of r e s u l t s d i f f i c u l t . The SSLS for patients on these hormonal treatments, f o r patients on no treatment and for controls was measured i n three experiments done on three d i f f e r e n t days. In each experiment sera was tested from 6 co n t r o l s , 3 patients with metastatic breast cancer on no treatment, 5 patients r e c e i v i n g Tamoxifen, 3 re c e i v i n g Aminoglutethimide, and 5 re c e i v i n g the combination. Sera from each patient was used only once. Therefore, i n a l l , sera from 18 controls, 9 patients on no treatment, 15 on Tamoxifen, 9 on Aminoglutethimide, and 15 on the combination were tested. ( i ) Analysis of Variance and x 2 a n ^ l y s i s : The r e s u l t s were analysed by a two-way analysis of variance with r e p l i c a t i o n s . The r e s u l t s are shown i n Table XVIII. The mean values of the SSLS were considerably lower f o r the groups of patients oh hormonal treatments than f o r controls and for patients on no treatment. However, because th e " v a r i a t i o n within each group was large, the analysis of variance performed on the logarithms of the SSLS data showed that the differences among the means of the groups were not s i g n i f i c a n t . However, i f the proportion of i n d i v i d u a l s with i n h i b i t o r y sera ( i . e . SSLS-I less than 30%) i n each group was considered, there was a s i g n i f i -cant diff e r e n c e among the proportions of i n h i b i t o r y sera f o r the f i v e TABLE XVIIT COMPARISON OF MEAN SSLS FOR CONTROL AND PATIENT GROUPS (PHASE 2) Mean SSLS Day- of Experiment Controls Untreated Patients on Patients on Patients Patients Tamoxifen Aminoglu-• . Tamoxifen tethimide + Amino-glutethimide Day 1 70,698 ± 6798* 62,505 ± 7470 74,876 ± 17,902 77,054 ± 7552 53,385 ± 8230 Day 2 70,856 ± 8728 83,964 ± 2049 27,186 ± 13,769 34,991 ± 19,131 28,353 ± 14,155 Day 3 78,374 ± 15,921 81,752 ±114;393 65,823 ± 16,642 42,082 ± 15,397 24,531 ± 8135 * Mean SSLS ± S.E. of patients i n each group h-1 treatment groups. There were no differences among the three hormonal treatment groups, b u t a l l of these had a s i g n i f i c a n t l y higher proportion of i n h i b i t o r y sera than the c o n t r o l or untreated patient groups. These r e s u l t s are shown i n Table XIX. Thus, although the mean values of SSLS do not d i f f e r s i g n i f i c a n t l y , there i s a s i g n i f i c a n t l y higher proportion of i n h i b i t o r y sera among the patients on hormonal treatments. ( i i ) Absence of an Immunosuppressive E f f e c t Caused by Hydrocortisone, Tamoxifen, or Aminoglutethimide Themselves: Hydrocortisone i s well known as an immunosuppressant. Therefore, one had to consider the p o s s i b i l i t y that the hydrocortisone ( c o r t i s o l ) administered as replacement therapy to patients receiving Aminoglutethi-mide might r a i s e the serum C o r t i s o l , even a f t e r d i a l y s i s , s u f f i c i e n t l y to cause immunosuppression. Therefore, serum C o r t i s o l was measured i n two control dialysed sera and i n four dialysed sera from patients receiving a combination of Tamoxifen, Aminoglutethimide, and hydrocortisone. The SSLS for a l l s i x sera had been measured previously, a l l i n the same experiment. Three of the patients' sera were i n h i b i t o r y . The r e s u l t s are shown i n Table XX. C l e a r l y , none of the serum C o r t i s o l l e v e l s was high. Normal serum C o r t i s o l l e v e l s by t h i s method are 7 to 25 yg/100 ml i n the morning and 2 to 9 yg/100 ml i n the evening,as established by the laboratory performing the analyses. A f t e r d i l u t i o n i n culture, the highest concentration of serum C o r t i s o l used here would be 1.24 yg/100 ml. There was no s i g n i f i c a n t c o r r e l a t i o n between C o r t i s o l l e v e l s i n dialysed sera and SSLS, although, of course, the number of sera studied was small. It has been shown (Heilman e_t a l . 1973) that the response of normal TABLE XIX • PROPORTION OF INHIBITORY SERA FROM CONTROL AND PATIENT GROUPS (PHASE 2) Proporti o n of I n d i v i d u a l s with I n h i b i t o r y Sera Con t r o l s Untreated P a t i e n t s P a t i e n t s on Tamoxifen P a t i e n t s on Aminogluteth-imide P a t i e n t s on Tamoxifen + Aminogluteth-imide P Value f o r D i f f e r e n c e s Among Pro p o r t i o n s * 1/18 0/9 3/15 3/9 6/15 p=0.05 * P value was determined by a x 2 a n a l y s i s . TABLE XX CORTISOL LEVELS IN DIALYSED SERA AND THEIR RELATION TO SSLS (PHASE 2) Serum Donor C o r t i s o l Concentration (yg/lOO ml)* SSLS (c.p.m.) C 27** 1.3 63,542 ± 2677 + C 28 2.1 91,858 ± 7825 P 63 3.1 21,895 ± 619 P 64 2.4 340 ± 122 P 65 2.8 88,837 ± 2348 P 66 3.1 755 ± 81 Correlation c o e f f i c i e n t between Cortisol concentration and log SSLS i s r = -0.361, not s i g n i f i c a n t . * Standard deviation i n t h i s method i s <11% of the mean. ** C 27 and 28 designate i n d i v i d u a l controls; P 63-66 designate i n d i v i d u a l patients. Each SSLS re s u l t i s the mean ± SE of 3 wells. human lymphocytes to PHA i s progressively i n h i b i t e d by 1 yg% to 100 yg% C o r t i s o l . The dose-response curve on a log-log p l o t i s approximately l i n e a r . A 50% i n h i b i t i o n of lymphocyte stimulation occurs at a f i n a l hydrocortisone concentration of approximately 30 yg% above that contained i n 20% human plasma and the medium 199 (Gibco) present. This i s consider-ably higher than the maximum 1.24 yg% i n our cultures. Therefore, the Cortisol l e v e l i n serum i s not an important f a c t o r i n the low SSLS seen i n sera of some breast cancer patients on Aminoglutethimide and hydro-cortisone. Tamoxifen and Aminoglutethimide were tested f or immunosuppressive a c t i v i t y i n the usual type of lymphocyte stimulation experiment. Each w e l l contained 10 5 normal lymphocytes; pooled normal human serum, dialysed against RPMI-1640, used at a p r o t e i n concentration of 14 mg/ml; and various concentrations of Tamoxifen, Aminoglutethimide, or both. The r e s u l t s are shown i n Table XXI. Aminoglutethimide and Tamoxifen themselves may each produce a s l i g h t decrease i n lymphocyte response to PHA, and they may even have a s y n e r g i s t i c e f f e c t i n t h i s i n h i b i t i o n . However, even with Tamoxifen, 1 yg/ml together with Aminoglutethimide, 200 yg/ml, the lympho-cyte stimulation with PHA was 60% of the co n t r o l without drugs. Studies have shown that the plasma l e v e l of Tamoxifen i n patients r e c e i v i n g 10 mg twice daily,as i n most of the patients i n t h i s study, was 0.09-0.20 yg/ml (Daniel et a l . 1979), and that the plasma l e v e l of Aminoglutethimide i n patients r e c e i v i n g 250 mg four times d a i l y was 3.0-44.2 yg/ml- (Murray e _ t a l . 197.9) . Therefore, these drugs i n the serum, e s p e c i a l l y a f t e r d i a l -y s i s , could not account for a l l of the suppression of lymphocyte response to PHA seen i n i n h i b i t o r y sera from breast cancer patients on these drugs. 166 TABLE, XXI: EFFECT OF TAMOXIFEN AND AMINOGLUTETHIMIDE ON' LYMPHOCYTE STIMULATION (PHASE 2) Concentration V Concentration Lymphocyte of AminoglutethiTJiide oof Tamoxifen Stimulation ((•'c.p.m.) (jig /ml ) ( ug /ml ) Q 0 •= 125,242 + 5923* 50 0 99,818 + 5675 100 0 106,305 + 4588 200 0 100,747 + 6594 300 0 89,157 + 7252 40.0 0 91,177 + 3545 0 0,25 121,373 + 2318 0 0.50 126,238 + 3168 0 1 118,167 + 4499 0 2 104,630 + 2880 0 4 120,655 + 828 100 1 87,295 + 4657 200 1 75,625 + 1033 *Each. r e s u l t i s the. mean ± S,E, of t r i p l i c a t e w ells. 167 ( i i i ) A Possible Source of Bias - Age of Patients and Controls: The mean ages of the controls and of patients i n the four groups, and the mean duration of storage of the sera before use i s shown i n Table XXII. The mean age of the patients i s nearly the same for the three hormonal treatment groups, and i s considerably higher than the mean age for the control and untreated patient groups. The proportion of i n h i b i t o r y sera f o r each age group among the patients r e c e i v i n g hormonal treatments i s shown i n Table XXIII. The increased proportion of i n h i b i t o r y sera with advancing age f a l l s j u s t short of s t a t i s t i c a l s i g n i f i c a n c e . Among the controls, the only i n h i b -i t o r y serum came from a woman of 75 years. This raised the p o s s i b i l i t y that the higher proportion of i n h i b i t o r y sera among patients on hormonal treatments might not have been found i f the control group had been more c l o s e l y matched for age with the treatment groups. To r u l e out t h i s p o s s i b i l i t y , SSLS-I was calculated for controls used i n a l l s u i t a b l e experiments throughout t h i s project. In each experiment, the controls represented a range of ages. Where a co n t r o l was used i n more than one experiment, the mean SSLS-I f o r the experiments was used. The r e s u l t s shown i n Figure 8 in d i c a t e no trend toward more i n h i b i t o r y sera with advancing age i n controls. Therefore, the SSLS r e s u l t s f o r the treatment versus control groups have apparently not been biased by the inexact age matching of controls and patients. ( The r e s u l t s used to calculate the values of SSLS-I i n Figure 8 came from Phase 1 experiments.) TABLE XXII MEAN AGE AND SERUM STORAGE TIME OF CONTROLS AND PATIENTS* (PHASE 2) Control or Patient Mean Age of Persons Mean Storage Time Group i n Years (range) of Serum i n Weeks (range) Controls 55.1 20.4 (38-75) (1-36) Untreated patients 54.7 22.3 (37-71) (11-66) Patients on Tamoxifen 62.7 24.1 (47-75) (3-47) Patients on Amino- 61.2 23.2 glutethimide (48-70) (5-44) Patients on Amino- 62.7 32.2 glutethimide and (48-73) (8-52) Tamoxifen *SSLS of sera of these controls and patients were tested i n the experiments shown i n Tables XVIII and XIX. 169 TABLE XXIII PROPORTION OF PATIENTS WITH INHIBITORY SERA AMONG PATIENTS OF DIFFERENT AGES ON HORMONAL TREATMENT (PHASE 2 ) * P r o p o r t i o n of P a t i e n t s w i t h I n h i b i t o r y Sera Age of P a t i e n t s <60 years 60-64 years £65 years P Value f o r Di f f e r e n c e s Among P r o p o r t i o n s * * 1/13 4/10 7/16 0.05<p<0.10 * SSLS values f o r these p a t i e n t s come from experiments shown i n Tables XVIII and XIX. ** P value was determined by a X a n a l y s i s . 170 • O O O 250 200 7 150 100 50 o o o o ooo £39 40-49 50-59 60-69 >J0 AGE OF CONTROLS (years) FIGURE 8: SSLS-I FOR HEALTHY CONTROLS OF DIFFERENT AGES (PHASE 1) Each po i n t represents the mean SSLS-I f o r an i n d i v i d u a l healthy c o n t r o l . This mean SSLS-I i s the mean of the values of SSLS-I obtained i n a l l the s u i t a b l e experiments i n which serum from that c o n t r o l was used. The h o r i z o n t a l bars rep-resent the means of the SSLS-I f o r c o n t r o l s i n each age group. 171 (iv) A Possible Source of Bias - Storage Time of Sera from Patients and Controls S i m i l a r l y , the mean storage time of the sera was higher for the patients on Aminoglutethimide plus Tamoxifen than for theoother groups. It was thought that SSLS might decrease upon prolonged storage of serum as control sera stored for very long periods (eg. 2 years) were often very i n h i b i t o r y and contained p r e c i p i t a t e . The sera from patients r e c e i v i n g hormonal treatments were s t r a t i f i e d by duration of storage. The proportion of i n h i b i t o r y sera i n each group i s shown i n Table XXIV. A somewhat higher proportion of i n h i b i t o r y sera was found among sera stored 40 weeks or longer, but these differences did not approach s t a t i s t i c a l s i g n i f i c a n c e . The control sera and a l l but one of the sera from untreated patients had been stored less than 40 weeks. The-)(2 analysis was repeated using only sera which had been stored le s s than 40 weeks. The r e s u l t s are shown i n Table XXV. Although the proportion of i n h i b i t o r y sera was s t i l l higher among patients receiving hormonal treatments than among controls or untreated patients, the differences were no longer s t a t i s t i c a l l y s i g n i f i c a n t . Some of the decline i n the; x value could be a t t r i b u t e d to the smaller number of sera included i n the a n a l y s i s , but some could be a t t r i b u t e d to the smaller proportion of i n h i b i t o r y sera among those stored less than 40 weeks. Data from some Phase 1 experiments were reviewed i n an attempt to c l a r i f y the r e l a t i o n s h i p between SSLS and serum storage time. There were no Phase 1 experiments- i n which the patients' sera used d i f f e r e d widely 172 TABLE XXIV PROPORTION OF INHIBITORY SERA AMONG SERA HAVING DIFFERENT STORAGE TIMES FROM PATIENTS ON HORMONAL TREATMENTS (PHASE 2) Proportion of Inh i b i t o r y Sera P Value for Storage Time of Serum Differences Among Proportions** <20 weeks 20-39 weeks >40 weeks 3/13 4/15 5/11 N.S. * SSLS values f o r these patients come from experiments shown i n Tables XVIII and XIX. ** P value was determined by a x 2 a n a l y s i s . 173 TABLE XXV PROPORTION OF INHIBITORY SERA IN CONTROL AND PATIENT GROUPS INCLUDING ONLY SERA STORED <40 WEEKS (PHASE 2)* Proportion of Individuals with I n h i b i t o r y Sera P Values for Controls Untreated Patients on Differences Among Patients Hormonal Proportions** Treatments 1/18 0/8 7/28 0.05<P<0.10 *SSLS values for these i n d i v i d u a l s come from experiments shown i n Tables XVIII and XIX. **P values were determined by a X 2 a n a l y s i s . ±n storage time within one experiment. However, there were two experi-ments i n which control sera had quite widely varying storage times. The storage times and SSLS for the control sera i n these two experiments are shown i n Table XXVI. Cor r e l a t i o n analysis performed using logarithms of the SSLS data showed r = 0.389 for Experiment 1 and r = -0.127 for Experiment 2. Neither was s i g n i f i c a n t . A Phase 2 experiment i n which pa t i e n t s ' sera spanned a wide range of storage times was analysed s i m i l a r l y . The data are shown i n Table XXVII. The c o r r e l a t i o n c o e f f i c i e n t between SSLS and serum storage time was r = 0.042 which was not s i g n i f i c a n t . Therefore i t appears that prolonged serum storage time up to almost 1 1/2 years, i s not strongly associated with i n h i b i t o r y serum. Thus the SSLS r e s u l t s f o r the treatment versus c o n t r o l groups have apparently not been biased by the lack of matching of controls and patients with respect to serum storage times. (v) E f f e c t of Tumour Burden on SSLS: The r e s u l t s shown i n Table XIX apparently indicated that i n h i b i t o r y dialysed sera were found with s i g n i f i c a n t l y higher frequency i n breast cancer patients r e c e i v i n g the newer hormonal treatments than i n controls or i n patients r e c e i v i n g no treatment. In fact,, no i n h i b i t o r y sera were found among the 9 sera tested from patients r e c e i v i n g no treatment. These r e s u l t s might indi c a t e that i t i s the hormonal treatments rather than the presence of breast cancer that i s re l a t e d to the f i n d i n g of i n h i b i t o r y serum. However, most of these; untreated patients had a smaller amount of tumour present, as expressed by the Disease Group, than did TABLE XXVT RELATION BETWEEN STORAGE TIME OF SERUM AND SSLS FOR CONTROL SERA. (PHASE 1) Experiment Storage Time of Serum (Weeks) SSLS (c.p.m.) Experiment 1 + July 29, 1975 27 65,082* ± 4131 32 88,780 ± 2904 14 113,640 ± 2707 35 80,545 ± 5553 14 124,750 ± 4570 14 117,543 ± 4078 29 43,308 ± 3971 21 29,485 ± 1951 14 84,448 ± 313 49 41,668 ± 3517 47 47,503 ± 3454 52 86,930 ± 5093 41 52,187 ± 5262 41 11,225 ± 157 41 22,625 ± 2460 41 105,560 ± 7248 22 130,138 ± 8916 Experiment 2 41 December 15, 1975 -^Correlation c o e f f i c i e n t r=-0.389, N..S. ^r=-0.127, N.S. 176 TABLE XXVII RELATION BETWEEN STORAGE TIME; OF SERUM AND SSLS FOR PATIENTS' SERA (PHASE 2) Storage Time of Serum (weeks) SSLS (c.p.m.) 72 187 ± 103* 72 4335 ± 472 71 27,205 ± 588 69 1043 ±- 247 65 315 ±..23.. 63 9225 ±.-8811 61 108 ± 44 59 3360 ± 333 59 7458 ± 2441 58 55 54 17,407 ± 169 53 14,435 ± 252 51 19,878 ± 1 7 0 2 48 803 ± 250 44 8530 ± 2468 33 30 ± 15 30 5060 ± 1251 19 1027 ± 98 14 49,572 ± 2072 177 TABLE XXVII (continued) Storage Time of Serum (weeks) SSLS (c.p.m.) 14 3460 ± 1151 6 .-.100 ± 70 r = 0.042, N. S. *Each. r e s u l t i s mean ± S.E. of t r i p l i c a t e w e l l s . 178 the t r e a t e d p a t i e n t s , suggesting the p o s s i b i l i t y that the presence of i n h i b i t o r y sera might be r e l a t e d to disease burden. This p o s s i b i l i t y was r u l e d out by the f i n d i n g that the p r o p o r t i o n of hormone-treated p a t i e n t s w i t h i n h i b i t o r y sera was greater f o r those w i t h Group I I and I I I disease than f o r those w i t h Group IV disease. The d i f f e r e n c e was s t a t i s -t i c a l l y s i g n i f i c a n t i f only p a t i e n t s over 65 years of age were considered. These r e s u l t s are shown i n Table XXVIII. When p a t i e n t s were s t r a t i f i e d by amount of disease and age, the p r o p o r t i o n of i n h i b i t o r y sera from p a t i e n t s w i t h Group I I I disease over 65 years was s i g n i f i c a n t l y higher than that from the other groups as shown i n Table XXIX. This was i n c o n t r a d i c t i o n to the r e s u l t s i n the Phase 1 experiment described i n I I I - I I I - l i n which p a t i e n t s w i t h the greatest disease burden had the highest p r o p o r t i o n of i n h i b i t o r y sera. When Group IV p a t i e n t s were s t r a t i f i e d according to whether one, two, of three of bones, s o f t t i s s u e s , and i n t e r n a l organs were in v o l v e d w i t h m e t a s t a t i c d i s e a s e , there was no s i g n i f i c a n t d i f f e r e n c e i n the p r o p o r t i o n of p a t i e n t s w i t h i n h i b i t o u y sera i n the three groups.. The r e s u l t s are shown i n Table XXX. There were only two p a t i e n t s w i t h a l l three s i t e s i n v o l v e d . ( v i ) R e l a t i o n of SSLS to Duration of Disease: Most of the untreated p a t i e n t s had been diagnosed as having breast cancer w i t h i n a few months of the t a k i n g of the blood sample. Many were women who had r e c e n t l y presented w i t h inoperable breast cancer.. In c o n t r a s t , the du r a t i o n of disease f o r most of the p a t i e n t s on hormonal treatments was considerably longer. I t was p o s s i b l e that the TABLE XXVIII PROPORTION OF INHIBITORY SERA AMONG PATIENTS WITH DIFFERENT AMOUNTS OF DISEASE ON HORMONAL TREATMENTS (PHASE 2)* Proport i o n of Pa t i e n t s w i t h I n h i b i t o r y Sera Age of P a t i e n t s ~ P V a l u e s f o r Disease Group D i f f e r e n c e s Among : — P r o p o r t i o n s * * I I - I I I IV A l l ages 5/10 7/29 N.S. ^65 years only 4/4 3/12 p<0.05 *SSLS values f o r these p a t i e n t s came from experiments shown i n Tables X V I I I arid XIX. *P values were determined by x 2 a n a l y s i s . TABLE XXIX PROPORTION OF INHIBITORY SERA AMONG PATIENTS OF DIFFERENT AGES AND WITH DIFFERENT AMOUNTS OF DISEASE ON HORMONAL TREATMENTS (PHASE-'2)* Proportion of Patients with Inhibitory Sera Age of Patients Amount of Disease Groups IT and I I I Group IV <65 years 1/6 4/19 >65 years 4/4 3/12 X 2 =< 10.85; d.f. = 3; P<0.025 *SSLS values for these patients came from experiments shown i n Tables XVIII and XIX. 181 TABLE XXX PROPORTION OF INHIBITORY SERA AMONG GROUP IV PATIENTS WITH DIFFERENT AMOUNTS OF DISEASE ON HORMONAL TREATMENTS (PHASE 2')* Proportion of Patients with I n h i b i t o r y Sera P Value for No. of Sites of Metastatic Disease Differences Among Proportions** 2/14 6/14 0/2 N.S. *SSLS values for these patients came from experiments shown i n Tables XVIII and XIX. **P values were determined by a x 2 a n a l y s i s . 182 diffe r e n c e i n frequency of i n h i b i t o r y sera between untreated patients and patients r e c e i v i n g hormonal treatments might be due to differences i n the duration of disease i n these two groups. I f that were the case, one would expect to f i n d a longer mean duration of disease i n patients on hormonal treatments with i n h i b i t o r y sera than i n those on hormonal treatments without i n h i b i t o r y sera as had been found i n the Phase 1 experiment. Similar data f o r t h i s Phase 2 experiment are shown i n Table XXXI. Cl e a r l y i n t h i s experiment there.was no s i g n i f i c a n t d i f f e r e n c e between the patients on hormones with i n h i b i t o r y and noninhibitory sera with respect to disease duration. Thus the Phase 2 experiment did not confirm the hypothesis suggested by the r e s u l t s of the Phase 1 experiment given i n I I I - I I I - l that patients with i n h i b i t o r y sera tended to be those with advanced disease which had been developing slowly over a long period of time. IV. DISCUSSION The Phase 1 study of the e f f e c t of various c l i n i c a l v a r i a b l e s on SSLS-I included patients r e c e i v i n g a v a r i e t y of treatments for metastatic breast cancer. There was a suggestion of a r e l a t i o n s h i p between some hormonal treatments and SSLS-I as treatment with c o r t i c o s t e r o i d s and androgens were associated wigh high values of SSLS-I whereas treatment with oestrogens was associated with low SSLS-I. The immunosuppressive e f f e c t of c o r t i c o s t e r o i d s i s w e l l known, and i s greater for cell-mediated than humoral immunity (David et a l . 1970; Haynes and Larner 1975). With a moderate dose of c o r t i c o s t e r o i d s , such as was administered to some patients i n t h i s study, the main e f f e c t of these hormones i s apparently a decrease i n the a v a i l a b i l i t y of T c e l l s and TABLE XXXI COMPARISON OF DURATION OF DISEASE FOR PATIENTS WITH INHIBITORY AND NONINHIBITORY SERA (PHASE 2)* Disease Interval Mean ± S. E. for Patients with Non-Inhibitory Sera Mean ± S.E. for Patients with Inhibitory Sera P value for difference between means** Mean disease free interval (months) 43 ± 6.9 48 ± 11,4 N.S. Mean duration of clinically apparent disease (months) 42 ± 6.7 42. ± 8.0 N.S. Mean time from in i t i a l treatment of breast cancer 85 ± 11.4 90 ± 15.1 ' N.S. *SSLS values for these patients come from experiments in Tables XVIII and XIX. *Means were compared using a t-test. 184 macrophages to i n i t i a t e c e l l mediated immune responses and to d e l i v e r help i n T cell-dependent antibody responses.(Fauci 1979). Most of the c o r t i c o s t e r o i d administered to patients would be removed from the serum by d i a l y s i s and would,.therefore, not exert a d i r e c t e f f e c t on the response of normal lymphocytes to PHA. However, i f , as has been suggested (Jenkins et a l . 1973; Burger et a l . 1974; Gahmberg and Andersson 1978), immunosuppressive a-globulins i n serum are produced i n response to prolonged immune stimulation, e.g. by cancer c e l l s , the administration of an immunosuppressive agent might decrease the production of such substances. C o r t i c o s t e r o i d administration might also decrease the production of Ag-Ab complexes which may have a nonspecific immuno-suppressive e f f e c t (Gorczynski et a l . 1975; Kilbur n et a l . 1976; Tanaka et a l . 1979). Treatment with chemotherapy had a s i g n i f i c a n t negative l i n e a r r e -l a t i o n s h i p to SSLS-I when a l l patients were considered. Like c o r t i -costeroids, chemotherapeutic agents are immunosuppressive but t h e i r r e l a t i o n s h i p with SSLS-I i s the opposite to that of c o r t i c o s t e r o i d s . The reason for t h i s i s unclear. It i s u n l i k e l y that the drugs would have been present i n the serum at a high enough concentration to exert a d i r e c t e f f e c t on lymphocytes, as blood was us u a l l y taken at le a s t one week a f t e r a course of chemotherapy. Patients with chemotherapy did not include an unusually high proportion of Group IV patients, 1 nor a s i g n i f -i c a n t l y higher proportion of patients with metastases at three s i t e s . There was no s i g n i f i c a n t c o r r e l a t i o n between chemotherapy and disease at any p a r t i c u l a r s i t e . I t may be that chemotherapy leads to a l t e r a t i o n s i n metabolism that may a l t e r the l e v e l s of some substances which stimulate of i n h i b i t immune functions. 185 As discussed i n I-VIII, oestrogen and androgen administration might be expected to have some effect on immune responses, and on the levels of certain serum proteins which may affect the response of normal lympho-cytes to PHA. However, the reported effects of oestrogens and androgens on immune responses have been very variable, and some possibly immuno-suppressive serum proteins are increased during oestrogen and androgen treatment whereas others are decreased. Therefore,.it would not be possible to predict the effect of oestrogens or androgens on the a b i l i t y of serum to support lymphocyte stimulation by PHA. The Phase 2 study indicated a s ignif icantly higher frequency of inhibitory sera in patients with metastatic breast cancer receiving the newer hormonal treatments, Tamoxifen and Aminoglutethimide, than i n un-treated patients with metastatic breast cancer or i n healthy controls. Greater mean patient age, mean storage time of serum, mean disease burden, and mean duration of disease i n treated than i n untreated patients did not appear.to be able to account for the increased frequency of inhibitory sera i n treated patients, although some interaction between these factors was not ruled out. It should be noted that patient age was s ignif icantly negatively related to log SSLS-I in Phase„1. Likewise the drugs, Tamoxifen and Aminoglutethimide and Hydrocortisone themselves were apparently not responsible for immunosuppression of the degree seen in some sera. As discussed i n I-VIII, the suppression of oestrogen pro-duction or effect might alter immune responses and might affect the levels of certain serum proteins which might i n turn affect the response of 186 normal lymphocytes to PHA. However one could not p r e d i c t the e f f e c t of such treatments as the e f f e c t s of oestrogens on the immune system are so v a r i a b l e . It would not be too s u r p r i s i n g f o r oestrogen antagonists and pharma-c o l o g i c a l doses of oestrogens to have s i m i l a r suppressive e f f e c t s on the immune system because there i s some evidence that physiologic doses of oestrogen are stimulatory whereas pharmacologic doses are i n h i b i t o r y to immune reactions. Aminoglutethimide also decreases the synthesis of androgens i n the body, and i f androgens are associated with stimulatory sera as i s suggested by the Phase 1 r e s u l t s , one might expect the lack of androgens to be associated with i n h i b i t o r y sera. In the Phase 1 study, there was a s i g n i f i c a n t r e l a t i o n s h i p between the number of s i t e s of metastases as a measure of disease burden and the degree of i n h i b i t i o n of PHA response by p a t i e n t s ' sera. Several studies demonstrating i n h i b i t i o n of normal lymphocyte functions by sera from patients with a v a r i e t y of cancers have indicated that the i n h i b i t i o n i s greater i n patients with more advanced disease ( F i e l d and Caspary 1972; Copeland et a l . 1974; Ueda et a l . 1978). The f i n d i n g of a p o s i t i v e r e l a t i o n s h i p between serum a l k a l i n e phosphataseiyand, SSL5-EI. i s d i f f i c u l t - to .explain, as,;.in.breast...cancer patients, the serum a l k a l i n e phosphatase i s usually elevated i n l i v e r and bone metastases, the presence of which i s negatively r e l a t e d to SSLS-I. On inspection of the data, i t appears that the presence of one patient with very high a l k a l i n e phosphatase, 392 o l d International Units (I.U.)/iL and with very stimulatory serum (SSLS-I = 190%) may have d i s t o r t e d the 187 r e s u l t . I f t h i s p a t i e n t was. o m i t t e d , the . r e l a t i o n s h i p between serum a l a k a l i n e phosphatase and SSLS-I was no l o n g e r s i g n i f i c a n t . As- d i s c u s s e d i n TIT-TTT-1, the f a c t t h a t p a t i e n t s w i t h i n h i b i t o r y s e r a tended to have a g r e a t e r d i s e a s e burden but not a s h o r t e r s u r v i v a l t ime than p a t i e n t s w i t h rioninhibitory s e r a would suggest t h a t p a t i e n t s w i t h i n h i b i t o r y s e r a might o f t e n be those w i t h advanced but s l o w l y growing tumours. T h i s h y p o t h e s i s was f u r t h e r supported by the f i n d i n g of a s i g n i f i c a n t l y g r e a t e r mean d u r a t i o n of d i s e a s e i n p a t i e n t s w i t h i n -h i b i t o r y s e r a . The f i n d i n g of an a s s o c i a t i o n between l e s s d i f f e r e n t i a t e d tumours and h i g h e r v a l u e s of SSLS-I i s a l s o i n support of the above h y p o t h e s i s , as p o o r l y d i f f e r e n t i a t e d tumours are f a s t e r growing. The h y p o t h e s i s t h a t p a t i e n t s w i t h i n h i b i t o r y s e r a tend to be those w i t h advanced but s l o w l y p r o g r e s s i n g d i s e a s e would be c o n s i s t e n t w i t h the v iew t h a t immunosuppressive f a c t o r s i n s e r a a r i s e because of an ongoing immune response ( J e n k i n s e t a l , 1973; Burger e_t a l , 1974; Gahmberg and Andersson 1978). One f a c t o r which might cause a tumour to grow s l o w l y would be e f f e c t i v e a n t i - t u m o u r immunity. However, d a t a from the Phase 2 experiment f a i l e d to support t h i s h y p o t h e s i s as they showed no d i f f e r e n c e i n d i s e a s e d u r a t i o n between p a t i e n t s w i t h i n h i b i t o r y and w i t h non i n h i b i t o r y s e r a , and showed a g r e a t e r frequency of i n h i b i t o r y s e r a i n Group I I I than i n Group IV p a t i e n t s . The d i f f e r e n c e s - i n r e s u l t s between Phase 1 and Phase 2 experiments might be e x p l a i n e d i n p a r t by the d i f f e r e n c e i n methods. I n s e r a used i n Phase 2 experiments, low molecular weight substances, (MW 3500 to 14,000), might be present which had been removed by d i a l y s i s from sera used i n Phase 1 experiments. I t may be that the important r e l a t i o n s h i p i n both Phase 1 and Phase 2 experiments i s that between treatment w i t h hormones and SSLS-I. Hormone-responsive tumours tend to be r e l a t i v e l y slow-growing tumours (Knight et a l . 1977; Meyer et a l . 1977). In a d d i t i o n , oestrogens are of t e n the l a s t i n a s e r i e s of hormones administered (see I - I I - 6 ) . There-f o r e , i n the Phase 1 experiment, which included p a t i e n t s on a v a r i e t y of treatments, those t r e a t e d w i t h oestrogens might have had a disease of longer d u r a t i o n than those r e c e i v i n g other treatments. In f a c t , there was a s i g n i f i c a n t c o r r e l a t i o n between oestrogen treatment and d u r a t i o n of c l i n i c a l l y apparent d i s e a s e , and between oestrogen treatment and time disease was present before treatment. Thus, i f oestrogen treatment were r e l a t e d to low SSLS-I, p a t i e n t s w i t h low SSLS-I would i n c l u d e many w i t h disease of long d u r a t i o n . At l e a s t i n younger women, androgens would u s u a l l y be given e a r l i e r i n the course of the d i s e a s e , and i f these are asso c i a t e d w i t h h i g h SSLS-I, p a t i e n t s w i t h .high SSLS-I might i n c l u d e many w i t h disease of shor t e r d u r a t i o n . However, there was not a s i g n i f i c a n t c o r r e l a t i o n between androgen treatment and disease d u r a t i o n . In the Phase 2 experiment, among those r e c e i v i n g hormone treatments there were many who had disease of long d u r a t i o n . However, mean disease d u r a t i o n d i d not d i f f e r between hormone—treated p a t i e n t s w i t h i n h i b i t o r y and n o n i n h i b i t ory sera. This would support the view that long disease d u r a t i o n i s not c a u s a l l y r e l a t e d to low SSLS i n Phase 1, but that both may be r e l a t e d to 189 oestrogen treatment. However, the r e l a t i o n between disease burden and SSLS-I seen i n Phase 1 cannot be explained s i m i l a r l y as there was no s i g n i f i c a n t r e l a t i o n s h i p between the amount of disease present and treatment. The degree of d i f f e r e n t i a t i o n of the tumour, perhaps s u r p r i s i n g l y , was not c o r r e l a t e d at a l l w i t h oestrogen treatment. C l e a r l y , treatment w i t h oestrogens or w i t h Tamoxifen and/or Amino-glute t h i m i d e or w i t h chemotherapy are not the only causes or s u f f i c i e n t . causes of i n h i b i t o r y sera as, i n Phase 1, not a l l p a t i e n t s w i t h i n h i b i t o r y sera were t r e a t e d w i t h oestrogens or chemotherapy, and i n Phase 2, not a l l p a t i e n t s r e c e i v i n g Tamoxifen and/or Aminoglutethimide had i n h i b i t o r y sera. Such f a c t o r s as i n d i v i d u a l s u s c e p t i b i l i t y to hormones or chemo-therapy might a l s o be i n v o l v e d . V. SUMMARY 1. The Phase 1 experiment included p a t i e n t s w i t h m e t a s t a t i c breast cancer r e c e i v i n g , v a r i o u s -treatments i n c l u d i n g radiotherapy,- chemotherapy, and the conventional hormones alone or i n combination. The d i a l y s i s of serum was c a r r i e d out w i t h o r d i n a r y d i a l y s i s membranes such that mole-cules of MW l e s s than 12,000 to 14,000 would pass through and would be removed. The r e s u l t s of t h i s experiment i n d i c a t e d t h a t : (1) SSLS-I d i d not appeat to be r e l a t e d to prognosis. (2) C l i n i c a l and p a t h o l o g i c a l stage at d i a g n o s i s , radiotherapy, p e r i p h e r a l blood lymphocyte count, and serum a l k a l i n e phosphatase were unrelated to SSLS-I. (3) V a r i a b l e s r e l a t e d to the du r a t i o n and r a t e of progression of disease were not r e l a t e d to SSLS-I as determined by the m u l t i p l e r e -gre s s i o n a n a l y s i s . However, the mean disease f r e e i n t e r v a l and the mean t o t a l d u r a t i o n of disease were greater f o r p a t i e n t s w i t h i n h i b i t o r y than w i t h n o n i n h i b i t o r y sera. (4) Higher p a t i e n t age was as s o c i a t e d w i t h low values of SSLS-I. (5) More d i f f e r e n t i a t e d tumours were as s o c i a t e d w i t h low values of SSLS-I. (6) Treatment w i t h chemotherapy and treatment w i t h oestrogens were a s s o c i a t e d w i t h low values of SSLS-I, and treatment w i t h androgens and treatment w i t h c o r t i c o s t e r o i d s were as s o c i a t e d w i t h high values of SSLS-I (7) The presence of more advanced disease was as s o c i a t e d w i t h low values of SSLS-I. 2. ..The Phase 2 experiment d i d not i n c l u d e any p a t i e n t s r e c e i v i n g chemo-therapy. P a t i e n t s were on no treatment or r e c e i v i n g Tamoxifen and/or Aminoglutethimide. The d i a l y s i s would have removed only molecules of MW l e s s than 3500. The r e s u l t s i n d i c a t e d t h a t : (1) There was a higher p r o p o r t i o n of p a t i e n t s w i t h i n h i b i t o r y sera among those r e c e i v i n g one of the hormonal treatments than among c o n t r o l s or p a t i e n t s on no treatment. However, there were no d i f f erencesiiamong'' the mean values of SSLS f o r the c o n t r o l and va r i o u s p a t i e n t groups. 191 (2) This experiment did not confirm the r e l a t i o n between amount, of disease or duration of disease, and SSLS', The only r e s u l t i n common from Phase 1 and Phase 2 experiments, was the f i n d i n g that various- hormonal treatments rcould.'.af feet -the 'SSLS. The e f f e c t of disease duration i n the Pase 1 experiment could be explained by i t s r e l a t i o n to hormone treatment. The inverse r e l a t i o n of disease burden to SSLS and the inverse r e l a t i o n between tumour d i f f e r e n t i a t i o n and SSLS could not be e n t i r e l y explained by t h e i r r e l a t i o n to treatments. In the Phase 2 experiment, i n which a l l the hormone treatments were f a i r l y s i m i l a r i n e f f e c t , no r e l a t i o n s h i p between low SSLS and disease duration was found, and i n h i b i t o r y sera were found more frequently i n Group ITT than i n Group IV disease. 192 CHAPTER IV THE CHEMICAL NATURE OF THE IMMUNOSUPPRESSIVE SUBSTANCE (S) IN THE SERA OF SOME BREAST CANCER PATIENTS I-. INTRODUCTION As was discussed i n I-VII, a number of d i f f e r e n t substances which are elevated i n the sera of cancer patients have been shown to cause de-creased lymphocyte response to PHA. In most studies, the r e l a t i o n s h i p between serum immunosuppresssion and the serum l e v e l s of the suspected immunosuppressive substances has not been investigated. Attempts to i s o l a t e an immunosuppressive substance: are described i n t h i s chapter. The re s u l t s suggested that no si n g l e immunosuppressive substance could be i s o l a t e d from a l l immunosuppressive sera from breast cancer patients, although a low molecular weight i n h i b i t o r appeared to be present i n some. The l e v e l s of c e r t a i n serum proteins which have been suggested as immunosuppressive substances i n cancer were measured i n sera of controls and patients, and t h e i r r e l a t i o n s h i p to SSLS was studied. I I . MATERIALS AND METHODS 1. Patients As described i n I I - I I and I I I - I I . 2. Controls As described i n I I - I I . The term control always refers to a woman over 30 years of age. In some experiments where indicated i n IV-II and IV-III sera from healthy males were studied. 193 3. Normal Lymphocyte Donors As described i n I I - I I . 4. Preparation of Sera for SSLS Measurement As described i n I I - I I . 5. Preparation of Lymphocytes As described i n I I - I I . 6. Lymphocyte Stimulation with PHA The methods used were described i n I I - I I . In experiments to determine the SSLS of unfractionated dialysed sera, the usual conditions of 10 5 lymphocytes per w e l l , 14 mg serum protein/ml, PHA 1:1500, and an incubation time of 72 hr before the addition of H-TdR were used. In experiments to test the e f f e c t s of serum f r a c t i o n s on the re s -ponse of normal lymphocytes to PHA, normal human serum (NHS) sometimes was added to the wells and various concentrations of PHA were used. The con-centrations of normal serum, serum f r a c t i o n , and PHA are given with the r e s u l t s . Each well contained 10 5 normal lymphocytes and the incubation time before the addi t i o n of 3H-TdR was 72 hr. In some cases, the con-centration of a serum f r a c t i o n was expressed as a percentage, based on the assumption that that f r a c t i o n reconstituted to the o r i g i n a l serum volume would have a concentration of 100%. In other experiments, the concentration of a serum f r a c t i o n was expressed as mg protein/ ml. 7. Fractionation of Serum on Sephadex G-150 Columns A 250-ml Sephadex G-150 column of diameter 2.74 cm and length approximately 40 cm (Pharmacia, Uppsala, Sweden) was washed with 0.01 M borate buffered normal s a l i n e , pH 8.4. Two ml.of serum from a con t r o l or a patient were applied to the top of the column. The serum was washed 194 through and eluted with borate buffered s a l i n e at a flow rate of approxi-mately 0.3 ml/min. Fractions of 3.4 ml were c o l l e c t e d . The protein con-centration was estimated by measurement of the o p t i c a l density at 280 nm. The f r a c t i o n a t i o n was c a r r i e d out at room temperature. This procedure yielded 3 protein peaks, designated Peaks I, I I , and I I I . A t y p i c a l e l u t i o n p r o f i l e i s shown i n Figure 9. The f r a c t i o n s comprising each peak were pooled and concentrated to a volume of 4 ml on UM -10 or PM -10 u l t r a f i l t e r s (Amicon Corp., Lexington, Mass.), and dialysed against PBS. The volume of PBS used was at l e a s t 30 times the volume of the s o l u t i o n to be dialysed. D i a l y s i s was c a r r i e d out for 24 hr with one change of PBS. 8. F r a c t i o n a t i o n of Serum by Ammonium Sulphate P r e c i p i t a t i o n Serum from patients or controls was divided into four f r a c t i o n s by the progressive addition of a saturated(NHi|) 2S0^ s o l u t i o n (SAS) following a modification of the methods described by Stelos (1967) and Sutherland (1967). B r i e f l y , SAS was added dropwise to serum with constant s t i r r i n g at room temperature u n t i l a concentration of 33% was reached. S t i r r i n g was continued for at l e a s t 15 min. The mixture was centrifuged at 40,000 g f o r 15 min. The p r e c i p i t a t e was washed once with 33% SAS and then dissolved i n a volume of PBS equal to the o r i g i n a l serum volume. This was c a l l e d the 33% SAS p r e c i p i t a t e (ppt) f r a c t i o n . To the 33% SAS supernatant, SAS was added as above u n t i l a concen-t r a t i o n of 50% SAS was reached. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i -fugation as above, washed once with 50% SAS, and redissolved i n a volume 195 FRACTION NUMBER FIGURE 9: TYPICAL ELUTION PROFILE OF SERUM PASSED THROUGH A SEPHADEX G-150 COLUMN (PHASE 1) Void volume approximately 70 ml. 196 of PBS equal to the o r i g i n a l serum volume. This was c a l l e d the 50% SAS ppt f r a c t i o n . S i m i l a r l y , SAS was added to the 50% SAS supernatant u n t i l a concen-t r a t i o n of 65% SAS was reached. The 65% SAS s o l u t i o n was so dense that the p r e c i p i t a t e could not be recovered by ce n t r i f u g a t i o n . Accordingly, the 65% SAS mixture was f i l t e r e d through a 0.45y membrane ( M i l l i p o r e '', . Corp., Bedford, Mass.). The p r e c i p i t a t e retained on the f i l t e r was dissolved i n a volume of PBS equal to the o r i g i n a l serum volume. This f r a c t i o n was c a l l e d the 65% SAS ppt. The f i l t r a t e f r a c t i o n was c a l l e d the 65% SAS supernatant (sup). Each f r a c t i o n was dialysed against a volume of RPMI-1640, at l e a s t 30 times the volume of s o l u t i o n being dialysed, with 2 changes of t h i s medium, s t e r i l i z e d by f i l t r a t i o n , and the protein concentration adjusted as required. The 65% SAS sup was concentrated by d i a l y s i s against dry F i c o l l 400 (Pharmacia). 9. I s o l a t i o n of Low Molecular Weight Substances Bound to the SAS Fractions The separation of low molecular weight components from larger serum proteins was c a r r i e d out by the method of Nimberg et a l (19 75). Each f r a c t i o n from the SAS f r a c t i o n a t i o n was adjusted to pH3 with 10% a c e t i c a c i d , agitated gently at 4° C, for 1-2 hr , centrifuged at 40,000 g for 15 min , and passed through a UM-10 Amicon f i l t e r followed by 3 3-ml aliquots at 1.5% a c e t i c acid (pH 1.5). F i l t r a t i o n was continued u n t i l the retentate volume was near the o r i g i n a l serum volume. The f i l t r a t e and retentate were then restored to pH near n e u t r a l i t y with NaOH. The 197 f i l t r a t e was then concentrated to the o r i g i n a l serum volume by passage through a UM-2 u l t r a f i l t e E : (Amicon). This procedure then y i e l d e d two f r a c t i o n s from each SAS f r a c t i o n , one of MW greater than 10,000 and one of MW 2,000 to .10,000. Each f r a c t i o n was centrifuged at 40,000 g f o r 15 min,, and d i a l y s e d approximately 40 hr against 30 volumes of RPMI-1640 with 3 changes of medium. The MW 2,000-10,000 f r a c t i o n s were dia l y s e d i n Spectrapor membranes r e t a i n i n g molecules greater than 3,500 MW (A. H. Thomas Co., Phila d e l p h i a , Pa.). The protein concentration was measured spectrophotometrically and adjusted as required. 10. Radioiodination of 33% SAS ppt, MW 2,000-10,000 Fr a c t i o n The 33% SAS ppt, MW 2,000 to 10,000 fr a c t i o n s from the normal serum and from the pooled i n h i b i t o r y sera were dial y s e d overnight against 30 volumes of PBS, with one change of PBS. The f r a c t i o n s were then radio-iodinated according to a modification of the Chloramine T method of Greenwood et. a l . (1963) . To 100-200 .ul of each of these f r a c t i o n s were added 500 jiCi °f sodium ( 1 2 5 I ) iodide (New England Nuclear Canada, Lachine, P. Q.) and 35 u l of chloramine T (Eastman Kodak Co., Rochester, N.Y.), 5 mg/ml, i n a fume hood at 0°C. This r e a c t i o n mixture was allowed to stand f o r 3 min. Then 75 U l of sodium metabisulphite, 12 mg/ ml i n PBS, were added. A f t e r 5 min,the reaction mixtures were placed into Spectapor d i a l y s i s bags r e t a i n i n g molecules larger than 3500 MW. The reaction tube was washed with.1% KI and the washings put i n t o the d i a l y s i s tube. D i a l y s i s was c a r r i e d out against 0.9% NaCl s o l u t i o n f o r several days with frequent changes of t h i s s o l u t i o n u n t i l the radio-a c t i v i t y outside the d i a l y s i s membrane was reduced to background l e v e l s . 198 10 y l . of each iodinated dialysed sample was counted i n a Beckman Bio-gamma Counter (Beckman Instruments Inc., F u l l t e r t o n , Ca.). 11. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)  and Autoradiography A 10% polyacrylamide slab separation g e l , 14.5 cm i n length and 1.5 mm thick, was prepared between two glass plates according to the method described by Laemli (19 70). The acrylamide and N, N'-methylene-bisa-crylamide were supplied by Eastman Kodak Co., Rochester, N. Y. This gel was o v e r l a i n by 1 cm of a 3% polyacrylamide stacking gel (Laemli 19 70). The gels contained 0.1% SDS. The top.; and bottom of the gel were i n contact with running buffer consisting of 0.05M T r i s glycine buffer, pH 8.4, containing 0.1% SDS. A volume . of radioiodinated f r a c t i o n containing 150,000 c.p.m. was d i l u t e d to 30 y l with normal s a l i n e . 5 y l of a s o l u t i o n of 0.07 M T r i s HCl buffer, pH 6.8, containing 7% SDS and 7% 2-mercaptoethanol was added to each sample. To allow determination of the molecular weight of radioiodinated molecules, 10 y l of a standard mixture of 6 proteins of known molecular weight, the low molecular weight electrophoresis c a l i b r a t i o n k i t (Pharmacia, Uppsala, Sweden), dissolved i n a s o l u t i o n consisting of ()..'..; 0.01 M T r i s HCl buffer, pH 6.8, containing 1% 2-mercapthoethanol, was u used. The iodinated samples and the standard proteins were treated i n a b o i l i n g water bath for one hour to d i s s o c i a t e any protein aggregates. Then :3yl of bromphenol blue tracking dye was added to each sample. A few 199 grains of sucrose were added to each to increase the density. The samples were applied to indentations i n the top of the stacking gel. Electrophoresis was c a r r i e d out at 150 v o l t s for approximately 1 hr u n t i l the tracking dye had entered the separating gel. Then the voltage was increased to 300 v o l t s and the electrophoresis continued u n t i l the tracking dye neared the bottom of the gel (approximately 3-4 h r ) . The gel was immediately f i x e d and stained i n a s o l u t i o n of 0.25% Coomassie B r i l l i a n t Blue, 25% isopropyl alcohol, and 10% a c e t i c a c i d , and subsequently destained i n a s o l u t i o n of 10% isopropyl alcohol and 10% a c e t i c a c i d . The gel was then soaked for 30 min i n a s o l u t i o n of 7% a c e t i c acid and 2% g l y c e r o l , and dried by a combination of vacuum and heat using a g e l dryer (Bio-Rad Laboratories, Richmond, Ca.). The distance of migration of each known protein from the beginning of the separating gel was measured and was plotted against the logarithm of the molecular weight to give a s t r a i g h t l i n e (Shapiro e_t a l . 1967; Weber and Osborn 1969). The r e s u l t s are shown i n Figure 10. The p o s i t i o n of the radioiodinated material was determined by auto-radiography following a modification of the method of Fairbanks et: a l . (1965). The dried gel was placed against a photographic plate (Kodak X-Omat-R f i l m , Kodak Canada Ltd., Toronto, Ont.), and wrapped i n f o i l for 7 days. The photographic plate was then developed and the presence of radioiodinated material noted as dark bands. The distance of migration of any radioiodinated band was measured, and i t s MW estimated from the standard curve as shown i n Figure 10. 200 MIGRATION FROM ORIGIN (cm) FIGURE 10: PLOT OF THE LOGARITHM OF THE MOLECULAR WEIGHT OF KNOWN PROT-EINS VERSUS THEIR DISTANCE OF MIGRATION FROM THE ORIGIN IN SDS-PAGE (PHASE 1) Each c i r c l e , • , represents an i n d i v i d u a l known protein used to construct the standard l i n e . The square, • , represents the main band of r a d i o a c t i v i t y from the radioiodinated 33% SAS ppt, MW 2,000-10,000 f r a c t i o n of pooled i n h i b i t o r y sera whose MW i s to be determined from the standard l i n e . 201 12. Determination of the Concentration of Serum Proteins The concentrations of a number of serum proteins were determined by immunodiffusion using immunodiffusion plates with 12 wells per plate (Behring I n s t i t u t , Marburg, W. Germany) and appropriate standards (Behring). Nine serum samples could be tested per plate, with three wells used for three different concentrations of standard. A plot of concentration versus the square of the diameter of the p r e c i p i t a t i o n ring for the standard wells gave a straight l i n e from whigh the con-centrations of the par t i c u l a r protein i n sera could be read using the measured ring diameter. 13. S t a t i s t i c a l Methods The mean lymphocyte stimulations of controls and patients i n various concentrations of Peak I and Peak I I I from Sephadex G-150 fractionations were compared using an analysis of variance with replications (Snedecor and Cochran 1967), (Tables XXXIV and XXXV). This was done by computer using logarithms of the stimulations. The r e l a t i o n between SSLS of unfractionated dialysed patient sera and the lymphocyte stimulation i n various concentrations of Peak I and Peak I I I was studied by correlation analysis (Snedecor and Cochran 1967), (Tables XXXIV and XXXV). The mean protein concentrations of Peak I and Peak I I I for patients and controls were compared by a t-test (Snedecor and Cochran 1967), (Tables XXXIV and XXXV). The r e l a t i o n between protein concentrations of Peaks I and I I I and the effect of the peak on lymphocyte stimulation was studied by correlation analysis (Snedecor and Cochran 1967), (Tables XXXIV and XXXV) 202 In the preliminary experiment the r e l a t i o n between SSLS and the levels of 6 serum proteins i n 9 sera was assessed by correlation analysis (Snedecor and Cochran 1967). The re p r o d u c i b i l i t y of measure-ments of serum proteins on different immunodiffusion plates was assessed by computer by a components of variance analysis (Kempthorne 1952; Snedecor and Cochran 1967). The r e l a t i o n between SSLS and the levels of albumin, haptoglobin, a i - a n t i t r y p s i n , and CRP i n 33 sera was analysed by multiple regression using logarithms of the SSLS data (Snedecor and Cochran 1967). This analysis was performed by computer. The mean value of SSLS for individuals with measurable CRP was compared with that of individuals with CRP below measurable levels by an analysis of variance with replications (Snedecor and Cochran 1967) , (Table XXXVIII). This analysis was performed by computer using the the logarithms of the SSLS data. The mean values of the levels of albumin, haptoglobin, and a i -antitrypsin for controls and patients on various treatments were compared by an analysis of variance (Snedecor and Cochran 1967), (Table XXXIX). The mean values of the levels of these substances for patients with Group I and I I disease and for those with Group I I I and IV disease were compared using a t-test (Snedecor and Cochran 1967), (Table XL). I I I . RESULTS 1. Fractionation of Serum on Sephadex G-150 Columns (Phase 1) The fractionation .pf serum on Sephadex G-150 yielded 3 protein peaks designated I , I I , and I I I , as shown i n Figure 9. In a p r e l i m i n a r y experiment us i n g 2 c o n t r o l sera and 3 p a t i e n t s ' sera which had been i n h i b i t o r y i n a previous experiment, Peak I from c o n t r o l s and p a t i e n t s was always very i n h i b i t o r y to the response of normal lymphocytes to PHA i n the absence of added normal serum. In f a c t , Peak I (mainly I g ) , at a conc e n t r a t i o n of 8%, supported lymphocyte s t i m u l a t i o n l e s s than 2% as w e l l as d i d 1% FCS. Peak I I I from c o n t r o l s and p a t i e n t s , at concent-r a t i o n s of 10%, 20%, and 30%, supported lymphocyte s t i m u l a t i o n w e l l (much b e t t e r than d i d 1% FCS). Peak I I had a v a r i a b l e and intermediate e f f e c t , always s t i m u l a t o r y at 10% c o n c e n t r a t i o n , but o f t e n i n h i b i t o r y at 30%. I t was shown that a mixture of the three peaks, i n the r a t i o i n which they were found i n serum, was very i n h i b i t o r y i n p a t i e n t s and c o n t r o l s . These r e s u l t s are shown i n Table XXXII. I t was found that the i n h i b i t i o n of lymphocyte s t i m u l a t i o n by Peak I could be overcome by normal human serum. When normal human serum was f i l t e r e d through a UM-10 u l t r a f i l t e r , both the concentrate and the f i l t r a t e supported lymphocyte s t i m u l a t i o n by PHA, but the f i l t r a t e could not over-come i n h i b i t i o n by Peak I. These r e s u l t s are shown i n Table XXXIII. FCS d i d not overcome i n h i b i t i o n by Peak I ( r e s u l t s not shown). A c c o r d i n g l y , a l a r g e r number of c o n t r o l and p a t i e n t sera were f r a c t i o n a t e d on a 250 ml Sephadex G-150 column. The Peak I m a t e r i a l s from the sera were t e s t e d f o r t h e i r e f f e c t of the PHA response of normal lymphocytes i n the presence of 5% normal human serum. The PHA concen-TABLE XXXII LYMPHOCYTE STIMULATION IN PEAKS I, II, AND III OF SEPHADEX G-150 FRACTIONATION: A PRELIMINARY EXPERIMENT (PHASE 1) Lymphocyte Stimulation (c.p.m.) Serum Donor Peak I Peak II Peak III Peaks I+II+III, 10% of each 1% 4% 8% 10% 20% .30% 1.0% 20% 30% C 12* Sample 2 19,585+ ± 948 1208 ± 207 592 ± 44 106,490 ± 4283 31,873 ± 3069 1275 ± 188 126,740 ± 1674 131,247 ± .1.030 123,545 ± 3150 832 ± .1.91. C 21 .14,930 + 394 2790 ± 188 960 ± 100 59,208 ± 1695 1230 ± 188 870 + 196 141,677 + 2259 136,565 ± 3354 127,962 ± 882 1362 ± 226 P 26, Sample 2 18,075 ± 558 1002 ± 111 757 ± 38 126,018 ± 2224 20,990 ± 3583 973 ± 214 139,267 ± 1141 142,445 ± 7659 133,555' ± 3200 1257 ± 203 P 45 67,183 ± 2946 1350 ± 419 660 ± 93 123,098 ± 4616 68,960 ± 3204 12,653 ± 1228 118,330 + 6605 125,362 ± 649 122,513 + 2724 920 ± 147 P 39, Sample 2 61,228 ± 4518 1303 + 323 855 ± 259 124,177 ± 1363 119,117 ± 4653 103,152 ± 3250 132,198 ± 2292 131,722 + 1848 131,387 . ± 1854 1023 ± 160 1% FCS In the same experiment 70,907 ± 6748 41,563 + 1321 41,563 + 1321 80,915 ± 3553 * C 12 and 21 designate individual controls; P 26-45 designate individual patients. Sample 2 indicates the second serum sample from that person. v Each result is the mean ± SE of 3 wells. PHA concentration used was 1:1500. o TABLE XXXIII ABILITY OF NORMAL HUMAN SERUM TO OVERCOME INHIBITION BY 8% PEAK I MATERIAL FROM SEPHADEX G-150 FRACTIONATION (PHASE 1) Lymphocyte S t i m u l a t i o n (c.p.m.) Donor of ~ " 8% Peak I Pooled Normal Human Serum (PNHS) Component Added No PNHS 5% PNHS 10% PNHS 20% PNHS 10% fPNHS* 20% fPNHS No Peak I 9370 58,477 55,727 27,590 72,377 82,997 ± 224 ± 3376 ± 4314 ± 2043 + 1489 ± 1424 C 22** 1185 43,732 32,998 24,125 2798 2253 ± 165 ± 1243 ± 1867 ± 2619 + 808 ± 572 C 23 362 25,335 34,872 20,683 817 772 - ± 43 ± 854 ± 2334 ± 2754 + 305 ± 38 P 46 908 38,528 35,573 13,213 710 520 ± 285 ± 1178 ± 2853 ± 560 + 178 ± 55 P 47 523 51,063 41,763 25,238 555 602 ± 136 ± 2102 ± 4072 ± 3150 + 228 ± 57 * fPNHS i s an u l t r a f i l t r a t e of PNHS passed through a UM-10 f i l t e r (Amicon). C 22 and 23 designate i n d i v i d u a l c o n t r o l s ; _ P 46 and 47 designate i n d i v i d u a l p a t i e n t s . Each r e s u l t i s the mean ± SE of 3 w e l l s . PHA concentration used was 1:3750. O On t r a t i o n used was 1:3750. The Peaks i l l m a t e r i a l s were t e s t e d i n the absence of normal human serum, and w i t h PHA 1:3750. The r e s u l t s are shown i n Tables XXXIV and XXXV. The i n h i b i t o r y Peak I was s l i g h t l y l e s s . i n h i b i t o r y i n p a t i e n t s than i n c o n t r o l s , and there was a b s o l u t e l y no suggestion of a c o r r e l a t i o n between lymphocyte s t i m u l a t i o n i n d i a l y s e d u n f r a c t i o n a t e d serum and that i n Peak I w i t h NHS. At the highest c o n c e n t r a t i o n used, there was a s i g n i f i c a n t c o r r e l a t i o n between p r o t e i n c o n c e n t r a t i o n i n Peak I and the i n h i b i t i o n caused by Peak I . T h i s might suggest t h a t the i n h i b i t o r y substance(s) i n Peak I was q u a n t i t a t i v e l y a major component of Peak I. Peak I I I at 2.5% only was s i g n i f i c a n t l y more s t i m u l a t o r y i n c o n t r o l s than i n p a t i e n t s (p<0.05). However, there was no s i g n i f i c a n t c o r r e l a t i o n between lymphocyte s t i m u l a t i o n i n d i a l y s e d u n f r a c t i o n a t e d serum and that i n Peak I I I . At concentrations of 5% and 10% Peak I I I , there was a s i g n i f i c a n t c o r r e l a t i o n between p r o t e i n c o n c e n t r a t i o n i n Peak I I I and lymphocyte s t i m u l a t i o n i n Peak I I I . Again t h i s might suggest that the s t i m u l a t o r y substance(s) i n Peak I I I was q u a n t i t a t i v e l y a major component of Peak I I I . I t was c l e a r from the above r e s u l t s that the e f f e c t of serum on the response of normal lymphocytes to PHA was not r e f l e c t e d i n the e f f e c t s of peaks obtained by f r a c t i o n a t i o n on Sephadex G-150. Therefore, t h i s technique was abandoned. TABLE XXXIV LYMPHOCYTE STIMULATION IN SERUM OR (PHASE 1) PEAK I Lymphocyte Stimulation (c.p.m.) Serum Donor Type of Serum or Fraction and PHA Concentration Protein of Peak Concentration I (mg/ml)** 20% Dialysed Serum; PHA 1:1500 Peak I, 1.25% and 5% NHS; PHA 1:3750 Peak I, 2.5% and 5% NHS; PHA 1:3750 Peak I, 5% NHS; 3.75% and PHA 1:3750 C 24 + 19,448 ± 1676* 6745 ± 1284 2498 ± 436 5.5 C 6, Sample 2 16,417 ± 1698 14,375 ± 471 4573 ± 800 5.3 C 3, S.-imp 11- 2 25,765 ± 1307 19,082 ± 4200 12,570 ± 438 3.9 C 25 19,210 + 81.3 7985 ± 362 3658 ± 370 6.2 Mean=20,210 Mean=12,047 Mean=5824 Mean=5.2 P 48 802 ± 292 25,390 ± 867 20,202 ± 2594 21,182 ± 570 2.8 P 49 31,937 ± 4278 23,122 ± 2196 13,145 + 1586 10,473 ± 1890 6.7 P 50 63,788 + 2242 17,513 ± 2155 11,002 ± 255 5620 ± 1123 8.6 P 51 46,950 ± 6805 17,745 ± 2438 16,793 ± 1548 7482 ± 1223 5.6 P 52 48,728 ± 13,302 17,262 ± 2028 10,148 ± 1242 6280 ± 462 7.4 P 53 43,520 + 2815 30,685 + 875 14,838 ± 975 6068 ± 1344 8.6 P 54 40,468 ± 1758 23,765 ± 750 16,958 ± 451 11,752 ± 2066 6.8 K3 TABLE XXXIV (continued) Lymphocyte Stimulation (c.p.m.) Serum Donor Type of Serum or Fraction and PHA Concentration Protein Concentration pf Peak I (mg/ml)** 20% Dialysed Serum; PHA 1:1500 Peak I, 1.25% and 5% NHS; PHA 1:3750 Peak I, 2.5% and 5% NHS; PHA 1:3750 Peak I, 3.75% and 5% NHS; PHA 1:3750 P 24 1 23,092 ± 1835 17,625 ± 1953 12,838 ± 1066 3.7 P 55 54,098 ± 9178 20,635 ± 406 17,090 ± 1595 15,057 ± 2093 5.4 P 56 7025 + 1440 13,048 ± 586 4118 ± 611 1163 ± 127 6.7 P 57 14,348 ± 3222 20,695 ± 253 13,955 ± 604 7740 ± 1383' 7.0 P 58 30,408 + 13,622 19,930 ± 2970 15,165 ± 1678 11,672 + 1258 6.3 Mean= 34,734 Mean=21,074 Mean=14,253 ' Mean=9777 Mean=6.3 * Each value of lymphocyte stimulation i s the mean ± SE of 3 wells. + C 3-25 desif.nntn individual controls; P 24-58 designate individual patients. Sample 2 indicates the second sample from that person. ** Concentration of Peak I i f i t were resuspended in the original serum volume. S t a t i s t i c a l Analyses: No significant difference between the mean Peak I protein concentrations for patients and controls. Correlation between lymphocyte stimulations in various concentrations of Peak I and i n 20% dialysed serum (using logarithms); 20% dialysed serum with 1.25% Peak I: r = -0.080, N.S. 20% dialysed serum with 2.5% Peak I: r = -0.039, N.S. 20% dialysed serum with 3.75% Peak I: r = -0.097, N.S. K3 O OO TABLE XXXIV (continued) Correlation between logarithms of .lymphocyte stiimilnti.ons in vnrlmin concentrnticms of Peak I and protein concentration In Peak I: Peak I protein concentration with stimulation in 1.25% Peak I: r = -0.159, N.S. Peak I protein concentration with stimulation in 2.5% Peak I: r = -0.414, N.S. Peak I protein concentration with stimulation in 3.75% Peak I: r = -0.542, p<0.05. Peak I was not s i g n i f i c a n t l y more inhibitory i n patients than i n controls at any concentration. ho O' VO TABLE XXXV LYMPHOCYTE STIMULATION IN SERUM OR • (PHASE 1) PEAK III Lymphocyte Stimulation (c.p.m.) Serum Donor Type of Serum or Fraction and PHA Concentration Protein Concentration of Pcnk T.I.I - (mp,/m1,)** 20% Dialysed Serum; PHA 1:1500 Peak III, 2.5%; PHA 1:3750 Peak III, 5%; PHA 1:3750 Peak I I I , 10%; PHA 1:3750 C 24 + 19,265 ± 567* 17,282 + 238 25,365 ± 613 17.6 C 6, Sample 19,667 + 994 23,415 ± 1205 29,145 + 709 24.6 C 3, Sample 2 23,058 ± 661 28,298 ± 674 43,055 ± 569 21.4 C 25 18,308 + 1304 18,073 ± 589 17,290 ± 210 15.0 C 26 19,135 i 561 17,042 ± 1671 24,213 ± 1028 20.4 Mean=19,887 Mean=20,822 Mean=27,814 Mean=19.8 P 48 802 ± 292 21,602 + 255 20,323 ± 565 24,618 + 814 18.2 P 49 31,937 ± 4278 14,360 ± 1570 16,665 ± 560 19,010 ± 79 21.2 P 50 63,788 ± 2242 15,932 + 935 16,990 ± 181 24,550 ± 530 22.8 P 51 46,950 ± 6805 15,968 ± 223 14,280 + 751 14,918 ± 630 15.2 P 52 48,728 ± 13,302 19,296 + 381 24,820 ± 1090 33,335 ± 1123 21.2 TABLE XXXV (continued) Lymphocyte Stimulation (c.p.m.) Serum Type of Serum or Fraction and PHA Concentration Protein Concentration Donor of Peak III (mg/ml) 20% Dialysed Peak III, 2.5%; Peak III, 5%; Peak III, 10%; Serum; PHA 1:1500 PHA 1:3750 PHA 1:3750 PHA 1:3750 P 54 40,468 ± 1758 14,148 ± 409 12,767 ± 1015 13,753 ± 405 13.0 P 24 18,513 ± 683 23,892 ± 1384 40,308 ± 1379 25.8 P 55 54,098 ± 'J1.78 lf>,620 * 4 78 17,085 ± 957 21,667 ± 310 19.4 P 56 7025 ± 1440 14,730 ± 1355 13,972 + 1047 17,080 + 437 14.4 P 57 14,348 ± 3222 14,737 ± 262 14,397 + 188 18,727 ± 174 23.0 P 58 30,408 ± 13,622 16,967 ± 1835 14,028 ± 332 14,528 ± 428 17.8 Mean=33,855 Mean=16,624 Mean=17,202 Mean=22,045 Mean=19.3 * Each value of lymphocyte stimulation i s the mean ± SE of 3 wells. + C 3-26 designate individual controls; P 24-58 designate individual patients. Sample 2 indicates the second sample from that person. ** Concentration of Peak III i f i t were resuspended in the or i g i n a l serum volume. S t a t i s t i c a l Analyses: No significant difference between the mean Peak III protein concentrations for patients and controls. Correlation between lymphocyte stimulations i n various concentrations of Peak III and i n 20% dialysed serum (using logarithms of data): 20% dialysed serum with 2.5% Peak III: r =.-0.480, N.S. TABLE XXXV (continued) 20% dialysed serum with 5% Peak III: r = -0.154, N.S. 20% dialysed serum with 10% Peak III: r = -0.104, N.S. Correlation between logarithms of lymphocyte stimulations in various concentrations of Peak III and protein con-centration i n Peak III: Peak III protein concentration with stimulation in 2.5% Peak III : r = 0.290, N.S. Peak III protein concentration with stimulation in 5% Peak III: r = 0.656, p<0.05. Peak III protein concentration with stimulation in 10% Peak III: r = 0.765, p<0.01. Peak III at 2.5% was s i g n i f i c a n t l y more stimulatory i n controls than i n patients, p<0.05. 213 2. F r a c t i o n a t i o n of Three Serum Samples by Ammonium Sulphate P r e d - i c a t i o n : I n i t i a l Experiment (Phase 1) Serum from a he a l t h y male, a pool of sera ( n o n i n h i b i t o r y when d i a l y s e d and tes t e d i n a previous experiment) from 2 breast cancer p a t i e n t s , and a pool of two p a t i e n t s ' sera ( I n h i b i t o r y when d i a l y s e d and teste d i n a previous experiment) were f r a c t i o n a t e d by p r e c i p i t a t i o n w i t h SAS. The d i a l y s e d f r a c t i o n s were t e s t e d f o r t h e i r a b i l i t y to support lymphocyte s t i m u l a t i o n w i t h PHA i n the presence of 5% NHS. The u n f r a c t i o n a t e d d i a l y s e d sera were a l s o t e s t e d without NHS. The PHA con c e n t r a t i o n used was 1:4000. The r e s u l t s are shown i n Table XXXVI. I t was seen that i n h i b i t o r y m a t e r i a l from the pooled i n h i b i t o r y sera was d i s t r i b u t e d i n a l l the SAS f r a c t i o n s whereas only the 65% SAS ppt from the normal male was i n h i b i t o r y ( i . e . <30% of s t i m u l a t i o n i n 5% NHS) at 2 mg protein/ml. I t had p r e v i o u s l y been shown that a mixture of the f r a c t i o n s of the normal serum without NHS were l e s s s t i m u l a t o r y than u n f r a c t i o n a t e d serum but not seve r e l y i n h i b i t o r y as w i t h Sephadex G-150 f r a c t i o n s . The 50% SAS ppt from the n o n i n h i b i t o r y p a t i e n t serum pool was i n h i b i t o r y at 3 mg protein/ml. The d i s t r i b u t i o n of i n h i b i t o r y a c t i v i t y i n a l l f r a c t i o n s from the i n h i b i t o r y p a t i e n t s ' sera was s i m i l a r to the p a t t e r n seen by Nimberg e_t a l . (1975) who f r a c t i o n a t e d serum on ion-exchange columns. These researchers found that the i n h i b i t o r y m a t e r i a l was a low molecular weight peptide which was c a r r i e d by a v a r i e t y of serum p r o t e i n s i n cancer p a t i e n t s and could be d i s s o c i a t e d TABLE XXXVI LYMPHOCYTE STIMULATION IN SERUM OR SAS FRACTIONS (PHASE 1) NHS Serin coo Fractions ceo-tra-tion _ unfraction-ated Serua Fraction Protein Con-centration (•g/ml) Support of Lymphocyte Stimulation (c.p.m.) Normal Male Serum 54,637 t 319T Type of Pooled Serum 51,047 48,332 t 14S1 t 2762 "~ Noninhibitory Patients Sera 17,555 4 380 12,737 t 573 9340 t 1076 Pooled Inhibitory Patients' Sera 4240 t 120 307 ± 75 78 1 19 5Z 33Z SAS ppt 0.5 1 2 3 86,467 83.213 81,193 94,267 1 2950 t 4428 t 3259 t 2592 75,440 t 2337 74,832 t 3034 60,702 t 1085 34,308 t 2185 78,070 1 5153 69,168 t 828 43,078 t 1519 6515 1 1312 5Z 50Z SAS ppt 0.5 1 2 3 88,356 84,300 74,035 67,675 t 1616 t 3078 t 3593 t 10.270 78.998 ± 2463 67,720 t 4566 44,890 1 1303 13,327 t 549 78,645 t 1965 62,567 t 653 15,912 t 364 175 ± 66 TABLE XXXVI.(continued) NHS Serum con- Fractions cen-tra-tlon 6SZ SAS ppt 5Z 65Z SAS sup Serum Fraction Protein Con-centration (mg/ml) 0.5 1 2 0.5 1 2 3 Support of Lymphocyte Stimulation (c.p.m.) Normal Male Serum 66.810 ± 2809 41,042 t 1297 5002 t 132 89,095 t 1944 85,500 t 3619 80,763 t 374 78,480 i 4624 Type of • 1 !'B7 o f t r l p U c a t e w e l l s c o n t a l n l n 8 5 X " • o n l y * • a 7 - 1 5 3 * c . p . . . + Bach value 1. * s.K. of triplicate wells. Pooled Nonlnhlbltory Patients' Sera 72,975 ± 2999 47.487 t 3112 32.643 ± 1425 90,940 ± 2216 73.900 t 3039 56,283 t 707 52,747 t 971 Serum Pooled Inhibitory Patients' Sera 27,735 t 9535 3187 t 728 152 * 17 76,897 t 322 49,798 * 371 31.155 * 987 11,752 * 544 ro I—1 216 at low pH. A c c o r d i n g l y , an attempt was made to i s o l a t e such a peptide from the SAS f r a c t i o n s of the i n h i b i t o r y p a t i e n t s ' sera. 3. F r a c t i o n a t i o n of SAS F r a c t i o n s From Three Serum Samples by U l t r a - f i l t r a t i o n on Amicon Membranes (Phase 1) The 33% and 50% SAS ppt f r a c t i o n s were separated i n t o >10,000 MW and 2,000 to 10,000 MW f r a c t i o n s by the method described i n I V - I I . The d i a l y s e d f r a c t i o n s were then t e s t e d f o r t h e i r a b i l i t y to support normal lymphocyte s t i m u l a t i o n w i t h PHA i n the presence of 3% NHS. The r e s u l t s are shown i n Table XXXVII. The 33% SAS ppt, MW 2,000 to 10,000 f r a c t i o n from the pooled i n h i b i t o r y p a t i e n t s ' sera was i n h i b i t o r y compared to 3% NHS alone, w h i l e the corresponding f r a c t i o n from normal serum had l i t t l e e f f e c t on the response of lymphocytes to PHA i n 3% NHS. 4. SDS-PAGE of Radioiodinated 33% SAS Ppt, MW 2,000 to 10,000 F r a c t i o n s  From a Normal Serum and Pooled I n h i b i t o r y Sera (Phase 1) Because of the i n d i c a t i o n that the 33% SAS ppt, MW 2,000 to 10,000 f r a c t i o n from the pooled i n h i b i t o r y sera might c o n t a i n an i n h i b i t o r y substance absent from,.or i n much-lower, con c e n t r a t i o n i n , ' the corresponding f r a c t i o n of the normal serum, . t h i s . f r a c t i o n of normal and i n h i b i t o r y sera was r a d i o i o d i n a t e d and analysed by SDS-PAGE. The r e s u l t s are shown i n Figures 10 and 11. The autoradiograph showed a prominent band of approximate MW 10,100 from the f r a c t i o n of i n h i b i t o r y sera w i t h only a f a i n t band i n t h i s p o s i t i o n from the normal serum f r a c t i o n . TABLE XXXVII LYMPHOCYTE STIMULATION IN ULTRAFILTRATION FRACTIONS OF SAS FRACTIONS OF SERUM (PHASE 1) Lymphocyte Stimu-lation (c.p.m.) with 3'Z NHS only ln experiment  Support of Lymphocyte Stimulation (c.p.m.) Type of Serum Fraction Serum Fraction Concen-tration Normal Male Sena Nonlnhlbltory Patients Sera Serum Pooled Inhibitory Patients' Sera 78,730 ±2290 33Z SAS ppt, MW 2,000 -10,000 20Z (v/v) 40Z (v/v) 60Z JJZ SAS ppt, MM > 10,000 80,928 t 3257* 72,060 ± 225 71,403 t 985 Not available 0.56 mg/ml 1.12 mg/ml 1.68 mg/ml 83,028 t 3122 76,093 1 1048 65,172 t 1012 Not available 82,830 t 1040 46,033 t 9S4 19,473 t 1066 61,225 t 594 34,692 1 609 3420 t 329 97,709 ± 2336 50Z SAS ppt, MW 2,000 -10,000 20Z (v/v) 40Z (v/v) 60Z (v/v) Not available 86,362 t 1999 76,482 ± 68 67,317 t 1859 77,983 t 2551 74,928 t 3131 62,178 t 1334 TABU XXXVII (continued) Support of Lymphocyte Stimulation Type of Serum (c.p.m.) Lymphocyte Stimu-lation (c.p.m.) with Serum 3 Z NHS only la same Fraction experiment Serum Fraction Concen-tration Normal Malm Serum fooled Noninhibitory Patients'Sera^ Pooled Inhibitory Patients' Sera SOX SAS ppt MP 10,000 .35 mg/ml 88,485 1 1480 83,963 t 2296 .7 mg/ml 78,508 x 1825 100733 i 1071 76,313 x 1405 1.2 mg/ml 76,468 x 1010 90,090 i 970 63,245 t 1953 2.0 mg/ml 69,143 x 1785 * Bach value la the mean t 8.R. of triplicate we11a. 219 10,100 MW band Normal F r a c t i o n Pooled I n h i b i t o r y P a t i e n t s ' F r a c t i o n FIGURE 11: AUTORADIOGRAPH OF THE GEL FROM THE SDS-PAGE OF RADIOIODINATED 33% SAS PPT, MW 2,000-10,000 FRACTIONS FROM A NORMAL SERUM AND FROM POOLED INHIBITORY PATIENTS' SERA (PHASE 1) 220 5. Fractionation of Other Sera by SAS P r e c i p i t a t i o n (Phase 2) The SAS f r a c t i o n a t i o n procedure was repeated with two normal sera and pooled i n h i b i t o r y serum samples taken from one breast cancer patient on d i f f e r e n t occasions. In t h i s case, only the 65% SAS ppt f r a c t i o n of the i n h i b i t o r y serum pool was i n h i b i t o r y compared with the corresponding f r a c t i o n of normal sera. However, no i n h i b i t o r y a c t i v i t y was recovered i n the 2,000 to 10,000 MW f r a c t i o n of t h i s 65% ppt f r a c t i o n . Accordingly, i t was concluded that, while a low molecular weight i n h i b i t o r might contribute to immune suppression i n some p a t i e n t s ' sera, i t was not uniformly present i n i n h i b i t o r y p a t i ents' sera. The possible immune i n h i b i t o r found i n one pool of two i n h i b i t o r y sera had a MW of 10,100 and appeared to be present i n normal sera but i n lower concen-t r a t i o n . 6. Relationship Between Levels of Certain Serum Proteins and  SSLS (Phase 2) In a preliminary experiment the l e v e l s of s i x serum proteins were measured i n nine sera whose SSLS had previously been determined. Three c o n t r o l sera, two i n h i b i t o r y patients' ser.a, and four non-i n h i b i t o r y p a t i e n t s ' s e r a were used. The sera had been dialysed against RPMI-1640 and adjusted to 35 mg protein/ml.^before the serum protein concentrations were measured. The serum proteins measured were albumin, a i - a n t i t r y p s i n , d i - a c i d glycoprotein, caeruloplasmin, haptoglobin and CRP. These were chosen because of reports i n the l i t e r a t u r e i n d i c a t i n g t h e i r possible e f f e c t s on lymphocyte response to PHA as discussed i n I-VII. Albumin, a i - a n t i t r y p s i n and haptoglobin l e v e l s showed a s i g n i f i c a n t 221 or borderline s i g n i f i c a n t c o r r e l a t i o n with SSLS. CRP was undetectable by immunodiffusion i n 6 of 9 sera. CRP was measurable i n one patient with i n h i b i t o r y sera, i n one patient with borderline i n h i b i t o r y sera, and i n one with noninhibitory sera (data not shown). Accordingly, the four serum proteins, albumin, d i - a n t i t r y p s i n , haptoglobin, and CRP were selected for further study. The r e p r o d u c i b i l i t y of measurements of these four serum proteins was assessed. For each of the four serum proteins, two patients' sera and a pool of normal sera were tested i n three wells of each of three p l a t e s . The v a r i a b i l i t y of serum protein measurements between wells within one plate and the v a r i a b i l i t y between plates was analysed by a components of variance a n a l y s i s . The standard error of the mean of r e p l i c a t e measurements with-i n one plate was always less than 13% of the mean, and usually much less than 10% of the mean. The v a r i a b i l i t y between plates was s i g n i f i c a n t l y greater than the v a r i a b i l i t y between wells within plates only i n the case of albumin. The albumin measurements on one of the plates were s i g n i f i c -a ntly lower than the measurements for the same sera on the other two p l a t e s . Because only 1 of the 3 sera gave a p o s i t i v e CRP measurement, the compon-ents of variance analysis could not be done f or CRP. However, the standard error of the mean CRP values f o r the one serum on the three plates was 10.5% of the mean which was not greatly d i f f e r e n t from the standard error of the mean f o r each p l a t e . The SSLS f o r sera from 22 controls and 30 breast cancer patients was measured on three separate occasions using lymphocytes from the same normal donor. On each occasion, the SSLS for each serum was designated 222 h i g h , medium, or low. A high SSLS was greater than or equal to 70% of the c o n t r o l mean on that day, a medium was 30% to 70% of the c o n t r o l mean, and a low SSLS was l e s s than 30% of the c o n t r o l mean. For 33 s e r a , the SSLS remained i n the same category, i . e . h i g h , medium, or low, on a l l three occasions. Each of the four serum p r o t e i n s , albumin, a i - a n t i -t r y p s i n , h a p t o g l o b i n , and CRP was measured f o r each of these 33a sera. The serum p r o t e i n measurements were made usin g sera d i a l y s e d against RPMI-1640 and adjusted to 35 mg protein/ml. The samples used i n the t h i r d SSLS experiment were those used f o r serum p r o t e i n measurements. The r e l a t i o n s h i p between SSLS on the t h i r d occasion and the serum l e v e l s of the four serum p r o t e i n s was analysed by m u l t i p l e r e g r e s s i o n a n a l y s i s , using logarithms of the SSLS data. In a stepwise m u l t i p l e r e g r e s s i o n a n a l y s i s , only the c o n c e n t r a t i o n of a i - a n t i t r y p s i n was b o r d e r l i n e s i g n i f i c a n t l y l i n e a r l y r e l a t e d to In SSLS, p=0.0865. The r e g r e s s i o n equation was: In SSLS = 9.97-0.063 [ a i - a n t i t r y p s i n ] Thus high values of serum Cii-antitrypsin were associated w i t h low values of SSLS. The c o r r e l a t i o n c o e f f i c i e n t of In SSLS w i t h [ a i - a n t i t r y p s i n ] was r = -0.303. Thus r 2 = 0.092 which i n d i c a t e d that 9.2% of the v a r i a b i l i t y i n In SSLS could be explained by v a r i a b i l i t y i n a i - a n t i t r y p s i n c o n c e n t r a t i o n . CRP was too low to measure by immunodiffusion i n most sera. In the stepwise m u l t i p l e r e g r e s s i o n a n a l y s i s , CRP was the next most important v a r i a b l e a f t e r C4i-antitrypsin, w i t h p = 0.158, which was not s t a t i s t i c a l l y s i g n i f i c a n t . There was a c o n s i d e r a b l y higher mean SSLS among persons without measurable CRP than among those w i t h measurable CRP, as shown i n Table XXXVIII. However, t h i s d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f -TABLE XXXVIII MEAN SSLS IN INDIVIDUALS WITH MEASURABLE AND NONMEASURABLE LEVELS OF CRP (PHASE 2) Mean SSLS (c.p.m.) 4 Individuals with 29 Individuals with Measurable CRP Nonmeasurable CRP 6734 ± 5578* + 23,732 ± 4647* + * Each r e s u l t i s the mean ± SE of the SSLS values f o r each i n d i v i d u a l i n that group. In turn, the SSLS value f o r each i n d i v i d u a l i s the mean of 3 wells. + These means are not s i g n i f i c a n t l y d i f f e r e n t , i . e . p>0.05. 224 icant, but the number of persons with measurable CRP was very small. There was a f a i r l y high p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t , r=0.470, between the concentrations of CRP and a i - a n t i t r y p s i n , making i t d i f f i c u l t to specify the e f f e c t of each on SSLS. In the o v e r a l l multiple regression a n a l y s i s , including a l l four serum proteins, none was s i g n i f i c a n t l y l i n e a r l y r e l a t e d to l n SSLS at pO.05, but again a i - a n t i t r y p s i n and CRP came closest to s i g n i f i c a n c e . The mean values of albumin, haptoglobin, and oti-antitrypsin for controls and patients on various treatments are shown i n Table XXXIX. The mean values of these serum proteins for patients with Group I and II disease and for patients with Group I I I and IV disease are shown: i n Table XL. Mean albumin l e v e l s were s i g n i f i c a n t l y lower i n patients on Tamoxifen than i n controls. Haptoglobin l e v e l s were s i g n i f i c a n t l y lower i n patients on Tamoxifen, with and without Aminoglutethimide, than i n controls. There were no s i g n i f i c a n t differences i n the l e v e l s of any of these serum proteins between patients with l i t t l e disease and those with much disease. IV. DISCUSSION The f r a c t i o n a t i o n of serum by Sephadex G-150 columns was found not to be s u i t a b l e as a method for p u r i f y i n g immunosuppressive substances, as immunosuppressive a c t i v i t y appeared to be generated (or perhaps simply stim-ulatory substances l o s t ) by t h i s procedure. Other researchers (Matthews and Whitehead 1976) found that the passage of serum from breast cancer patients through U l t r o g e l AcA34 columns (LKB Produkter AB, Bromma, Sweden) or through ion-exchange columns caused the IgG-rich f r a c t i o n s to become i n -h i b i t o r y toward ADCC of human K c e l l s . ' This was a t t r i b u t e d to p a r t i a l de-naturation of IgG. Presumably, t h i s denatured IgG would bind to Fc receptors on K c e l l s to prevent t h e i r binding to Ab-which was coating target : c e l l s . TABLE"XXXIX MEAN LEVELS OF SERUM PROTEINS IN CONTROLS AND PATIENTS ON VARIOUS HORMONAL TREATMENTS (PHASE 2)  Control or Patient Group Xi (mg/dl) (Mean :±. SE) X 2 (mg/dl) (Mean ± SEP X 3 : (mg/dl) >(Mean ± SE> X<4 Pro- j portion P o s i t i v e Controls Tamoxifen Aminoglutethimide Tamoxifen + Aminoglutethimide 1780 ± 51 1 1485 ± 67 1 1602 ± 75 1422 ± 422 21.2 ± 2.1 14.3 ± 2.53 36.2 ± 5.1 15.0 + 5.0 2 2. 3 21.4 ± 2.6 30.0 ± 3.6 28.3 ± 9.5 27.3 ± 2.5 2/15 1/12 1/4 Xi ' albumin, X 2 = haptoglobin, X 3 = a 1 - ant 11 r y p s i n , X14= CRP i> 2,3 Sig.. d i f f . , P<0.05 * Significance not tested. to to TABLE XL MEAN LEVELS OF SERUM PROTEINS IN PATIENTS WITH DIFFERENT AMOUNTS OF DISEASE (PHASE 2) Amount of Disease X x (mg/dl) X 2(mg/dl) X 3 (mg/dl) (Mean ± SE) (Mean ± SE) (Mean ± SE) Group I and II 1614 ± 107 21.8 ± 5.0 23.8 ± 4.7 Group III and IV 1462 ± 70.2 18.2 ± 3.7 31.4 ± 3.8 No differences between means for Group I and II compared with Group I I I and IV are s i g n i f i c a n t . K3 2 2 7 I t i s not c l e a r whether such denatured IgG could i n h i b i t the response of lymphocytes to PHA, although i t has been shown that t h i s r e a c t i o n can be i n h i b i t e d by Ag-Ab complexes (see I-VII). The i n h i b i t o r y 33% SAS ppt, MW 2,000-10,000 f r a c t i o n from two patients with extremely i n h i b i t o r y sera apparently contained a substance of MW approximately 10,000 i n higher concentration than d i d the corres-ponding f r a c t i o n from a normal male. This might represent the immuno-suppressive peptide i s o l a t e d from cancer patients' sera by Nimberg et a l . (1975). However, while they obtained a s i m i l a r pattern of i n h i b i t o r y a c t i v i t y present i n a l l f r a c t i o n s of ion exchange chromatography from v i r t u a l l y a l l i n h i b i t o r y cancer sera, l e s s severely i n h i b i t o r y sera i n the present study did not show a consistent pattern of i n h i b i t o r y a c t i v i t y i n the f r a c t i o n s from SAS f r a c t i o n a t i o n . An i n h i b i t o r y 65% SAS ppt f a i l e d to y i e l d an i n h i b i t o r y low MW f r a c t i o n . Therefore, i t appears that some but not a l l breast cancer patients with i n h i b i t o r y sera have a low MW i n h i b i t o r . The experiment measuring various serum proteins showed a s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between log SSLS and serum a i - a n t i t r y p s i n - c o n c e n -t r a t i o n and an almost s i g n i f i c a n t r e l a t i o n s h i p between log SSLS and CRP concentration. However, v a r i a t i o n i n these proteins accounted f o r l i t t l e more than.110% of the v a r i a b i l i t y i n SSLS. Of course, other possible :;. immunosuppressive substances such as PAM, and t r a n s c o r t i n could have been measured. The general conclusion to be drawn from these experiments i s that there i s no s i n g l e cause of immunosuppressive sera i n breast cancer 228 patients, but that the immunosuppressive substances may d i f f e r somewhat from one patient to another. In each i n d i v i d u a l the SSLS probably depends on the i n t e r a c t i o n of a number of d i f f e r e n t substances of which a i - a n t i t r y p s i n , CRP, and a low MW substance may be important. CHAPTER V 229 CONCLUDING REMARKS When t h i s project was undertaken, i t was thought that patients with immunosuppressive serum might have a poor prognosis. It was hoped that, i f the nature of the p r i n c i p a l immunosuppressive substance could be determined, i t might be possible to counteract i t or to prevent i t s synthesis so as to ben e f i t the patient. These expectations have not been r e a l i z e d . No sin g l e immunosuppressive substance could be i d e n t i f i e d i n a l l i n h i b i t o r y - s e r a . A pool of two extremely i n h i b i t o r y patients' sera appeared to contain a low MW i n h i b i t o r such as that reported by Nimberg et al.(1975). This could not be found i n several other i n h i b i t o r y sera. Measurement of a number of serum proteins revealed that only a i - a n t i t r y p s i n and perhaps CRP had even a borderline s i g n i f i c a n t negative l i n e a r r e l a t i o n s h i p with SSLS, and v a r i a b i l i t y i n these proteins would account for l e s s than 10% of the v a r i a b i l i t y of SSLS. Thus, i f there i s a sing l e major immuno-suppressive factor i n most i n h i b i t o r y sera, i t has not been i s o l a t e d . Another p o s s i b i l i t y i s that the SSLS i s the r e s u l t of l e v e l s of a number of d i f f e r e n t f a c t o r s , one of which may be a i - a n t i t r y p s i n , and one a low MW substance. Of course, these experiments did not measure every serum protein that has ever been reported to be immunosuppressive. In p a r t i c u l a r , PAM and t r a n s c o r t i n were not measured. Thus, no way of a l t e r i n g immuno-suppressive a c t i v i t y i n serum was suggested by the r e s u l t s . Furthermore, the presence of immunosuppressive serum does not appear to a f f e c t a patient's prognosis.adversely. Studies by others had found 230 immunosuppressive serum more commonly i n patients with advanced cancer ( F i e l d and Caspary 1972; Copeland et a l . 1974; Buda e t a l . 1975; Ueda et a l . 1978). 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