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UBC Theses and Dissertations

Studies on tumour antigens associated with human bronchogenic carcinomata Watson, Robert Dale 1975

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STUDIES ON TUMOUR ANTIGENS ASSOCIATED WITH HUMAN BRONCHOGENIC CARCINOMATA by ROBERT DALE WATSON B.Sc. University of British Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ... In the Department of Microbiology We accept this thesis as conforming, to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C olumbia, I agree t h a t the 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 the Head o f my Department o r by h i s 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 not 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 The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Bronchogenic carcinomata of various pathologies were obtained either at post-mortem or post surgically for the purpose of this study. Normal lung tissue as control material was obtained from individuals who died-from non-malignant causes. Antigenic materials were extracted from both normal and tumour tissue with 3.0 M KC1 i n order to determine whether bronchogenic tumours contained either tumour specific transplantation antigens (TSTA -c e l l surface antigens recognized as non-self by the host immune system) or tumour associated antigens (TAA - components not found in normal tissue which may or may not be antigenic i n the host but which are antigenic in a heterologous species). Attempts to identify tumour associated antigens by direct chemical comparisons between normal and tumour extracts were made using disc gel electrophoresis i n conjunction with ion exchange and gel f i l t r a t i o n chroma-tography of the extracts. A tumour associated antigen was detected i n this way but i t was not found in other tumour extracts and therefore i t s significance was limited. Autoradiographic studies using sections of tumour or normal lung, 125 patients' or normal sera, and I-labelled anti-human Ig indicated that patients with bronchogenic carcinoma may produce antibodies reactive with their own and other tumours. Skin test reactions to fractions of homo-logous tumour extract also suggested that patients may react to tumour antigens, but neither of these methods were found to be suitable as an assay for tumour antigens. Efforts to raise antisera against tumour antigens by rendering rabbits neonatally tolerant to normal lung antigens followed by immunization with tumour extract proved unsuccessful. Greater success was obtained by i i hyperimmunizing rabbits to tumour extracts and passing resultant antisera through solid immunoadsorbents of normal lung extracts. The absorbed anti-sera, in many instances, reacted specifically with antigens in tumour extracts. Broad studies using immunodiffusion with absorbed antisera and a panel of tumour extracts indicated that bronchogenic carcinomata may contain at least two tumour associated antigens which cross-react widely. One of these antigens appeared to share properties with an antigen in foetal lung extracts, but was neither carcinoembryonic antigen nor cti-foetoprotein. When summarized, the results suggested that some TAA were associated with tumour pathology and possibly aetiology; squamous c e l l , oat c e l l and ana-plastic carcinomas (carcinogen induced) appeared to contain related TAA which were distinguished from TAA in adenocarcinomas and alveolar c e l l carcinomas (not known to be associated with carcinogens). i i i TABLE OF CONTENTS INTRODUCTION Page I. Historical Development 1 II. TSTA in Human Neoplasms 2 III. The Nature of TSTA and TAA 3 1. Cell Membrane Chemistry 4 2. TSTA and Normal Histocompatibility Antigens 7 3. Mutational Basis for the Origins of TAA 9 4. Tumour-Associated Foetal (Embryonic) Antigens 11 5. Viral Antigens 12 IV. TSTA and Tumour Rejection 13 V. TSTA and the Roles of Antibody in Tumour Rejection 15 1. Immunologic Enhancement 15 2. Manipulation of Immune Responses by Modified Antigen 18 3. Tumour Rejection Augmented by Antibody 20 VI. Experimental Approaches to the Identification of TSTA and TAA • 21 1. Detection of Cell Mediated Immunity to Tumour Cells . . 21 2. Detection of Antibodies Against TSTA or TAA 22 3. Detection of TSTA and TAA in Human Systems 23 4. The Importance of Detecting Human TSTA and TAA . . . . 24 5. The Uses of Specific Antibodies Against TAA 26 VII. Objectives of the Present Study 26 VIII. Experimental Approaches to the Present Study 27 1. Extraction of Tumour Cells 27 2. Detection of TAA 28 i v TABLE OF CONTENTS MATERIALS AND METHODS Page I. Tissue Preparation and Extraction 32 1. Preparation of Tumour Tissue 32 2. Normal Lung Tissue . . . . . . . 32 3. Embryonic Lung Tissue 32 4. Tissue Extraction . . 33 II. Neonatal Tolerization of Rabbits 34 III. Immunization of Rabbits . . . . . 35 1. Hyperimmunization 35 2. Alum Precipitate *••.•• " 37 IV. Immunodiffusion 37 1. Semi-Micro 37 2. Micro • • 3 8 3. Staining 38 4. Micro Radioimmunodiffusion • 38 V. Immunoelectrophoresis 39 VI. DEAE Cellulose Chromatography 39 1. Gradient Elution 40 2. Step Elution 40 3. Treatment of Fractions 40 VII. Gel Chromatography 41 VIII. Ammonium Sulfate Precipitation 41 IX. Disc Gel Electrophoresis • 42 X. Migration Inhibition Factor Test with Human Peripheral Blood Leukocytes . . . . 42 V TABLE OF CONTENTS (Continued) MATERIALS AND METHODS Page XI. Guinea Pig Skin Tests 44 XII. Agarose Immunoadsorbent Columns 44 XIII. Glutaraldehyde Cross-Linked Immunoadsorbents 46 1. Human Immunoglobulin Immunoadsorbent 46 2. Normal Lung Extract Immunoadsorbent 46 XIV. ",. Other Antiserum Absorption Methods 48 1. Precipitation of Immune Complexes 48 2. Absorption with Cells 50 XV. Purification of Immunoglobulin 51 1. Human Immunoglobulin 51 2. Rabbit Immunoglobulin • 51 XVI. Human Serum Fractionation 52 XVII. Indirect Immunofluorescence 52 1. Absorbed Anti (Tumour Extract) Serum 52 2. Human Serum 55 XVIII. Indirect Immunoautoradiography • • • 55 1. Experimental Techniques 55 2. Quantitation 57 3. Testing Human Serum and Fractions 58 4. Testing Absorbed Rabbit Anti (Tumour Extract) Serum • • 58 XIV. Iodination 59 XX. St a t i s t i c a l Analyses 6 1 XXI. Skin Testing of Cancer Patients 6 1 XXII. Analysis of Extracts for CEA 6 2 v i TABLE OF CONTENTS RESULTS AND DISCUSSION Page I. Neonatal T o l e r i z a t i o n of Rabbits to Antigens of Normal Lung Extracts 63 I I . Migration I n h i b i t i o n of Allogeneic Human Leukocytes by Extracts of Patients' Tumours , 64 I I I . Disc Gel Electrophoresis of the Extracts and Their Fractions 68 IV. Immunodiffusion Studies 76 1. The Detection of TAA i n Extracts Using Absorbed Rabbit Antisera 76 A. The Use of Immunoadsorbents 77 B. The Detection of Tumour Associated Antigens . . . . 83 2. cti-f oetoprotein 112 3. Studies on Patients' Sera for Detection of TAA Using Absorbed Antisera 114 4. Studies on Patients' Sera for Detection of A n t i -bodies to TAA 114 5. Studies on Radioimmunodiffusion f or Improvement of Resolution 115 V. Carcinoembryonic Antigen Assay 117 VI. Immunofluorescence 124 VII. Indirect Autoradiography . 126 1. Studies with Absorbed Rabbit Antisera 126 2. Studies with Human Serum 132 VIII. Skin Tests of Patients with Extracts of Their Own Tumours . . 140 v i i TABLE OF CONTENTS Page GENERAL DISCUSSION 155 BIBLIOGRAPHY 160 APPENDIX A 171 APPENDIX B 172 APPENDIX C 174 LIST OF TABLES Immunodiffusion results using antisera to lung tumour extracts of 5 types of tumour pathology against extracts of the following pathologies: a. squamous c e l l carcinoma b. adenocarcinoma and anaplastic carcinoma c. oat c e l l and alveolar c e l l carcinoma, a variety of less common lung tumour, and foetal lung. A summary of Table I showing the percentage of positive results. Quantitation of CEA in extracts of lung tumour and normal appearing lung from the same person, by radioimmunoassay. Quantitation of CEA in extracts according to pathology. Indirect autoradiography survey for the binding of antibody from patients' sera to tumour and normal tissue sections. ix LIST OF FIGURES Figure Page 1. Procedure for the neonatal tolerization of 36 rabbits. 2. The test of the absorption of rabbit anti-human 47 Ig by immunodiffusion. 3. Precipitin reaction with antiserum against C-41. 49 4. Fractionation of patient serum on Sephadex G-150 53 equilibrated in acetic acid. 5. The testing of human serum fractions by immuno- 54 diffusion against anti-human Ig. 125 125 6. The removal of free I from I conjugated Ig 60 by Sephadex G-25. 7. Human peripheral blood MIF results with extracts. 66 8. Fractionation of a tumour extract by DEAE 69 cellulose gradient chromatography. 9. Further fractionation of a tumour extract fraction. 70 by Sephadex G-75 chromatography. 10. Disc gel electrophoresis results with fractions 71 of C-26 and N-pool extracts. 11. Immunodiffusion results using absorbed antiserum 73 to C-26-I against fractions of C-26. 12. Disc gel electrophoresis results with: a. fractions of tumour extracts 74 b. fractions of pooled foetal extracts. 75 13. a. Immunodiffusion with whole anti-C-26. 78 b. Immunodiffusion with absorbed anti-C-26 78 c. Immunoelectrophoresis with absorbed anti-C-26 78 d. Immunodiffusion with 'anti-normal' antibodies 79 from anti-C-26 serum. 14. a. Immunodiffusion with absorbed anti-C-40 81 b. Immunoelectrophoresis with absorbed anti-C-40 81 15. Immunodiffusion with anti-C-67: a. absorbed by a Sepharose 4B immunoadsorbent 82 b. absorbed by a Sepharose 4B and a glutaraldehyde 82 insolubilized immunoadsorbent LIST OF FIGURES (Continued) Figure Page 16. Immunodiffusion with absorbed anti-C -6 89 17. Immunodiffusion with absorbed anti-C-53 90 18. Immunodiffusion with absorbed anti-C-57 91 19. Immunodiffusion with absorbed anti-C-63 93 20. Immunodiffusion with absorbed anti-C-71 94 21. Immunodiffusion with absorbed anti-C-24 95 22. Immunodiffusion with absorbed anti-C-26 96 23. Immunodiffusion with absorbed anti-C-41 98 24. Immunodiffusion with absorbed anti-C-46 99 25. Immunodiffusion with absorbed anti-C-62 I Q O 26. Immunodiffusion with absorbed anti-C-62 101 against extracts which were stored under different conditions. 27. Immunodiffusion with absorbed anti-C-67 103 28. Immunodiffusion with absorbed anti-C-40 104, 29. Immunodiffusion with absorbed anti-C-78: a. against different extracts 105 b. against dilutions of C-94 105 30. a. Immunodiffusion with anti-C-30 absorbed 107 using N-pool insolubilized by both Sepharose 4B and glutaraldehyde. b. Immunodiffusion with anti-C-30 absorbed 108 twice as in part a. and re-absorbed using N-83 insolubilized by Sepharose 4B. 31. Immunodiffusion with purified anti-cii-foeto- 113 protein. 32. Immunodiffusion with patient serum and fractions. 116 33. a. Micro-radioimmunodiffusion and - 119 b. Micro-immunodiffusion test with anti-human Ig 119 against serial dilutions of human Ig LIST OF FIGURES (Continued) Semi-micro-immunodiffusion tests with anti-human-Ig against serial dilutions of human Ig. Test of the specificity of the sheep anti-rabbit Ig by Immunoelectrophoresis. The results of testing absorbed anti (tumour extract) serum for the binding of antibody to tumour and normal lung tissue sections by indirect autoradiography. Sources of high background grain counts in indirect autoradiography: a. by a single c e l l , and b. by large patches with uneven distribution of grains. The results of testing cancer patients' sera and fractions for the binding of antibody to tumour and normal lung tissue sections by indirect autoradiography. ~ Examples of indirect autoradiography results testing serum 68 against: a. C-68 tumour tissue section and b. N-86 normal tissue section Skin test results from an osteogenic sarcoma patient (C-68) with extract and fractions of autochthonous tumour, measuring: a. the diameter of erythema with undiluted materials b. the diameter of induration with undiluted materials c. the diameter of erythema with 1/10 diluted materials - ' , d. the diameter of induration with 1/10 diluted materials Skin test results from an osteogenic sarcoma patient (C-73) with extract and fractions of autochthonous tumour, measuring: a. the diameter of erythema b. the diameter of induration Skin test results from a non-immune guinea pig with C-87 extract and fractions. x i i LIST OF FIGURES (Continued) Figure Page 43. Skin test results from four bronchogenic carcinoma patients with extract and fractions from autochthonous tumours: a. patient 87 148 b. patient 85 148 c. patient 76 148 d. patient 72 149 44. Skin test results from five bronchogenic carcinoma patients with 1:2 dilutions of extract and fractions, and a 1:1:1 mixture of the fractions of autochthonous tumour: a. patient 57 153 b. patient 93 153 c. patient 87 153 d. patient 88 153 e. patient 60 153 X11X i F o r H e a t h e r ACKNOWLEDGEMENTS I wo u l d l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e t o Dr. J u l i a L e v y f o r h e r encouragement, d i r e c t i o n , a s s i s t a n c e and c o n s t r u c t i v e c r i t i c i s m t h r o u g h o u t t h i s p r o j e c t . I g r e a t l y a p p r e c i a t e t h e a s s i s t a n c e g i v e n by Anaj a n e S m i t h . I woul d l i k e t o than k Dr. D. K i l b u r n and t h e o t h e r members o f my committee f o r t h e i r a d v i c e i n t h e e d i t i n g o f t h i s t h e s i s . I w o u l d l i k e t o than k t h e f o l l o w i n g p e r s o n s f o r t h e i r a s s i s t a n c e as mentioned i n t h e t e x t o f t h i s t h e s i s . Dr. J . B u r t o n , Dr. P. Coy, Dr. J . W. Thomas, Dr. B. P o l a n d , Dr. E. M i n c e y , B e t t y - L o u A r c h i b a l d , Dr. S. 0. Freedman, Mr. C. C u l l i n g , D a l e G r e g e r s o n , and Dr. J . Yensen. L a s t l y , I would l i k e t o thank Rosemary Morgan f o r h e r p a t i e n c e i n t h e t y p i n g o f t h i s m a n u s c r i p t . 1 INTRODUCTION I. Historical Development The attitude towards immunity and malignant disease has changed radically during this century. As pointed out by Fairley (34) there have been three phases in the approach to tumour immunology: the optimistic, followed by the pessimistic, and f i n a l l y the r e a l i s t i c . The optimistic phase occurred around the turn of the century. Lymphocyte i n f i l t r a t i o n of tumours was found to be important in the rejection of transplanted tumours. Later, Woglom (140) i n his literature review of six hundred papers published between 1913 and 1929 concluded: "Nothing may be hoped for at present in respect to a successful therapy from this direction". The pessimistic phase was ushered out with the breeding of syngeneic mice. Only with syngeneic animals could the presence of tumour-specific transplantation antigens (TSTA) be proven, as opposed to allo-transplantation antigens, thus suggesting the f e a s i b i l i t y of immunization against some types of carcinogenesis. TSTA can be defined as those antigens against which the immune mechanisms of the host are directed in the rejection of syngeneic tumours. Gross (39) and then Lewis and Aptekman (69) demonstrated that tumour immunity could be induced in syngeneic animals to transplanted isologous tumours. However, these results could also be explained either i n terms of heterogeneity of the inbred animals or a mutation in the tumour lines as a result of transplantation. Foley (35) subsequently showed that tumour immunity could be induced by tumour cells which had not been repeatedly transplanted. Prehn and Main (102) proved that tumour immunity was not a result of heterogeneity of the inbred animals by showing that only tumour tissue and not normal tissue from the same inbred animal, could immunize against isologous tumour challenge. 2 II. TSTA in Human Neoplasms It has not been proven that TSTA in man exist, although there i s evidence supporting this possibility (20, 34, 65). This evidence may be summarized as follows: 1. Patients with abnormal immune systems have an increased incidence of malignant diseases (37). For example, patients with hypogamma-globulinaemia have an increased incidence of leukaemia and lymphoma (95 ), autoimmune haemolytic anaemia i s not infrequently associated with chronic lymphocytic leukaemia and lymphosarcoma (27), and renal transplant patients who have been treated with immunosuppressive drugs have a higher than normal incidence of reticulosarcomas (63). The evidence i s consistent with the requirement for an intact immune system to effect the elimination of malig-nant cells arising i n the body, as postulated in Burnet's (20 ) immune surveillance theory. 2. Southam (122), by injecting autochthonous (from the same individual) tumour c e l l suspensions, found that subcutaneous tumour nodules would be produced i f the c e l l dose were high enough. This dose i s related to the immunological status of the host. 3. Histological evidence (14) demonstrates that for carcinoma of the breast and stomach, lymphoid i n f i l t r a t i o n and sinus histiocytosis in the regional lymph nodes are associated with a favourable prognosis. 4. Specific lymphoid c e l l reactivity against autochthonous cultured tumour cells can be detected in vitro by colony inhibition techniques (47). 3 Peripheral blood lymphoid cells from patients even with progressive malig-nant disease, can have a cytostatic effect upon either autochthonous or tumour ce l l s of a similar c e l l type to the patient's neoplasm. These observations indicate not only the presence of TSTA on human neoplasms but also the possibility that common antigens may be present on tumours of similar morphology. 5. Some cancer patients have antibodies in their serum, which appear to be specific for their tumour. Burkitt's lymphoma patients (64) can produce at least two specific populations of antibodies: one against the Epstein-Barr virus, thought to be responsible for the disease, and one against an antigen'present on the tumour c e l l membrane. Many patients with other types of tumours have anti-tumour antibodies in their sera, either in the form of blocking factors or cytotoxic antibodies (48). 6. Everson (33) has reported 130 cases of diagnosed cancer dis-appearing spontaneously or following t r i v i a l treatment. The tumours which undergo spontaneous remission w i l l often induce inflammation in the v i c i n i t y of the tumour lesions upon immune stimulation. The simplest explanation for these phenomena is anti-tumour immunity. III. The Nature of TSTA and Tumour-Associated Antigens (TAA) TAA can be defined as any material predominantly associated with neo-plastic disease, which can be detected by an immunological method. This material i s not necessarily antigenic in the tumour-bearer and includes TSTA.' Information on the nature of TAA can possibly shed light on the little-known process of oncogenesis. 4 1. Cell Membrane Chemistry Emmelot (32) has reviewed c e l l membrane chemistry with emphasis on the comparison of different tumour c e l l membranes with each other and normal c e l l membranes for their content of various phospholipids and carbohydrates. There was great variation between different tumours but no tumour-specific changes in content were observed although reaction with plant agglutinins was frequently increased (81). According to the f l u i d mosaic model of the structure of membranes (119), the surface membranes of cells are dynamic flu i d structures in which membrane components may migrate. Malignant transformation is closely associated with a greatly increased a b i l i t y of the cells to be agglutinated by plant lectins. Lectins (such as concanavalin A) are capable of agglutinating cells by binding to specific sugar residues (such as a-D-glucopyranosyl or ct-D-mannopyranosyl residues). It has been suggested (119) that a difference between normal and transformed cells could be the membrane f l u i d i t y . The lectin receptors would be more mobile and produce clusters so that the l e c t i n molecules can become locally concentrated and thus able to agglutinate the c e l l s . Mild treatment of normal cells with proteolytic enzymes can induce agglutinability similar to that of transformed c e l l s . Perhaps the most obvious explanation for this finding i s that the enzyme cleaves protein and exposes lectin binding receptors which were previously buried in cryptic sites. However, i t i s also possible to explain this finding in terms of f l u i d i t y differences between normal and transformed cells (119). The proteo-l y t i c enzyme would preferentially cleave polar peptides from the membrane proteins, thus making the proteins more hydrophobic and resulting i n aggregation of lectin receptors. 5 I t i s p o s s i b l e t o make f i r m l y e s t a b l i s h e d tumours r e g r e s s i n s y n g e n e i c m i c e i f t h e h o s t i s c h a l l e n g e d w i t h tumour c e l l s w h i c h have been r e a c t e d w i t h V i b r i o c h o l e r a e n e u r a m i n i d a s e i n v i t r o (118) • The e f f e c t i s augmented i f non-s p e c i f i c i m m u n o s t i m u l a n t s s u c h as M y c o b a c t e r i u m b o v i s ( s t r a i n BCG) i s i n j e c t e d a l s o . Tumour r e g r e s s i o n c o u l d n o t be i n d u c e d w i t h c e l l s t r e a t e d w i t h i n a c t i -v a t e d n e u r a m i n i d a s e o r w i t h tumour c e l l s o f a d i f f e r e n t t y p e , s u g g e s t i n g t h a t t h e n e u r a m i n i d a s e i s making the c e l l s more immunogenic. N e u r a m i n i d a s e c l e a v e s a p o r t i o n o f t h e s i a l i c a c i d from c e l l membrane g a n g l i o s i d e s . T h i s r e s u l t s i n a d e c r e a s e i n n e g a t i v e charge and c o u l d l e a d t o i n c r e a s e d i m m u n o g e n i c i t y by d e c r e a s i n g t h e s t e r i c h i n d r a n c e t o a n t i g e n , o r i n c r e a s i n g c e l l d e f o r m a b i l i t y o r s u s c e p t i b i l i t y t o p h a g o c y t o s i s Q18)• C o n c a n a v a l i n A t r e a t e d tumour c e l l s can i n d u c e tumour r e g r e s s i o n s i m i l a r t o n e u r a m i n i d a s e t r e a t e d c e l l s . There i s c o n s i d e r a b l e p a r a l l e l i s m between t h e e f f e c t s o f t h e s e a g e n t s even though c o n c a n a v a l i n A does n o t remove groups from t h e membrane ( l i k e n e u r a m i n i d a s e ) b u t i s bound t o s p e c i f i c s u g a r r e s i d u e s on t h e membrane. T h e r e f o r e , i t i s p o s s i b l e t h e i n c r e a s e d immuno-g e n i c i t y i n d u c e d by t h e s e a g e n t s i s v i a t h e same mechanism. B o t h a g e n t s have a m i t o g e n i c e f f e c t on l y m p h o c y t e s . I f m i t o g e n s were c r e a t e d on tumour c e l l membranes, r e a c t i o n s w i t h i m m u n o r e a c t i v e c e l l s c o u l d be supplemented. B o t h a g e n t s c o u l d d i s r u p t c e l l s u r f a c e s n o n - s p e c i f i c a l l y , t h e r e b y e x p o s i n g neo-a n t i g e n s 018). I t i s a l s o p o s s i b l e t h a t b o t h a g e n t s c o u l d r e d u c e s t e r i c h i n d r a n c e t o a n t i g e n s by d i f f e r e n t mechanisms: n e u r a m i n i d a s e by removing s i a l i c a c i d r e s i d u e s , and c o n c a n a v a l i n A by a g g r e g a t i n g g l y c o p r o t e i n s and l e a v i n g t h e a n t i g e n s exposed. A wide v a r i e t y o f enzymes has been t e s t e d f o r t h e a b i l i t y t o m o d i f y tumour c e l l s and i n d u c e tumour r e j e c t i o n (101). B e s i d e s n e u r a m i n i d a s e , o n l y t r y p s i n had any e f f e c t , , and t h i s was augmented by r e a c t i n g t h e t r y p s i n i z e d c e l l s w i t h n e u r a m i n i d a s e ( n e u r a m i n i d a s e f o l l o w e d by t r y p s i n gave no p r o t e c t i o n ) . 6 It i s possible to gain a further understanding of membrane chemistry by studying the effects of modifying membranes. Besides modification with enzymes, chemicals capable of reacting with specific chemical"groups have also been used. Prager and Baechtel (101). have reviewed several catagories of specific chemical group modifications and their a b i l i t y to augment tumour rejection. In general, only the addition of small, non-haptenic reagents are effective. Alteration of sulfhydryl groups with reagents such as iodo-acetate have been shown to make murine tumour cells more immunogenic. Sodium periodate, which cleaves certain monosaccharide units, results in a decrease or loss of tumour c e l l immunogenicity. (It i s of interest to note that like concanavalin A and neuraminidase, lymphocytes reacted with periodate undergo blastogenesis as i f they were reacted with a mitogen. However, only tumour cells reacted with concanavalin A or neuraminidase can induce tumour reject-ion (118), suggesting that the increased immunogenicity of these agents is not by the creation of mitogens on tumour c e l l surfaces). Reagents capable of reacting with amino groups have given a range of results. Reaction with fluorodinitrobenzene has generally not induced tumour rejection while reaction with formaldehyde can frequently give some protection 001). Tumour extract reacted with diketene has been shown to induce tumour rejection (70). Because of the interdependence of membrane components for their location, configuration and exposure, changes in tumour antigenicity induced by specific reagents does not necessarily mean that these groups are part of the antigen i t s e l f . Most of these studies (LOI) have been done by modi-fying tumour cells and looking for increased immunogenicity, whereas more definitive studies on the nature of tumour antigens could be gained by trying to diminish the antigenicity of soluble tumour antigen. 7 2. TSTA and Normal Histocompatibility Antigens TSTA are almost certainly associated with the c e l l membrane. It i s d i f f i c u l t to conceive how intracellular antigens could function i n immuno-logical rejection, since the tumour c e l l membrane i s largely impermeable to antibody, and c e l l - c e l l contact interactions are thought to be required for tumour c e l l k i l l i n g mediated by sensitized lymphocytes (5). Normal histo-compatibility antigens are known to reside on the c e l l membrane (58) and are capable of stimulating an immunological response analogous to (although more effective than) that stimulated by TSTA. Methods used for the extraction and purification of normal alloantigens have been successfully applied to studies of TSTA. We might therefore expect to find similarities between normal alloantigens and TSTA. Considerable work has already been done on the chemical characterization of normal alloantigens. Mouse H-2 alloantigens have been extracted from membrane fractions by papain, purified, and found to be glycoproteins with molecular weights of 65,000 to 75,000, 90% of which i s protein and 10% i s a variety of different carbohydrates (117). Although the antigenic determinants have not yet been characterized, their susceptibility to protein denaturants and prolonged proteolytic enzyme digestion implies that the protein configurations are important. Treatment with different glycosidases produces no loss in antigenicity. The loss of antibody binding a b i l i t y by chemical modifications of the glycoprotein molecules with rea-gents specific for tyrosine residues or amino groups provides further evidence that the protein structure determines H-2 reactivity (96). Guinea pig transplantation antigens (MW 15,000) extracted by sonication contain no more than one carbohydrate residue per molecule and HL-A antigens (MW 34,600) extracted by 3M KC1 contain no more than two carbohydrate residues (107). 8 Singer and Nicholson (119) have categorized membrane proteins as integral or peripheral. Briefly, peripheral proteins are bound by weak, non-covalent forces and are easily solubilized free of l i p i d . Integral proteins are much more closely associated with membrane l i p i d s , more d i f f i c u l t to dissociate from membranes, and then usually insoluble or aggregated. These proteins possibly have appreciable a-helical content and a significant portion of their amino acids buried in l i p i d by hydrophobic interactions. It has been suggested Q33) that H-2' alloantigens are integral; proteins. However, this i s only on the basis of the close association of H-2' antigens with membrane l i p i d as determined by the lack of shedding of these antigens by the cell s . This was compared to the rapid shedding of peripheral molecules such as immunoglobulin and 9 antigen. It i s also possible that HCL-A antigens are integral proteins in spite of the relatively large quantities of HL-A antigens detected in the spent medium of cultured lymphocytes (99). This could be due to membrane fragments of dead cells rather than shedding from viable ce l l s . HL-A antigens solubilized by papain have a molecular weight of 48,000 and can be dissociated into 33,000 and 11,000 MW fragments. The smaller fragment i s f^-roicroglobulin which has also been found in urine and is the same for a l l HL-A specificities studied 026). It has 28% sequence homology with parts of the Y chain of IgG-1 and i s not required for the reaction of HL-A antigen with antibody (99). (This protein i s also suspected as a possible link between histocompatibility and immune response gene l o c i (99,103)). B^-roicroglobulin i s not essential for the binding of HL-A anti-gens to membranes because HL-A antigens (33,000 MW ) can be solubilized with papain, from the Daudi c e l l line. This c e l l line does not produce or contain B^-microglobulin (103). Thus, i t appears that the 33,000 MW." fragment (which contains the HL-A antigenicity) could be the antigenic part 9 o f t h e p r o t e i n bound t o t h e c e l l membrane and c l e a v e d by p a p a i n . HL-A a n t i g e n s s o l u b l i z e d by d e t e r g e n t (NP-40) have a m o l e c u l a r w e i g h t o f about 150,000 w h i c h upon f u r t h e r p u r i f i c a t i o n and r e m o v a l o f t h e d e t e r g e n t i s r e d u c e d t o about 70,000. The B2 - m i c r°gl°bulin i s a s s o c i a t e d w i t h t h e s e f r a c t i o n s . I f t h i s s m a l l e r fragment i s r e d u c e d and a l k y l a t e d , t h e p r e d o m i n -a n t component i s i n d i s t i n g u i s h a b l e i n s i z e (by g e l chromatography) from IgG heavy c h a i n s (24,103). T h i s e v i d e n c e s u g g e s t s t h a t HL-A a n t i g e n s may f o r m a complex s i m i l a r t o i m m u n o g l o b u l i n m o l e c u l e s . S i m i l a r f i n d i n g s a r e r e p o r t e d f o r H-2 a n t i g e n s (103) . The 33,000 MW . fragment w h i c h r e t a i n s t h e HL-A a n t i g e n i c i t y has a v e r y s i m i l a r amino a c i d a n a l y s i s , r e g a r d l e s s o f t h e HL-A t y p e . However, some HL-A s p e c i f i c i t i e s c a n be s e p a r a t e d by i s o e l e c t r i c f o c u s i n g 026). T h e r e f o r e , t h e w i d e range o f HL-A a n t i g e n s p e c i f i c i t i e s i s d e t e r m i n e d by mi n o r changes i n t h e p r o t e i n . C h a r a c t e r i z a t i o n o f TSTA i s s t i l l a t a p r i m i t i v e s t a g e o f development and t h e r e f o r e i t i s d i f f i c u l t t o draw sound a n a l o g i e s between them and n o r m a l a l l o a n t i g e n s . There i s e v i d e n c e t h a t i n r a t hepatomas, p r o t e i n , c a r b o h y d r a t e , and l i p i d m o i e t i e s a r e a l l i n v o l v e d i n t h e arrangement o f t h e TSTA (MW*-55,000) i n t h e c e l l membrane. However, t h e p u r i f i e d a n t i g e n i c a l l y a c t i v e component i s p r o t e i n w i t h o n l y a t r a c e o f c a r b o h y d r a t e (5 )• 3. M u t a t i o n a l B a s i s f o r t h e O r i g i n s o f TAA I n o r d e r t o e x p l a i n t h e d i f f e r e n c e i n a n t i g e n i c i t y between s p o n t a n e -o u s l y a r i s i n g tumours and m e t h y l c h o l a n t h r e n e i n d u c e d tumours, P r e h n and M a i n CL02) s u g g e s t e d t h a t a p o t e n t mutagen wou l d be more l i k e l y t o p r o d u c e s i m u l t -aneous a n t i g e n i c and n e o p l a s t i c changes t h a n t h e a g e n t ( s ) r e s p o n s i b l e f o r th e i n d u c t i o n o f o c c a s i o n a l spontaneous tumours. The e x p r e s s i o n o f TSTA was 10 not concommitant with the malignant process per se since one of the tumour lines lost i t s antigenicity after numerous transplants. The possibility that TSTA represent a replacement.of or modification of normal alloantigens is unlikely since the H-2 alloantigens from mouse spleen and tumour cells are indistinguishable (79). However, i t i s possible that TSTA might result from manipulations on weak histocompatibility gene l o c i (58). Since a large portion of a cell's genetic material i s probably involved in regulatory functions, a genetic change capable.of interfering with the control of c e l l division could also interfere with protein synthesis control. This genetic change could occur at a location in the cell's DNA specific for the particular agent and result in TSTA specific for the onco-genic agent. Oncogenic virus induced tumours possess common group-specific TSTA (62). Alternatively, the genetic change could occur at random, in which case only rarely would tumours possess common TSTA. Tumours induced by chemical carcinogens or physical agents only rarely cross-react immuno-logically (62). The genetic changes associated with oncogenesis could lead to production of molecules not normally produced, an increase in production of normally produced molecules or even a change in existing molecules. Neoantigens associated with oncogenesis could be either immunogenic or non-immunogenic in the tumour bearer. Even molecules normally produced by a c e l l could be immunogenic i f more was produced or i f exposed to the immune system in a different manner (40). Examples of TAA which can also be detected in non-malignant conditions are the various polypeptide factors associated with cancers, particularly lung cancer (92), and the T-globulin which can be detected in the sera of cancer patients and pregnant women (125) . Another TAA i n normal tissue i s the brain-associated tumour antigen. Lymphocytes from cancer patients are capable of responding specifically 11 to heterologous basic protein of myelin (30). A similar antigen can be detected on rat tumours by immunofluorescence with xenoantiserum against rat brain. This antigen i s not the theta antigen (129). Other TAA could be produced by normal tissue in response to neoplastic disease. An example of such a substance, although not tumour specific, i s alpha 1-antitrypsin. High levels can be detected in the serum of patients with obstructive lung disease and lung cancer (44). The carcinogens themselves do not constitute TAA (at least in experi-mental animal tumours) since the tumours can be transplanted over many generations without a loss of antigenicity. 4. Tumour-associated Foetal (embryonic) Antigens Some TAA can also be found in normal foetal tissue. A suggested name for them i s 'onco-foetal antigens' (OFA) (1). 0FA include carcino-embryonic antigen (CEA), originally thought to be specifically associated with human colon carcinoma, alpha-foetoprotein (AFP), associated with rat and human hepatomas, and foetal alkaline phosphotase (Regan iso-enzyme). These TAA could be produced by the tumour ce l l s as a result of derepression associated with oncogenesis or by normal tissues in response to neoplastic growth or tissue damage. Although originally thought to be highly specific for tumours of the digestive tract and hepatomas (1), CEA and AFP respect-ively, were found widely associated with non-malignant diseases when highly sensitive radioimmunoassays were developed. While no longer thought to be useful for the mass-screening of cancer, ascertaining the level of these foetal antigens can be useful c l i n i c a l l y by aiding prognostication (82,143). Other OFA have been found in experimental animals with tumours. These tumours also possess TSTA which are distinct from the OFA in both 12 rats ( 8 ) and guinea pigs (17). OFA can be associated with lymphocyte mediated cytotoxicity (104) and can stimulate cell-mediated immunity in cancer patients as detected by delayed cutaneous hypersensitivity (52). Thus i t appears that OFA may function as TSTA as well as serving as aiding the clinician as an index of neoplastic disease. 5. Vi r a l Antigens Although i t is not yet proven whether the viruses associated with human cancers are oncogenic or opportunistic, there is considerable evidence to suggest that viruses can play a causative role in cancer. Much of this evidence is based on immunological studies on the viral-associated antigens and antibodies found associated with certain tumours. Whether viruses are a primary or secondary oncogenic agent, or merely a neoplastic tissue-specific passenger, the virus-associated antigens and antibodies are important to study like any other TAA. For this reason, I w i l l summarize the types of antigens associated with the viruses which are most l i k e l y oncogenic in humans. Of the DNA viruses, Herpesviruses are most lik e l y to be oncogenic in man. The Epstein-Barr virus (EBV) is closely associated with Burkitt's lymphoma and nasopharyngeal carcinoma (80). The virus i s very common in humans and many people develop antibodies against the v i r a l capsid antigen. However, patients with Burkitt's lymphoma have 5 to 10 times as much antibody as do matched normal controls. Early antigens are another EBV-related antigen complex and are so-called because they appear early in the v i r a l replication cycle when cultured cells are infected with EBV. Antibodies against early antigen are rarely detected in healthy people but reach high levels in patients with Burkitt's lymphoma. If, after chemotherapy the 13 antibodies disappear or decline, the prognosis i s generally good; i f the antibody concentration does not decline, the prognosis i s generally poor. These antibody populations may be present even though no EBV particles can be detected. The antigens against which these antigens are directed must either be or have been present in the patient, and are probably coded by the v i r a l DNA which can also be detected (80). Burkitt's lymphoma cells also produce an EBV associated nuclear antigen that may be analogous to the T-antigens produced in cells infected with oncogenic animal viruses such as SV40. T-antigen, an early v i r a l gene product, is present in the nucleus of a trans-formed c e l l but not in the virus i t s e l f (2). Burkitt's lymphoma patients can also produce antibodies against this nuclear antigen. A c e l l membrane antigen can be detected in biopsies from these patients as well as specific antibody against this non-virion antigen (49). Other herpesviruses also are possibly oncogenic. Herpes simplex (hominis) viruses types 1 and 2, cause most oral and cutaneous herpetic infections, and genital infections, respectively. Patients with carcinoma of the uterine cervix frequently have in their serum, antibodies which react with early antigen from type 2 infected cultured c e l l s . There have also been reports that patients with l i p and cervical carcinomas have antibodies capable of reacting with herpesvirus non-virion antigens (112) 'and that the tumours contained these antigens (54). These findings could provide strong evidence for oncogenesis by herpes simplex viruses, but the results were later not confirmed (111). IV. TSTA and Tumour Rejection If many tumours possess TSTA and the host organisms have immunity against these antigens (45), why are the tumours not rejected? Burnet's 14 immune s u r v e i l l a n c e t h e o r y (20) s u g g e s t s t h a t most m a l i g n a n t c e l l s a r e r e -j e c t e d . Tumours t h a t do a r i s e have escaped t h e immune s u r v e i l l a n c e by a d e f e c t i n t h e immune r e s p o n s e o f t h e h o s t t o t h e m a l i g n a n t c e l l s . On t h e b a s i s o f t h e f i n d i n g t h a t tumours w h i c h a r i s e i n immunosuppressed mice a r e no more a n t i g e n i c t h a n tumours a r i s i n g i n n o r m a l m i c e , t h e immune s u r v e i l l a n c e t h e o r y has been c h a l l e n g e d , a l t h o u g h t h e i m p l i c a t i o n s a r e n o t f u l l y u n d e r s t o o d ( 8 8 ) . There c o u l d be a number o f r e a s o n s f o r a d e f e c t i v e immune r e s p o n s e w h i c h a l l o w s m a l i g n a n t c e l l s t o escape immune s u r v e i l l a n c e and tumours t o p r o g r e s s and m e t a s t a s i z e . 1. The TSTA p r o d u c e d c o u l d be weak, o r a t a low c o n c e n t r a t i o n on t h e tumour c e l l membrane (1 0 9 ) . T h i s c o u l d i m p a i r i m m u n i z a t i o n and/or k i l l i n g by s e n s i t i z e d l y m p h o c y t e s o r c y t o t o x i c a n t i b o d y . I t i s a l s o p o s s i b l e t h a t t h e r e i s o n l y a s m a l l number o f l y m p h o c y t e s c a p a b l e o f r e s p o n d i n g t o tumour a n t i g e n s . 2. The c a r c i n o g e n i n v o l v e d may be immunosuppressive. Some onco-g e n i c v i r u s e s have been shown t o d e p r e s s c e l l - m e d i a t e d immunity (90) and many c h e m i c a l c a r c i n o g e n s a r e a l s o immunosuppressive ( 5 ) . 3. The n e o p l a s t i c c e l l s c o u l d p r o d u c e an immunosuppressive f a c t o r (19). 4. The immune s y s t e m c o u l d be t e m p o r a r i l y i n c a p a c i t a t e d by a cause u n r e l a t e d t o the m a l i g n a n c y . Thus, i n r e n a l t r a n s p l a n t r e c i p i e n t s , because o f t h e r e q u i r e d i mmunosuppression, c a n c e r c o u l d be an i a t r o g e n i c ( r e l a t e d t o t r e a t m e n t ) d i s e a s e ( 9 7 ) . 5. The i n d i v i d u a l may have a g e n e t i c i n c a p a c i t y t o r e s p o n d immuno-l o g i c a l l y t o c e r t a i n groups o r t y p e s o f TSTA. 15 6. Neonatal tolerance to TSTA can be developed. Tolerance has been demonstrated in the case of several tumours induced by verti c a l l y transmitted viruses, such as lymphomas induced by the Gross virus, and leukaemias induced by the Moloney virus (62) . By transplanting the tumours into adult animals of the same inbred strain, the tumours were shown to possess TSTA and were rejected, while animals infected neonatally with the viruses were incapable of reacting against them. 7. A simple explanation for the escape from immune defences i s that the immune system cannot cope with the rapid growth of the tumour;; Closely associated with rapid growth i s the tumour load. As the tumours get larger, anti-tumour immunity and even general immunity decreases (139, 45). This could be related to any of the factors already mentioned. V. TSTA and the Roles of Antibody in Tumour Rejection. 1. Immunologic Enhancement Immunological enhancement of tumour development has been a subject of much study and controversy. This phenomenon was originally observed as enhanced tumour growth in animals which had been either actively immunized with the same tumour or passively immunized with hetero or isoantiserum. There were two main mechanisms suggested to explain enhancement. The f i r s t suggestion was that contact with antibody stimulated tumour growth either directly or by release of stimulatory c e l l products. The other suggestion embraces the 'blocking' phenomenon. Antibody binding to the tumour c e l l membrane would 'wall o f f the tumour antigens so they would be inaccess-ible to sensitized lymphoid cells capable of tumour c e l l k i l l i n g (60), 16 The Hellstrb'ms (46) demonstrated immunological enhancement in vitro using the colony inhibition assay. Briefly, the method involves the plating of target tumour cells and counting the tumour c e l l colonies which arise in the presence and absence of specifically sensitized lymphoid cel l s . Incu-bation of the target cells with lymphocytes from animals or patients bearing even progressively growing tumours of the same types as the target cells, w i l l frequently result i n an inhibition of colony formation. Lymphocytes from animals which have not had a tumour or from animals with a different type of tumour, do not have this effect, thus demonstrating immunologic specificity. If serum from the tumour-bearing donor i s added to the target cells, the cytostatic effect of the sensitized lymphocytes may be abrogated. The factor in the serum which results in the inhibition of colony formation i s called 'blocking factor' and is specific for the particular tumour-lympho-cyte system being used. The cytostatic a b i l i t y of the lymphocytes was similar for tumour-bearing animals and for animals whose tumours were resected. Serum from animals which had become tumour-free, was frequently capable of inhibiting colony formation of the respective target cells i n the presence of complement.,. This indicates that blocking factor may not be free antibody. Indeed, the relatively simple hypothesis that tumour-specific antibodies on their own may cause enhancement of tumour growth by shielding tumour cells from destruction by sensitized lymphocytes i s no longer tenable in the light of recent experiments. Blocking serum has been shown to exert i t s effect either upon the target cells or the sensitized lymphocytes. Blocking activity can also be absorbed out of serum by either target cells or lymphocytes and may be eluted from tumour cells at low pH. Blocking factor has been characterized to the extent that i t i s known to be a high molecular weight moiety. By u l t r a f i l t r a t i o n at pH 3.1, blocking factor can be dissociated into a large 17 (MW > 100,000) component and a small (MW < 100,000) component (121). When mixed with target c e l l s , neither component alone can block lymphocyte mediated cytostatic effects, but significant blocking can be seen when the two components are mixed. When mixed with sensitized lymphocytes, the larger component, which i s known to contain antibody, cannot block, but the smaller component alone or the mixture can block. If blocking factor were antigen-antibody complexes, i t would also explain the disappearance of blocking factor upon tumour removal and the appearance of complement dependent cytotoxic antibody (121). When rat hepatoma antigens and specific antibodies are mixed with excess antibody and incubated with target c e l l s , lymphocyte cytostatic effects can be blocked. If the mixture contains antigen excess or i f tumour antigen alone i s added to the blocking serum, the blocking effect i s not obtained (10). If antigen i s pre-incubated with the lymphocytes however, i t can block by i t s e l f (11). Soluble tumour antigen, either free or in immune complexes in antigen excess, have been detected in the serum of sarcoma bearing rats. Antigen becomes undetectable 48 hours after complete tumour excision, which corresponds to the time of disappearance of blocking factor and the appearance of cytotoxic antibody (127). This evidence implies that the primary effect of blocking factor i s on the effector lymphocytes. With hyperimmune anti-tumour serum, i t appears possible to produce efferent blocking, but the central blocking effect, which acts upon the lymphocytes, is probably more important (45). In order to differentiate between tumour enhancement mediated by antibody and that mediated by complexes or antigen, the terms 'blocking' and 'inhibition' have been used respectively (5). It has been suggested (31) that antibody-mediated and antigen-mediated immune suppression may act by a similar mechanism. The mechanism has been postulated to be a concentration or cross-linking of antigen on 18 the lymphocyte plasma membrane. Thus a large dose of polymerized f l a g e l l i n from Salmonella adelaide or small doses of monomeric f l a g e l l i n plus specific antibody can produce immunological unresponsiveness (31). By analogy, tumour-specific antibody could cross-link the antigens on lymphocyte surface receptors and render them non-functional. It i s postulated from this that the antibody part of blocking antigen-antibody complexes acts by carrying the antigen to the lymphocyte and cross-linking the antigen to the surface receptors (45,110). This explanation of immunologic enhancement of tumour growth is similar to an explanation of allograft tolerance in rats and mice rendered neonatally tolerant to allogeneic lymphoid ce l l s CtLO) • Lymphoid cells from the graft' recipient were found to be cytotoxic to the tolerated graft in"vitro. Serum from the tolerant animal abrogated the cytotoxicity. Similar findings were obtained from studies of tetraparental mice, which had been established as chimeras by joining eight c e l l stage embryos from different strains (98). Spleen cells from these mice were cytotoxic to cultured fibroblasts from either parent strain, and serum from these mice would block the mixed leukocyte reaction between the parental strains. The blocking factor could be removed from the serum by absorption with anti-mouse immunoglobulin. It would thus appear that immunologic enhancement of tumour growth i s probably a complex interaction between sensitized lymphoid cells and circulating antibody-antigen complexes which possess the a b i l i t y to 'paralyze' those lymphoid cells which carry the potential to destroy the tumour ce l l s . 2. Manipulation of the Immune Responses by Modified Antigen. I have already described (Section 111:1) findings that immunization with syngeneic, chemically modified tumour ce l l s , can induce tumour re-19 j e c t i o n . I t i s t h o u g h t t h a t m o d i f i e d a n t i g e n s s t i m u l a t e c e l l - m e d i a t e d immunity b u t n o t humoral immunity a g a i n s t t h e tumour a n t i g e n s , t h u s d i m i n i s h -i n g t h e p o s s i b i l i t y o f tumour enhancement G D I ) • There a r e two p l a u s i b l e models f o r t h i s m a n i p u l a t i o n o f t h e immune r e s p o n s e . A. By c o n j u g a t i n g f a t t y a c i d s t o p r o t e i n s , D a i l e y and H u n t e r (28) have p r e s e n t e d e v i d e n c e t h a t t h e p r o p e r t i e s o f an a n t i g e n w h i c h i n d u c e c e l l - m e d i a t e d immunity a r e n o t i n t h e n a t u r e o f t h e a n t i g e n i c d e t e r m i n a n t s b u t i n t h e l i p o p h i l i c . n a t u r e o f t h e whole immunogen. T h i s mechanism i s a p p e a l i n g because i t e x p l a i n s t h e predominance o f t h e c e l l m e d i a t e d immune-r e s p o n s e a g a i n s t c e l l membrane a n t i g e n s . I t i s p o s s i b l e t h a t t h e c h e m i c a l m o d i f i c a t i o n ( e s p e c i a l l y o f s u l f h y d r y l g roups) r e s u l t s i n a g r o s s c o n f i g u r -a t i o n change so t h a t i n t e r n a l h y d r o p h o b i c r e g i o n s o f t h e p r o t e i n m o l e c u l e s a r e exposed. The r e a s o n why r e a g e n t s s p e c i f i c f o r s u l f h y d r y l groups appear t o work b e t t e r t h a n o t h e r r e a g e n t s o r m o d i f i c a t i o n s s u c h as h e a t d e n a t u r a t i o n c o u l d be due t o d e n a t u r a t i o n o f t h e a n t i g e n i c d e t e r m i n a n t s . I n summary, t h i s model s u g g e s t s a p r e f e r e n t i a l s t i m u l a t i o n o f c e l l m e d i a t e d immunity by m o d i f i c a t i o n o f p a r t s o f t h e a n t i g e n o t h e r t h a n t h e a n t i g e n i c d e t e r m i n a n t . B. The o t h e r model s u g g e s t s t h a t t h e a n t i g e n i c d e t e r m i n a n t s a r e m o d i f i e d so t h a t t h e immune r e s p o n s e s a r e d i r e c t e d a g a i n s t t h e s e a l t e r e d d e t e r m i n a n t s . R e c o g n i t i o n i s d i f f e r e n t f o r c e l l - m e d i a t e d and humoral immunity, c e l l m e d i a t e d immunity h a v i n g a h i g h e r degree o f c r o s s r e a c t i v i t y GDI). I m m u n i z a t i o n w i t h c h e m i c a l l y m o d i f i e d a n t i g e n can i n d u c e a n t i b o d i e s a g a i n s t t h e m o d i f i e d a n t i g e n i c d e t e r m i n a n t s and w h i c h do n o t c r o s s r e a c t w i t h n a t i v e d e t e r m i n a n t s , w h i l e c e l l - m e d i a t e d r e s p o n s e s do c r o s s - r e a c t (H3)• 20 T h i s e v i d e n c e s t r o n g l y s u p p o r t s t h e l a t t e r model, b u t t h e models a r e n o t m u t u a l l y e x c l u s i v e . 3. Tumour R e j e c t i o n Augmented by A n t i b o d y A l l i s n o t as s i m p l e as i t may seem: t h e hum o r a l r e s p o n s e i s n o t n e c e s s a r i l y h a r m f u l i n terms of tumour immunity t o neoplasms. S e r a f r o m some mi c e and humans w i t h spontaneous tumour r e m i s s i o n s o r s u c c e s s f u l tumour r e m o v a l , have been shown n o t o n l y t o l a c k b l o c k i n g f a c t o r s , b u t t o a l s o c o n t a i n f a c t o r s c a p a b l e o f s p e c i f i c a l l y ' u n b l o c k i n g ' o r a b r o g a t i n g t h e b l o c k i n g e f f e c t o f b l o c k i n g serum (12). These u n b l o c k i n g a n t i b o d i e s can a l s o be p r o d u c e d by i m m u n i z a t i o n o f M y c o b a c t e r i u m b o v i s ( s t r a i n BCG) p r i m e d a n i m a l s (even a n o t h e r s p e c i e s ) w i t h tumour c e l l s . A p o s s i b l e e x p l a n a t i o n f o r u n b l o c k i n g i s t h a t t h e a n t i b o d i e s a r e d i r e c t e d a g a i n s t b o t h a n t i b o d y and a n t i g e n components o f t h e b l o c k i n g complexes. I t has a l s o been shown t h a t s e r a f r o m some a n i m a l s and p a t i e n t s i n r e m i s s i o n a r e c a p a b l e o f i n c r e a s i n g t h e c y t o t o x i c e f f e c t o f l y m p h o i d c e l l s (45). A p o s s i b l e mechan-i s m i s t h e 'arming', by a n t i b o d y , o f n o n - s e n s i t i z e d c e l l s . C e r t a i n immune s e r a a r e c a p a b l e o f ar m i n g n o n - s e n s i t i z e d l y m p h o i d c e l l s so t h a t t h e y can s p e c i f i c a l l y k i l l s y n g e n e i c tumour c e l l s . The k i l l i n g i s m e d i a t e d by B l y m p h o c y t e s c a l l e d 'K' ( k i l l e r ) l y m p h o c y t e s . A n o t h e r p o s s i b l e b e n e f i t from a humoral immune r e s p o n s e a g a i n s t tumours i s complement dependent c y t o t o x i c a n t i b o d y (45). T h i s o f t e n a p p e a r s a f t e r tumour r e m o v a l and i s p o s s i b l y b l o c k i n g complexes minus tumour a n t i g e n . I t may f a c i l i t a t e e l i m i n a t i o n o f r e s i d u a l tumour c e l l s by k i l l i n g d i r e c t l y o r by augmenting t h e c e l l - m e d i a t e d r e s p o n s e as i n b a c t e r i a l immunity. We can now see t h a t tumour development may be r e l a t e d t o immuno-21 s u p p r e s s i o n o r n o n - r e s p o n s i v e n e s s and/or a r e l a t i o n s h i p between humoral and c e l l - m e d i a t e d immunity. These f a c t o r s w i l l be d e t e r m i n e d by p r o p e r t i e s o f t h e h o s t , s u c h as i t s g e n e t i c and a c q u i r e d c a p a c i t y t o r e s p o n d t o c e r t a i n a n t i g e n s , hormone l e v e l s and any number o f p r o p e r t i e s t h a t may be a s s o c i a t e d w i t h age: and p r o p e r t i e s o f t h e n e o p l a s t i c c e l l s , s u c h as l o c a t i o n , r a t e o f g rowth, i n v a s i v e p o t e n t i a l and q u a l i t y and q u a n t i t y o f tumour a n t i g e n s -Tumour a n t i g e n s , t h e n , a r e a l i k e l y s p o t upon w h i c h t o f o c u s a t t e n t i o n i n t h e s t u d y o f i n t e r a c t i o n s o f h o s t and tumour. V I . E x p e r i m e n t a l Approaches t o t h e I d e n t i f i c a t i o n o f TSTA and TAA The i d e n t i f i c a t i o n o f TSTA and TAA i n a n i m a l models has been a c h i e v e d by a number o f e x p e r i m e n t a l a p p roaches and t e c h n i q u e s . The m a j o r t e c h n i q u e s , and s e v e r a l o f t h e i r v a r i a t i o n s w i l l be r e v i e w e d h e r e . 1. D e t e c t i o n o f C e l l - M e d i a t e d Immunity t o Tumour C e l l s . The f o l l o w i n g p r o c e d u r e s d e t e c t c e l l - m e d i a t e d immunity i n the tumour b e a r e r t o tumour c e l l s ( o r e x t r a c t s ) and t h e r e f o r e p r o b a b l y d e t e c t t h e p r e s e n c e o f TSTA. A. A n i m a l s o f an i n b r e d s t r a i n can be p r o t e c t e d f r o m tumour development upon c h a l l e n g e w i t h tumour c e l l s i f t h e y have p r e v i o u s l y been immunized w i t h tumour c e l l s , o r c e r t a i n tumour e x t r a c t s . A l s o , a n i m a l s f r o m whom s m a l l tumours have been removed o r l i g a t e d a r e f r e q u e n t l y immune t o subsequent c h a l l e n g e w i t h a t u m o u r i g e n i c dose o f tumour c e l l s . B. D e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s t o tumour a n t i g e n s can be 22 measured by skin test or foot pad swelling, with tumour extract in tumour-immune or tumour-bearing animals. C. The reactivity of tumour immune lymphoid cells with TSTA can be assayed by several methods: a. Adoptive transfer, or incubation of sensitized lymphocytes with tumour cells before inoculation into susceptible animals can protect the recipient from tumour development. b. The blastogenic response can be measured when sensitized lymphoid cells are cultured with mitomycin-treated tumour cells or tumour extracts. c. The supernatant from this culture (from part b) can be tested for immune lymphocyte mediators such as migration inhibitory factor (MIF) by incubation with guinea pig spleen cells for an indicator system. d. The sensitized cells can be directly tested for the production of mediators such as MIF when incubated with tumour extract. e. Sensitized lymphoid cells can be cytotoxic for target tumour cells when they are cultured together. This method i s the basis of the colony inhibition and micro-cytotoxicity techniques. 2. Detection of Antibodies Against TSTA or TAA. Antigens detected by antibody mediated immunity would not necessarily 23 be expected to be TSTA since tumour rejection i s probably mediated mainly by cellular immunity. However, i t may be possible to detect either TAA or TSTA by these techniques. A. An Arthus reaction developing at the site of skin testing with tumour extract would detect antigens by complement dependent antibody response. B. Tumour-bearer serum may be tested for i t s a b i l i t y to block lymphocyte-mediated tumour c e l l cytotoxicity at the lymphocyte level. C. Tumour-bearer or regressor serum, or serum from syngeneic animals immunized with tumour ce l l s , or allogeneic or xenogeneic antisera may be tested for antibody against TAA by several methods. The latter antisera would need to have antibodies against normal tissue antigens removed. a. Complement dependent cytotoxicity of tumour cells. b. Fixation of complement upon incubation with tumour antigen. c. Immune precipitate formation. d. Immunofluorescence. e. Autoradiography or radioimmunoassay. 3. Detection of TSTA and TAA in Human Systems. In the study of human tumour antigens, many of these methods are not 24 possible for ethical reasons and some are not practical for other reasons. It i s often d i f f i c u l t to get and store large numbers of human tumour cells in single c e l l suspensions and retain their s t e r i l i t y and v i a b i l i t y as required for cytotoxicity reactions. Tumour cells must be continued by long-term jLn vitro culture, which for many tumours is very d i f f i c u l t i f not impossible. In experimental animals, the tumour c e l l lines can be carried by serial transplants in syngeneic animals. In human c e l l lines, where only continuous i n vitro cultures i s possible, the probability that anti-genic changes and selective pressures w i l l change the characteristics of the c e l l line, makes this approach questionable. With human experiment-ation i t i s also very often d i f f i c u l t to provide a good control for many methods. Other problems in studying TAA are their apparently low concentrations, relatively weak antigenicity and the large quantities of contaminating normal c e l l antigens (58). Also, i t i s not known and d i f f i c u l t to ascertain, whether antigen changes by neoplastic cells are qualitative or quantitative. 4. The Importance of Detecting Human TSTA and TAA. There are many possible uses for TAA which make their study and purification worthwhile, in spite of the problems involved. Although some of the uses of TAA described here (F, G.) are best applied in the study of experimental animal tumours, the results of these studies must be evaluated for correlation with human malignant disease before the cancer patient w i l l benefit. A. With purified TAA, a high t i t r e of specific anti TAA would be easy to obtain in experimental animals. 25 B. Assays for TAA could serve as a diagnostic and prognostic aid to c l i n i c a l medicine. Trace amounts of anti TAA could be detected by radio-immunoassay. Such an assay could also be used to screen large numbers of people for the presence of early malignant disease. C. TSTA could be used for specific immunotherapy, or even in the prevention of neoplastic disease by immunization. These antigens would possibly have to be modified to increase antigenicity or to preferentially stimulate cell-mediated immunity. Perhaps other TAA could be made immuno-genic in the cancer patient, and function as TSTA. Methods which could augment the immunogenicity of TAA might also make normal tissue components immunogenic, so purified TAA should be used to eliminate the possibility of inducing autoimmunity. D. TSTA could be used to monitor the effects of immunotherapy on the patients' immune responses. E. The presence of-common or related TAA could provide information about the etiology of malignant disease. F. The relationship between antigen and antibody in blocking factor could be studied, as could the relative importance of blocking factor to cell-mediated defenses against cancer. G. Information about the biochemistry of TAA could supply a key to understanding oncogenesis and the basis for the weak antigenicity. 26 5. The Uses of Specific Antibodies Against TAA. A. Anti TAA could be used in a radioimmunoassay to detect trace amounts of TAA in serum for diagnostic and prognostic purposes. Such methods are already i n use for the detection of alpha-feotoprotein and carcino-embryonic antigen although the tumour specificity i s not as good as i t was earlier hoped (82). B. The antibodies might be used for passive immunotherapy either directly i f they f i x complement and are cytotoxic, or by 'arming' them with l 31 cytotoxic drugs or I. A better understanding of tumour antigen catabolism and tumour enhancement in humans could greatly f a c i l i t a t e such studies. C. If the antibodies were made radiopaque, they could aid radio- . logical methods in locating and diagnosing a tumour. VII. Objectives of the Present Study The original goal of this research was to attempt to purify TSTA from different bronchial carcinomas, and to study the immunochemical relationships of these antigens. Although these studies would be much easier to perform in experimental animals, the possible c l i n i c a l value from studies of TSTA or TAA associated with human malignant disease j u s t i f i e s the concomitant experimental complexity. Studies on syngeneic animal tumours would not give information about naturally occurring immunochemical relationships between tumour antigens. Also, results of experimental animal tumour studies must be evaluated by human experimentation before the c l i n i c a l significance can be established, and so far, animal experimentation has contributed l i t t l e to 27 human immunotherapy. There a r e a number of r e a s o n s f o r s t u d y i n g l u n g c a n c e r . Lung c a n c e r i s a v e r y common tumour, has been i n c r e a s i n g r a p i d l y , and has a v e r y h i g h m o r t a l i t y . A t l e a s t i n p a r t , t h e poor p r o g n o s i s o f t h e s e p a t i e n t s i s r e l a t e d t o t h e l a t e d i a g n o s i s , a t w h i c h t i m e t h e tumours a r e o f t e n l a r g e and m e t a s t a s i z e d . These f a c t o r s a r e s u i t a b l e f o r tumour a n t i g e n s t u d i e s s i n c e l a r g e tumours wo u l d mean more t i s s u e t o use i n t h e s t u d i e s and one o f th e more p r o m i s i n g a s p e c t s o f tumour immunology i s e a r l y d i a g n o s i s by radioimmunoassay f o r common TAA. The l a t t e r p o i n t i s e s p e c i a l l y i m p o r t a n t s i n c e t h e r e a r e a l r e a d y h i g h r i s k i n d i v i d u a l s , and i n heavy c i g a r e t t e smokers the d i s e a s e i s more d i f f i c u l t t o d e t e c t . A c t i v e immunotherapy i s p r o b a b l y more s u i t a b l e f o r m a l i g n a n t d i s e a s e s i n w h i c h the major method o f t h e r a p y i s s u r g e r y (such as l u n g c a n c e r ) r a t h e r t h a n i r r a d i a t i o n o r chemo-t h e r a p y w h i c h a r e immunosuppressive. Because t h e d i s e a s e i s so common and the m o r t a l i t y so h i g h , even a s l i g h t improvement by immunotherapy would be s i g n i f i c a n t and d e t e c t a b l e . V I I I . E x p e r i m e n t a l Approaches t o the P r e s e n t Study 1. E x t r a c t i o n o f t h e Tumour C e l l s Because o f t h e e x p e c t e d s i m i l a r i t i e s between TSTA and a l l o a n t i g e n s , a p r o c e d u r e known t o be s u c c e s s f u l i n t h e e x t r a c t i o n o f a l l o a n t i g e n s was used t h r o u g h o u t t h e p r e s e n t s t u d y . Three M KC1 e x t r a c t i o n i s r e l a t i v e l y e a s y t o p e r f o r m , can h a n d l e l a r g e q u a n t i t i e s o f t i s s u e , and can produce a good y i e l d o f a l l o a n t i g e n s f r o m c u l t u r e d c e l l s ( 4 6 ) . L e s s l i p o p r o t e i n , w h i c h g r e a t l y i n t e r f e r e s w i t h p r o t e i n p u r i f i c a t i o n ' s l i b e r a t e d t h a n by many o t h e r e x t r a c t i o n methods. I t t a k e s s e v e r a l days t o co m p l e t e t h e e x t r a c t i o n and 28 causes considerable destruction of internal cytoarchitecture of the c e l l with subsequent release of large amounts of nuclear DNA, nucleoprotein, and protein. However, these materials do not greatly interfere with alloantigen purification (106). 2. Detection of TAA A. Our f i r s t attempt to show the presence of TAA in our tumour extracts was by producing an antiserum against the TAA. The method of Gold and Freedman (38) for the demonstration of CEA was chosen. A state of immunological tolerance to the antigens i n an extract of normal human lungs, was to be induced in neonatal rabbits so that subsequent immunization with lung tumour extract would induce antibodies only against TAA. Autochthonous normal and tumour tissue extracts could have been used, but i f TAA were released from the tumour (like CEA), we could also induce tolerance against these TAA. A pool of extracts from the lungs of six tumour free individuals (at autopsy) was used to minimize any chance of a reaction against histo-compatibility antigens. The antisera were tested by double diffusion in gel against the tumour and normal extracts. B. Another test for TAA was an in vitro assay of cell-mediated immunity of lung cancer patients. Peripheral blood leukocytes from cancer patients and healthy donors were tested for MIF production upon incubation with normal lung and tumour extracts. Antigens detected by this method would possibly be TSTA. Unfortunately, during the time that these experi-ments were being performed, we did riot receive any surgical tumour specimens so that autochthonous leukocytes and tumour extract could not be tested. 29 C. A d i r e c t , non-immunological test for tumour associated materials was c a r r i e d out by disc gel electrophoresis. Normal lung and tumour extracts were compared i n t h i s manner. To lower the background protein concentrations and further test f o r tumour s p e c i f i c i t y , normal lung and tumour extracts were fractionated by DEAE c e l l u l o s e chromatography followed by Sephadex chromatography and the f r a c t i o n s were analyzed and compared f o r the presence of tumour associated bands on d i s c gel e l e c t r o -phoresis. D. Instead of eliminating antibodies against normal t i s s u e components by the induction of tolerance, t h e i r removal from the serum of rabbits hyperimmunized with tumour extract was t r i e d . Immunoadsorbents were prepared by i n s o l u b i l i z a t i o n of the normal lung extracts by the co-valent coupling to agarose beads and by the polymerization with g l u t a r -aldehyde. The absorbed an t i s e r a were tested for anti-TAA antibodies to double d i f f u s i o n i n agar gel and Immunoelectrophoresis against normal lung and tumour extracts. Some were l a t e r tested by i n d i r e c t immunofluorescence and immunoautoradiography on tiss u e sections. The absorbed antisera were used to test a wide v a r i e t y of normal and tumour extracts f o r TAA by immuno-d i f f u s i o n . These a n t i s e r a were also used to test patients' serum samples for TAA by immunodiffusion. E. Several cancer patients' sera were tested f o r the presence of tumour antibody and/or tumour antigen-antibody complexes. Sera were tested against tumour extracts d i r e c t l y by immunodiffusion, f o r tumour antibody. The pH of serum samples was lowered to dis s o c i a t e d immune complexes, and the serum chromatographed on an a c i d i f i e d Sephadex G-150 column to separate high molecular weight components (antibody?) from low molecular weight components (antigen?). These components were tested against each other and the high molecular weight components were also tested against several tumour extracts, by immunodiffusion. F. The TAA detected i n the extracts with the absorbed antisera are probably not TSTA because the immunodiffusion technique i s not s e n s i -t i v e enough to detect the expected quantities of TSTA. The p o s s i b i l i t y that the TAA detected i s f o e t a l or embryonic antigen was tested two ways. a. Samples of the extracts were assayed f o r CEA by rad i o -immunoassay by the D i v i s i o n of Nuclear Medicine at the Vancouver General Hospital (VGH). Several extracts were also tested f o r cti-foetoprotein by immunodiffusion against anti-Cii-foetoprotein donated by Dr. S. 0. Freedman. b. Extracts of f o e t a l lungs were tested by immunodiffusion against the absorbed antisera and compared with tumour extracts by dis c gel electrophoresis. G. Since the immunodiffusion technique was being used near i t s l i m i t of s e n s i t i v i t y , attempts were made to e s t a b l i s h a more s e n s i t i v e assay method. Indirect immunofluorescence techniques were t r i e d f i r s t because of t h e i r widely reported use i n the l i t e r a t u r e . An i n d i r e c t immunoautoradiography technique was vigorously pursued because of the p o t e n t i a l s e n s i t i v i t y of the technique and because i t allowed accurate quantitation. I t was also hoped to give an i n d i c a t i o n of the l o c a t i o n of antigens within the tiss u e and c e l l s by using ti s s u e sections. Tissue sections were used instead of c e l l suspensions.for several reasons: 31 s i n g l e c e l l s u s p e n s i o n s o f n o r m a l l u n g and tumour t i s s u e a r e v e r y d i f f i c u l t t o - p r o d u c e , s t o r e and m a i n t a i n r e p r o d u c i b i l i t y , whereas p i e c e s o f t i s s u e can e a s i l y be s t o r e d f r o z e n and t h e n made i n t o c r y o s t a t microtome s e c t i o n s . These methods were used t o t e s t t h e a b s o r b e d a n t i s e r a , and c a n c e r p a t i e n t s e r a and a c i d - d i s s o c i a t e d h i g h m o l e c u l a r w e i g h t f r a c t i o n s , f o r a n t i b o d y b i n d i n g t o t h e t i s s u e s e c t i o n s . H. A n o t h e r q u e s t i o n r a i s e d about t h e TAA d e t e c t e d i n t h e e x t r a c t s by i m m u n o d i f f u s i o n , p e r t a i n s t o t h e i r p o s s i b l e a n t i g e n i c i t y i n t h e c a n c e r p a t i e n t s . The e a s i e s t and most d i r e c t way t o t e s t t h i s (as w e l l as b e i n g a n o t h e r p o s s i b l e a s s a y f o r TAA) i s s k i n t e s t i n g o f c a n c e r p a t i e n t s w i t h e x t r a c t s shown t o c o n t a i n TAA by t h e i m m u n o d i f f u s i o n t e c h n i q u e s . Because o f t h e p o s s i b i l i t y o f t r a n s m i t t i n g an i n f e c t i v e agent t o o t h e r p a t i e n t s o r h e a l t h y p e r s o n s , o n l y c a n c e r p a t i e n t s were s k i n t e s t e d , and o n l y w i t h e x t r a c t s o f t h e i r own tumours. T h i s r e s t r i c t i o n makes i t i m p o s s i b l e t o c o m p l e t e l y c o n t r o l t h i s e x p e r i m e n t . However, t h e c o n t r o l s i n c l u d e d s k i n t e s t i n g w i t h r e c a l l a n t i g e n s and DEAE c e l l u l o s e f r a c t i o n s o f t h e tumour e x t r a c t s . 32 MATERIALS AND METHODS I . T i s s u e P r e p a r a t i o n and E x t r a c t i o n 1. P r e p a r a t i o n o f Tumour T i s s u e The tumours used i n t h i s s t u d y were e i t h e r s u r g i c a l o r post-mortem specimens. S u r g i c a l specimens were made a v a i l a b l e t h r o u g h t h e a r r a n g e m e n t s o f D r s . P. Coy and J . W. Thomas (B.C.C.I.)'. Dr. Coy a l s o s u p p l i e d the s u r g i c a l p a t h o l o g y r e p o r t s . Post-mortem specimens and a u t o p s y r e p o r t s were s u p p l i e d by Dr. J . B u r t o n (V.G.H.). A l l o f t h e t i s s u e s were s t o r e d a t 4°C f o r l e s s t h a n 5 h o u r s b e f o r e b e i n g minced and e x t r a c t e d . M a c r o s c o p i c a l l y n o r m a l t i s s u e was trimmed f r o m t h e tumours w h i c h were t h e n c u t i n t o s m a l l p i e c e s (1-2 cm d i a m e t e r ) , and m inced w i t h a t i s s u e g r i n d e r a t 4°C. These m a n i p u l a t i o n s were c a r r i e d out i n a s t e r i l e box e q u i p p e d w i t h a g e r m i c i d a l u l t r a v i o l e t l i g h t , as much as p o s s i b l e . 2. Normal Lung T i s s u e Two t y p e s o f n o r m a l l u n g t i s s u e were used. I n i t i a l l y , m a c r o s c o p i c a l l y t u m o u r - f r e e p i e c e s o f n o r m a l l u n g were removed from t h e l u n g s o f c a n c e r p a t i e n t s , as d i s t a l l y as p o s s i b l e from t h e tumour. Most n o r m a l l u n g t i s s u e was t a k e n a t p o s t mortems o f i n d i v i d u a l s f r e e o f m a l i g n a n t d i s e a s e . No a t t e m p t was made t o wash t h e t i s s u e f r e e o f b l o o d . 3. Embryonic Lung T i s s u e Lungs were removed f r o m embryos a t 12 t o 20 weeks o f g e s t a t i o n . 33 These specimens, f r o m s a l i n e - i n d u c e d a b o r t i o n s , were s u p p l i e d by Dr. B. P o l a n d (V.G.H.). F-48 i s a p o o l o f seven embryonic l u n g e x t r a c t s and F-49 i s a p o o l o f f o u r e x t r a c t s . 4. T i s s u e E x t r a c t i o n The t i s s u e s were e x t r a c t e d by a method based on an a l l o n t i g e n e x t r a c t -i o n method o f R e i s f e l d et^ a l _ ( 1 0 8 ) . The f i r s t specimens were minced i n p h y s i o l o g i c a l s a l i n e , t h e n s o l i d KC1 was added t o a f i n a l c o n c e n t r a t i o n o f 3.0 M. L a t e r specimens ( a f t e r C-30) were m i n c e d , and t h e n mixed w i t h a volume o f 3.5 M KC1 e q u a l t o 6 t i m e s t h a t o f t h e t i s s u e . T i s s u e s were m a i n t a i n e d a t 4°C t h r o u g h o u t t h e e x t r a c t i o n p r o c e d u r e . E x t r a c t i o n o f t h e t i s s u e was accom-p l i s h e d by g e n t l e a g i t a t i o n i n t h e 3.0 M K C l f o r 18 h r s . The e x t r a c t e d t i s s u e was t h e n c e n t r i f u g e d f o r 90 min a t 16,000 g and t h e s u p e r n a t a n t was s t r a i n e d t h r o u g h gauze. The s u p e r n a t a n t was d i a l y z e d e x h a u s t i v e l y a g a i n s t p h y s i o l o g i c a l s a l i n e and c e n t r i f u g e d a g a i n . The e x t r a c t was e i t h e r s t e r i l i z e d by f i l t r a t i o n t h r o u g h a S e i t z f i l t e r pad and s t o r e d a t 4°C, o r s t o r e d a t -20°C. The e x t r a c t i o n p r o c e d u r e was n o t p e r f o r m e d a s e p t i c a l l y . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by t h e s t a n d a r d Lowry method (74) and/or s p e c t r o p h o t o m e t r i c a l l y (67) ( A p pendix A ) . Because t h e r e s u l t s by t h e Lowry method were u s u a l l y w i t h 20% o f t h o s e by s p e c t r o p h o t o m e t r y methods ( f o r 20 samples w h i c h were compared), Lowry q u a n t i t a t i o n s were n o t r o u t i n e l y p e r f o r m e d . Tumour, n o r m a l and embryonic t i s s u e e x t r a c t s were l a b e l l e d w i t h t h e p r e f i x e s C-, N-, and F-, r e s p e c t i v e l y . The 3.0 M K C l e x t r a c t i o n method was chosen f o r a number o f r e a s o n s . The method has been s u c c e s s f u l l y a p p l i e d t o a l l o a n t i g e n e x t r a c t i o n , i s g e n t l e , r e p r o d u c i b l e , and can h a n d l e l a r g e volumes o f t i s s u e . The d i s a d v a n t a g e s a r e t h e l o n g t i m e r e q u i r e d and t h e r e q u i r e m e n t o f a v i a b l e s i n g l e c e l l s u s p e n s i o n 34 for a high yield of alloantigens from cultured ce l l s . Although much cytoplasmic protein i s released besides membrane protein, i t does not interfere with a l l o -antigen purification like lipoproteins. Since my study includes TAA besides TSTA, cytoplasmic protein extraction may even be an advantage. It i s thought that the chaotropic effects of KCl disrupt hydrogen bonding and break salt linkages (58). Other common methods of extracting antigens have more serious disadvant-ages. Tissue homogenization in saline does not extract alloantigens. Even methods used successfully for alloantigen extraction were f e l t to have draw-backs. Low intensity sonication requires very carefully controlled conditions, i s d i f f i c u l t to reproduce, and can only handle small volumes. Antigens extracted with detergents frequently aggregate and are only sparingly soluble in water. Papain can destroy alloantigens and leads to great va r i a b i l i t y (58). II. Neonatal Tolerlzation of Rabbits. The method of Gold and Freedman (38) was used to render rabbits tolerant to the normal tissue components. A pool of 6 different normal lung extracts (N pool), was used for the tolerization. These extracts were from malignant disease-free post mortem specimens. Although we could have used an autologous extract from normal lung from cancer patients, we suspected that such normal extracts could contain tumour associated antigens (TAA), and thus induce tolerance to these TAA. Tumour antigens could be present in adjacent normal lung tissue i f metastases not visible macroscopically were present, or i f TAA . were released into the blood, lik e carcinoembryonic antigen (CEA). This possibility of detecting histocompatibility differences between the hetero-logous normal extracts and tumour extracts, instead of tumour associated differences, was minimized by using a pool of 6 different heterologous lung 35 extracts. The use of pooled extracts also allows the preparation of a large volume of extracts and the use of the same normal extract for a l l of the experiments. Within 12 hrs of b i r t h , 6 rabbits (U.B.C. animal unit) were inj e c t e d with s t e r i l e N-pool (29 mg protein/ml). High protein concentrations were used so that minor components i n the extract would be present i n greater amounts. The t o l e r i z a t i o n protocol and the i n i t i a l immunization seri e s i s outlined i n F i g . 1. I I I . Immunization of Rabbits 1. Hyperimmunization Complete Freund's adjuvant (Difco) was used to hyperimmunize rabbits against tumour extracts. Tumour extracts were d i l u t e d 1:1 with adjuvant, emulsified, and 1.0 ml was i n j e c t e d into the rabbits weekly. 0.2 ml was i n j e c t e d intramuscularly (I.M.) into each limb and 0.2 ml i n t r a p e r i t o n e a l l y (I.P.). A f t e r 4 weeks of immunizations, the tumour extracts were d i l u t e d 2:1 with CFA to maximize the amount of antigen and minimize the granulomatous reactions of the r a b b i t s . At t h i s time also, the r a b b i t s were bled from the ear, the serum was removed from the blood, and the complement was i n a c t i v a t e d by heating the serum at 56°C for 30 min. The a n t i s e r a were tested by immuno-d i f f u s i o n (I.D.) and stored at -20°C. The rab b i t s were bled weekly and boosted for 5 to 10 weeks or u n t i l there was no increase i n the antibody detected by I.D. Many of the a n t i s e r a were also tested for maximum antibody production by p r e c i p i t i n reaction (XIV). This did not c o r r e l a t e with a n t i -body production any better than I.D., and since i t required more manipula-tions , was discontinued. ri2 ^ B L E E D 11 10 . B O O S T ( s k i n test). . I M M U N I Z E W I T H A L U M P R E C I P I T A T E D T U M O U R E X T R A C T o o Q. I Z I— u E 6 E o 1 wk. 6 RABBITS BORN FEB. 15,1972 Procedure f o r the neonatal t o l e r i z a t i o n of rabbits to the normal antigens i n lung extract. 37 2. Alum Precipitate. There are experimental conditions which preclude the use of CFA as the adjuvant for immunization. With CFA, i t i s possible to break immune tolerance and to induce the production of extraneous antibodies. When these factors may be important, an alum precipitate of the antigen was used for immunization instead of a CFA emulsion. The method of Campbell ^ t a l (21) was used. 1.0 ml of a suspension of alum precipitated extract (5.0 mg protein/ml) was used to immunize the rabbits neonatally tolerant to N-pool. A similar suspension (100 ug protein/ml) of purified human immunoglobulin (XV; ) was used to prepare clean antiserum against human immunoglobulin". IV. Immunodiffusion. 1. Semi-micro. The method used was similar to that presented by Clausen (22). In order to f a c i l i t a t e sticking of the gel, the clean 50 x 75 mm micro-scope slides (Fisher) were precoated with agar by dipping them in 0.2% Ionagar No.2 (Oxoid) and air drying. 10.0 ml of molten 0.85% Ionagar No. 2 in barbital buffered saline, was pipetted onto the microscope slide, allowed to set at room temperature, and allowed to become firm by storage at 4°C for 1 hr. Hole patterns (2 patterns per slide) were punched in the agar with a no. 2 (0.5 cm diameter) or 3 (0.7 cm diameter) cork borer and the agar plugs were removed by a Pasteur pipette on an aspirator. The wells were f i l l e d with 500 or 100 ul of antiserum and antigen. The precipitin bands were allowed to develop for up to one week at 4°C in a humidified chamber. 38 2. Micro. This technique i s similar to the semi-micro technique except that only 7.0 mis of jonagar No. 2 i s used per slide and the hole patterns were punched with a template. Six hole patterns (0.2 cm diameter) were used per slide. Each well could hold approximately 7 u l of antigen or antiserum. The reaction times were also shorter. 3. Staining. To remove the unbound protein, the slides were soaked in several changes of 0.9% saline at 4°C for 2 to 3 days. Then they were br i e f l y rinsed with d i s t i l l e d water, covered with a piece of f i l t e r paper and allowed to dry at room temperature overnight. The f i l t e r paper was removed and the slides were soaked in tap water for one minute and in Amido Black stain for 15 min. Destaining was carried out for 2 hrs with several changes of 3% acetic acid, and the slides were a i r dried. Photographs were taken with the agar side down on white paper, or with transmitted fluorescent light. 4. Micro Radioimmunodiffusion. This technique was developed in an attempt to increase the sensitivity of the I.D. technique. After washing micro I.D. slides free of unreacted protein, a dilution (from 1/5 to 1/50) of 1 2 5 i conjugated anti-immunoglobulin (XIX ) was added to either the central (antiserum) wells or a l l seven of the wells, and the slides were incubated overnight. After a thorough washing in 0.9% saline and drying, the slides were exposed to Kodak Blue Brand -14 X-ray film in the dark at 4°C for 1 tp 2 weeks. The film was developed with Kodak 39 X-ray film developer plus replenisher according to the developer instructions. The slides were then stained with Amido Black stain for comparison of results. V. Immunoelectrophoresis The method used was similar to that described by Campbell e_t al_ (21) . 4.0 ml of molten Ionagar No. 2 instead of 2.0 ml, was pipetted onto each microscope slide so that larger samples could be applied. The f i l t e r paper wick for electrophoresis was not impregnated in the agar but placed on top, at each end of the slide. After adding 100 y l of antiserum to the central trough (Fig. 13c), the slides were treated like immunodiffusion slides. VI. DEAE Cellulose Chromatography DEAE cellulose (Bio-Rad) was suspended in d i s t i l l e d water, allowed to settle and the fines removed by decanting the supernatant. After further washing, and packing i n a Buchner funnel, the DEAE cellulose was soaked twice in 0.2 N HCl for 15 min. each time. Several d i s t i l l e d water washes on a Buchner funnel were followed by two soakings in 0.5 N NaOH. After several more d i s t i l l e d water washes, the DEAE cellulose was soaked in 0.1 M phosphate buffer pH 7.5, then washed with starting buffer (0.01 M phosphate buffer pH 7.5). One ml of packed DEAE cellulose was used for 3 to 7 mg of protein to be fractionated. The slurry was packed into a column and equilibrated with at least 5 times the column volume of starting buffer. The sample, after dialysis against starting buffer, was washed through the column and the bound material was fractionated either by gradient or step elution. 40 1. Gradient Elution., A linear gradient, from starting buffer to starting buffer plus 1.0 M NaCl, was run. Both buffer reservoirs contained 3 times the column volume of their respective buffers. A small volume of 3.0 M NaCl was frequently run after the gradient, to check for complete elution. The remaining starting buffer was removed from above the cellulose and 0.05 M phosphate buffer pH 7.5 was flushed through the column until the A28O approached zero. The f i n a l elution buffer was 0.2 M phosphate buffer pH 7.5 plus 0.2 M NaCl. For our purposes, step elution was found to be superior to gradient elution because the three fractions were convenient to work with and the protein concentrations in each fraction were similar. Fractions were collected on an LKB fraction collector and the elution profile was monitored by reading the absorbance at 280 nm (A2B0) with a Beckman DB-G spectrophotometer. The fractions in each peak were pooled and concentrated to the original sample volume by dialysis against F i c o l l (Pharmacia), vaccuum dialysis, or u l t r a f i l t r a t i o n on a PM10 u l t r a f i l t r a t i o n membrane (Amicon). The peaks were labelled I, II and III in the order of elution, and stored at -20°C. 2. Step Elution. 3. Treatment of Fractions. 41 VII. Gel Chromatography. Both Sephadex G-75 and G-150 (Pharmacia) were used for gel chromato-graphy. 2.5 x 90 cm columns were packed and were usually run in 0.15 M NaCl. The columns were fi t t e d with a flow adaptor and run ascending at room tempera-ture. Sample and fraction volumes were 5.0 ml. The flow rate was maintained by gravity at 20 ml/hr for G-75 and 15ml/hr for G-150. The packing quality, sta b i l i t y , and the void volume of the column bed were determined by chromto-graphing 2 mg of Blue Dextran 2000 (Pharmacia). One of the G-75 columns was further calibrated by the chromatography of 2.0 mg of ovalbumin (M.W. 45,000) and chymotrypsinogen A (M.W. 25,000). Fractions were treated as were those from DEAE cellulose chromatography. VIII. Ammonium Sulfate Precipitation. Enough saturated (NH^) (Fisher) solution was very slowly added to protein solutions, at room temperature, with st i r r i n g , to make the desired concentration of (NH^^SO^. For example, 33%, 40% and 50% saturated solutions can be achieved by the addition of 1/2, 2/3 and 1 volume of saturated (NH^^SO to a volume of protein solution. A l l of these concentrations can be used to precipitate immunoglobulin, depending on the required purity or yield. The mixtures were stirred for 1 hr and allowed to settle for 3 hrs. For 67% and 100% saturation, solid (NH^^SO^ was slowly added, mixed 2 hrs and allowed to settle several hrs. The supernatants were decanted after centrifugation at 5,000 g for 15 min. and the pellets were dissolved in a minimum amount of d i s t i l l e d water. The dissolved precipitates were dialyzed against 0.15 M NaCl at 4°C and stored at -20°C. 42 IX. Disc Gel Electrophoresis. The method has been described in detail by Maizel (77). The resolving gels were 710% acrylamide (Kodak) and 0.55 x 11 cm. The high pH buffer system was used throughout. A 3.8% acrylamide spacer gel was also used i n i t i a l l y , but was not found to improve the results. Sample volumes were from 20 ul to 100 u l , since there was no difference in resolution within this range. Samples were diluted 1:1 with 30% sucrose before application to the gels. 350 to 500 yg protein and 200 to 350 yg protein were found to give optimum resolution for the whole extracts and DEAE cellulose frations, respectively. The gels were stained with Amido Black 10B (Merck) for 2 hrs and destained by overnight diffusion in 7.0% acetic acid at room temperature. Several attempts were made to increase the resolution with the use of Coomassie B r i l l i a n t Blue R250 (Colab). The gels were stained for 3 hrs at 37°C or overnight at room temp-erature in 0.25% Coomassie Blue in 7.0% HOAc, 5.0% MeOH. Destaining by diffusion in 7.0% HOAc, 5.0% MeOH or.7.0% HOAc, 5.0% MeOH required 2 to 3 days. Although the sensitivity may have been increased slightly, the resolution of the bands was not. X. Migration Inhibition Factor Test with Human Peripheral Blood Leukocytes. 100 units of heparin (Connaught) was used per ml of blood. Blood was taken from healthy volunteers and patients with bronchial carcinoma and incubated for 1 hr at 37°C with 20% Plasmagel (Laboratoire Roger Bellon, Neuilly, France) in an inverted syringe. Plasma and the buffy coat were transferred to a st e r i l e , siliconized (Siliclad; Canlab) centrifuge tube, through a bent needle. The cells were washed twice with phosphate buffered saline (PBS) plus 10% foetal calf serum (FCS) by centrifugation at 200 g for 43 10 min. The remaining red blood cells were lysed with 0.85% NH^Cl, and the leukocytes further washed twice with PBS. The c e l l pellet was suspended in 7 times i t s volume of RPMI 1640 tissue culture medium (Gibco) supplemented with 100 units of p e n i c i l l i n and 100 yg of streptomycin per ml. The migration inhibition factor (MIF) test was performed as described by Waterfield e_t a l (134) and a l l tests were done in quadruplicate. In order to determine the maximum amount of extract which would be incubated with the leukocytes, healthy donors' leukocytes were incubated with a 1/15, 1/60 and 1/200 dilution of N-pool (29 mg protein/ml). Only the 1/200 dilution (150 yg/ml) did not inhibit the migration of any of the leukocytes tested i n i t i a l l y . The finding that the leukocytes from one donor (J.L.), were not inhibited by even a 1/15 dilution of N-pool, strongly suggests that migration inhibition by the extract i s not non-specific cytotoxicity. Although we could have diluted the protein concentration of a l l of the extracts to 150 yg/ml, we chose instead to dilute a l l of the extracts 1/200. Appendix A shows that a l l the normal lung extracts have higher protein concentrations than the tumour extracts. Therefore, i f a l l of the extracts were similarly diluted, the normal extract control migration chambers would have more protein than the tumour extract migration chambers. Thus, any inhibition of leukocyte migration by tumour extract i s probably not due to non-specific inhibition by high protein concentration, and is more lik e l y to be specific inhibition than i f a l l of the protein concentrations were the same. Also, since the lungs contain a large quantity of blood, and the blood was not removed before extraction, much of the protein in the normal lung extract i s serum protein and not lung tissue protein. During the time these experiments were being done, we received no surgical tumour specimens. Therefore, i t was not possible to test autolo-gous tumour extract and leukocytes. 44 XI. Guinea Pig Skin Tests The backs of non-sensitized guinea pigs were clipped, depilated with Nair, washed, and skin tested with extracts and their DEAE cellulose fractions. Undiluted extract (0.1 ml) was injected intracutaneously and the diameter of erythema was measured at 2, 6, and 24 hours. XII. Agarose Immunoadsorbent Columns The method used was similar to that already outlined (136). The normal lung extract to.be coupled was dialyzed against borate buffered saline pH 8.45 (0.2 M borate, 0.3 M NaCl). One ml of packed Sepharose 4B (Pharmacia) was used per 6.0 mg of protein. The Sepharose was washed with d i s t i l l e d water and the pH was brought to 11.5 with 5.0 M NaOH. 40 mg of cyanogen bromide (J.T. Baker), dissolved in 1.0 ml d i s t i l l e d water per 25 mg, was added per ml of packed Sepharose. The pH was maintained at 11.2 to 11.4 for 10 to 15 mins., or u n t i l the pH became constant. After quickly (less than 2 min.) washing on a Buchner funnel with ice-cold d i s t i l l e d water and borate buffered saline, the activated Sepharose was transferred to a tube and the dialyzed normal lung extract was added. The tube was sealed and gently agitated on a rocker for 20 hrs at 4°C. The coupled Sepharose was transferred to a 1.6 x 80 cm water jacketed Pharm-acia column, cooled with running tap water, and f i t t e d with a flow adaptor (Pharmacia) to f a c i l i t a t e buffer changes. The column was flushed with 3 column volumes of t r i s buffered saline pH 7.6 (0.1 M t r i s , 0.4 M NaCl), 3 45 column volumes of Sorensen's glycine I buffer pH 2.6, and again with t r i s buffered saline. Some of the columns were reacted with 0.1 to 1.0 M ethanol-amine to block any remaining active groups. 2.0 to 4.0 ml of heat inactivated rabbit anti (tumour extract) serum was added and washed through with either t r i s buffered saline or 0. 15 M NaCl. The flow rate was maintained at 15 ml/hr with an LKB per i s t a l t i c pump. 10.0 ml fractions were collected on an LKB fraction collector and fractions containing A 2so were pooled. The pooled fractions were either concentrated by u l t r a f i l t r a t i o n on an XM100A membrane (Amicon) or re-absorbed with a glutaraldehyde precipitated normal lung extract. The concentrated absorbed antisera were stored at -20°C. The anti normal antibodies could be recovered and the columns regenerated by flushing with Sorensen's pH 2.6 buffer, and the columns neutralized with t r i s buffered saline. Whether the extracts were reacted at 6 mg.or 10 mg protein per ml of packed Sepharose, 2 to 3 mg protein was bound per ml. Thus for a 70 ml column, 140 to 210 mg of protein was bound. Since the protein concentrations of the normal extracts used were 30 mg/ml, such a column would be the equivalent of roughly 6 ml of normal extract. Since 4.0 mis of antiserum was usually absorbed at a time, i t would appear that each volume of antiserum was absorbed by 1.5 volumes of normal extract. However, i t must be remembered that many of the antigenic determinants w i l l not be accessible to antibody. The absorbed antisera were not any more specific i f only 1.0 or 2.0 mis of antisera were used. A problem encountered with these columns was the stripping of materials from the immunoadsorbents as detected by the loss of their red colour. This was not observed with any of the several buffers (including low pH) used. It was observed with ethanolamine, immune rabbit serum and even non-immune rabbit serum. Efforts were made to prevent this, since i t was suspected that many 46 proteins were being removed. However, the column capacity was not appreciably diminished with each use, although the red colour was completely eliminated. Recent communications with Dr. A. Jackson of Kent Laboratories, Vancouver, have suggested that haemoglobin can be removed from insoluble complexes by the avid binding of haptoglobin. XIII. Glutaraldehyde Cross-Linked Immunoadsorbents. 1. Human Immunoglobulin Immunoadsorbent. 150 mg of purified human immunoglobulin (see Section XV.1.) plus 100 mg of bovine serum albumin (Sigma) was insolubilized by polymerization with glut-araldehyde (J.T. Baker) according to the method of Avrameas and Teimynck (4 )• The protein recovered was 1.5 mg per ml of antiserum (Section III.2.). Much of the antibody activity was recovered and most of the antibody was removed from the antiserum as seen in Fig. 2. 2. Normal Lung Extract Immunoadsorbent. The method of Avrameas and Ternynck (4 ) for the insolubilization of serum proteins was not suitable for the insolubilization of normal lung extracts. Some modifications were required. 50 mg (instead of 10 mg) of glutaraldehyde was reacted per 100 mg of protein. Because the extract con-centration was 30 mg/protein/ml instead of 50 mg/ml, 25% glutaraldehyde was used instead of 2.5%, to keep volumes and protein dilutions to a minimum. Reaction times were lengthened to 4 hrs of mixing at room temperature and leaving overnight at 4°C. After thorough washing, the precipitate was sus-pended in PBS equal to the original volume of the' extract. 47 Fig. 2: Immunodiffusion results of tests for the absorption of rabbit anti-human Ig by glutaraldehyde i n s o l u b i l i z e d human Ig. The control w e l l contained human Ig (40.0 mg) and the outer wells contained anti-human Ig preparations as follows: 1. whole rabbit anti-human Ig diluted 1:1, and 4 undiluted 2. absorbed rabbit anti-human Ig diluted 1:1 and 5 undiluted 3. rabbit anti-human Ig eluted from immunoadsorbent diluted 1:1 and 6 undiluted. 48 Two volumes of glutaraldehyde immunoadsorbent suspensions were used to further absorb one volume of antiserum already abosrbed by an agarose column immunoadsorbent. Aft e r mixing and reac t i n g overnight at 4°C, the p r e c i p i t a t e was removed by ce n t r i f u g a t i o n at 2000 g for 15 min. and the supernatant was concentrated by p r e c i p i t a t i o n with 50% saturated (NH^^SO^ or by u l t r a f i l t r a t i o n on an XM100 A membrane. E a r l y attempts to regenerate these immunoadsorbents showed that the o r i g i n a l capacity was not nearly achieved. XIV. Other Antiserum Absorption Methods Even when used i n combination, the immunoadsorbent methods of sections XII and XIII.2. frequently could not completely eliminate the anti-normal antibodies from the ant i s e r a . Since both of these methods involve reactions with amino groups of proteins, i t i s possible that both methods could make the same proteins i n a c c e s s i b l e to protei n , or f a i l to i n s d l u b i l i z e the same proteins. Two methods were used to t r y to eliminate non-tumour s p e c i f i c antibody. 1. P r e c i p i t a t i o n of Immune Complexes Many of the ant i s e r a were tested weekly, by p r e c i p i t i n reaction i n order to ascertain when the ra b b i t s were maximally producing antibodies. 25 u l of doubling d i l u t i o n s of N-pool was incubated for 1 hr at 37°C with 25 u l of heat i n a c t i v a t e d antiserum. The p r e c i p i t a t e s were washed 3 times with PBS and dissolved i n 0.1 N NaOH. The A28O w a s determined f o r each sample and the d i l u t i o n at which maximum p r e c i p i t a t i o n occurred was determined (eg. F i g . 3). For the hyperimmune rabbit sera, the maximum p r e c i p i t a t e s occurred at a n t i -49 E c o .10 . CO CN .05 L 1 2 3 4 5 6 7 DOUBLING DILUTIONS OF N-pool Fi g . 3: P r e c i p i t i n reaction of rabbit whole antiserum against C-41 a f t e r 8 weeks of hyperimmunization, against doubling d i l u t i o n s of N-pool. 50 serum:extract ratios ranging from 4:1 to 64:1. No relationship was found between this t i t e r and the a b i l i t y of the antiserum to produce antibodies against TAA. With a single antigen, both specific antigen and antibody can be effectively precipitated from solution when incubated at their zone of optimal proportions. However, for a crude protein mixture there is no single zone of optimal proportions, and different antigens are optimally precipitated at different antibody:extract ratios. In order to absorb the antisera by precipitation of immune complexes, the antisera were mixed with N-pool at ratios from 4:1 to 64:1. When these absorbed antisera were subsequently re-absorbed by a solid-phase immunoadsorbent, the antibodies were s t i l l not tumour specific. 2. Absorption with Cells. This i s probably the most commonly reported method for the absorption of antisera. Although we expected our antisera to contain antibodies against internal c e l l antigens, which would not be absorbed by cells, we tried this method in the hope of complementing our other absorption methods, and for the sake of completion. Normal lung tissue was minced at 4°C i n PBS and the large.clumps of tissue were allowed to settle for 20 min. The cells in the supernatant were washed 5 to 7 times with PBS by centrifugation at 800 g. One volume of anti-serum was mixed with one volume of packed cells and diluted 10 fold. This mixture was incubated overnight at 4°C with gentle agitation, the cells were pelleted, and the antibody in the supernatant was concentrated by precipi-tation with 50% saturated (NH,)„S0,. This absorption procedure did not 4 2 4 result in specific anti tumour antibody, even when combined with the agarose 51 immunoadsorbent method. Very large amounts of tissue, time and manipulations were also required. XV. .. Purification of Immunoglobulin. 1. Human Immunoglobulin. The immunoglobulin (Ig) fraction was precipitated from 50.0 ml of normal human serum with 33% saturated (NH^^SO^ and further purified by passing the fraction through a DEAE cellulose column in 0.01 M phosphate buffer pH 7.5 (21). After concentrating the material by precipitation with 40% saturated (NH^^SO^, and dialysis against 0.15 M NaCl, the Ig was further purified by chromatographing on a 2.5 x 90 cm Sephadex G-150 column. Fractions in the major peak ( A 2 8 0 ) were pooled and concentrated on an XM100 A u l t r a f i l t r a t i o n membrane. Protein was quantitated spectrophotometrically, assuming the molar absorbancy index to be 1.38. The yield was 6.0 mgiVIg/ml of serum. 2. Rabbit Immunoglobulin. This was used for the immunization of a sheep, to get anti-rabbit Ig, and was supplied by D. Gregerson. He purified the Ig from rabbit serum by precipitation with 40% saturated (NH^^SO^, passing i t through a DEAE cellulose column, and re-precipitating the Ig fraction with 50% saturated (NH^^SO^. The sheep was immunized with 3.0 mg of Ig in 50% complete Freund's adjuvant 3 times, 10 days apart. 52 XVI'. . Human Serum Fractionation. Whole serum or a 40% saturated (NH,)„S0, cut was tested for free anti-4 2 4 tumour antibody. To dissociate any immune complexes which may have been present, the serum (2.0 ml diluted to 5.0 mis) or the (NH^^SO^, precipitate cut was chromatographed on Sephadex G-150 equilibrated in 0.1 M HOAc, 0.15 M NaCl. The effluent was immediately neutralized by dialysis against 0.2 M phosphate, 0.15 M NaCl pH 7.5. Fractions in the Ig peak (Fig. 4) were pooled and concentrated by either precipitation with 50% saturated (NH^) o r u l t r a f i l t r a t i o n on an XM100 A membrane. The remaining fractions of the column volume were concentrated by either precipitation with saturated (NH^)^SO^ or u l t r a f i l t r a t i o n on a PM10 membrane (Amicon). Although the separation (Fig. 4 ) i s not good, the results of Fig.5 show that most of the Ig was in peak 2. Both pre-operative and post-operative sera were tested. XVII. Indirect Immunofluorescence. The method used was described by Culling (26). Instructions, including the operation of a cryostat microtome (International, I.E.C.) were arranged by C.F.A. 'Culling (Department of Pathology, U.B.C). Pieces of tissue were snap frozen in liquid nitrogen and stored at -70°C 6 um sections were fixed to clean microscope slides by a i r drying. 1. Absorbed Anti(Tumour Extract) Serum. Either 10 or 25 Ul of absorbed antisera or normal rabbit serum, was incubated with tumour and normal lung tissue sections i n a humidified chamber at room temperature for 30 min. The slides were rinsed with PBS and up to 20 53 S - 8 9 - a I S-89-b FRACTION NUMBER (5 .0 ml ) Fig. 4: 2.0 ml of serum from patient 89 (S-89) fractionated on a Sephadex G-150 column (2.5 x 90 cm) equilibrated in 0.1 M HOAc, 0.15 M NaCl. The fractions in S-89-a were concentrated by- u l t r a f i l t r a t i o n on an Amicori X M100A membrane and in S-89-b on a PM10 membrane. Fig. 5: Immunodiffusion results using purified antibodies to human Ig (diluted 1:5) against human serum and fractions designated as follows: 1. Human Ig (200 yg) 2. S-68-a diluted 1:5 3. S-68-b diluted 1:5 4. S-89 diluted 1:5 5. S-89-b diluted 1:5 6. S-89-a diluted 1:5 The presence of a precipitin band in wells 3 and 5 shows that the Sephadex G-150 fractionation of the serum Ig was incomplete The heavy staining around wells 2,3,5 and 6 suggests that the Sephadex fractions contained poorly soluble components. 55 slides at a time were washed with 250 ml of PBS, 5 times with agitation for a total of 30 min. The tissue sections were then incubated with fluorescein conjugated goat anti (rabbit'IgG) IgG (Microbiological Associates), washed again, mounted in buffered glycerine mountant, and sealed with a coverslip. The sections were examined by a microscope with a dark ground condenser. Several dilutions of the reactants were also tried, but because of the great d i f f i c u l t y in quantitation and the more promising results by indirect immuno-autoradiography (being done concurrently), the dilutions of the reactants., required for optimum sensitivity were not determined. 2. Human Serum. Healthy donor and cancer patients' sera and globulin fractions were tested similarly. The labelled antibody was fluorescein conjugated rabbit anti (human globulin) globulin. XVIII. Indirect Immunoautoradiography. 1. Experimental Techniques. The method i n i t i a l l y used was analogous to the immunofluorescence method. 8 um tissue sections were used because i t was easier and faster to prepare large numbers of good sections. Instead of a fluorescein label, the anti Ig was conjugated with 1 2 5 I (Section XIX)• Bound radioactive label was assayed by the method described by Kelly et a l (61). Slides were fixed by soaking in absolute MeOH for 15 min., then soaked in water for 30 min. and allowed to dry. The following steps were carried out in a darkroom equipped with a No. 2 red safelight (Kodak). NTB-2 nuclear track emulsion (Kodak) 56 was heated to 43 C and transferred to a coplin jar. Two slides at a time (back to back) were slowly dipped 3 times for a total of 10 sec. The slides were drained and air dried vertically for 1.0 hr. Exposure to the emulsion was at 4°C in tape-sealed microscope slide boxes (containing a few grams of dessicant) and ranged from 36 hr to one week. I n i t i a l l y , the developing was done as described by Kelly e_t a l (61) • However, problems were encountered: some tissue sections and the nuclear track emulsion would often detach from the slide and the background grains were extremely high. Thoroughly cleaning the slides by washing with acid-alcohol or detergent had no noticeable effect. Precoating the slides by dipping them in 0.2% gelatin helped the emulsion stick more firmly but interfered with the sticking of the tissue sections. By fixing the tissue sections i n MeOH for one min., coating the slides with 0.2% gelatin, and keeping the developing times to a minimum and the temperature to 12°C, there were no further problems with the slipping of emulsion or tissue sections. (The problem with the emulsion varied with each batch). The emulsion was developed for 2.5 min. in f u l l strength Kodak Dektol Developer, rinsed in H^ O for 10 s e c , fixed in Edwal High-Speed Fixer with Hardener for 3.0 min., rinsed with H^ O for 5.0 min., and dried for 1.0 min. in MeOH. The cells were stained with 6% Geimsa solution for 2.5 min. and washed with ^ 0 for 2.5 min. After air drying, the slides were examined under o i l immersion. The changes in methodology had no noticeable effect on the high back-ground grains which were never eliminated in spite of several attempts. These attempts included: fixing the tissue sections to plastic instead of glass, adding FCS or adult bovine serum to the PBS used for rinsing the slides, preincubating of the tissue sections with non-conjugated anti-Ig, and adding activated charcoal to the 1 2 5I-conjugated anti-Ig. The labelled anti-Ig would bind to the glass and directly to the tissue sections even 57 i f there was no incubation with the intermediate antiserum. To determine which component of the conjugated a n t i - I g was^responsible f o r t h i s s t i c k i n e s s , i t was mixed with beef serum or g l o b u l i n , 1 2 7 I - c o n j u g a t e d beef g l o b u l i n , or KI. The KI produced a s i g n i f i c a n t i n h i b i t i o n of binding of the 1 2 5 l conju-gated a n t i - I g to the tissue sections, but iodide i s a chaotropic agent, and besides increasing the s o l u b i l i t y of proteins, can also d i s s o c i a t e antibody-antigen complexes (305). When the other materials were added, only a s l i g h t decrease i n background was observed. The 1 2 5 i conjugated a n t i - I g subsequently had 20% bovine serum added to i t . Dr. I. Ber c z i (Department of Immunology, University of Manitoba) has reported s i m i l a r problems with an analogous technique (personal communication). The problem with the high background was circumvented by incubating d i l u t i o n s of the 1 2 5 I - c o n j u g a t e d a n t i - I g with the tiss u e sections. The d i l u -t i o n which resulted i n the maximum to l e r a b l e background grain l e v e l was used for subsequent experiments. This d i l u t i o n was determined for each l a b e l l e d a n t i s e r a and could be estimated afterwards according to the 1 2 5 i h a l f l i f e . This d i l u t i o n ranged from 1/200 to 1/20. 2. Quantitation Two methods were used to quantitate the binding of l a b e l l e d antibody. The f i r s t was for a rough comparison i n order to determine the e f f e c t s of techniques to lower background grain counts and to determine the d i l u t i o n s that optimize tumour s p e c i f i c d i f f e r e n c e s . The whole t i s s u e sections were quickly scanned and 3 or 4 representative areas were v i s u a l l y compared. The other method was s e n s i t i v e but was very tedious and time consuming to per-form, and had to be b l i n d c o n t r o l l e d because of i t s subjective nature. The whole tis s u e sections were c a r e f u l l y scanned, noting any areas of exceptionally 58 high background to be avoided later. The numbers of grains on one represent-ative c e l l was counted for 50 microscope fields. This large number was required because of the high background. 3. Testing Human Serum and Fractions 10 y l of cancer patient or healthy donor serum or fractions (Section XVI) were incubated on each tissue section for 30 min. in a humidified chamber at room temperature. The slides were rinsed and washed as in Section XVII), dried in MeOH for 1 min. and incubated with a pre-determined dilution of 1 2 5 i conju-gated rabbit anti (human Ig) globulin for 30 min. The slides were again rinsed, washed, and dried, and then treated as in part 1. A further attempt to minimize background grains was done by iodinating immunoadsorbent purified antibody (Section XIII.1.) instead of the ammonium sulfate globulin fraction of the rabbit antiserum. 4. Testing Absorbed Rabbit Anti (Tumour Extract) Serum 10 y l of absorbed antiserum and a pre-determined dilution of 1 2 5 I conjugated sheep anti (rabbit Ig) globulin were reacted with tissue sections as i n part 1. The grain counts were far too high to count on both normal and tumour sections, indicating that antibodies against normal tissue antigens were s t i l l present. Several antisera were re-absorbed (Section XII, XIII.2.) and tested again. One particular antiserum (C-66) was re-absorbed several times until no antibodies were detectable by immunodiffusion, and s t i l l required a 1/50 dilution before the grain count was low enough to,be con-veniently counted. A convenient dilution for a l l of the absorbed antisera was determined for subsequent experiments. 59 XIX. Iodination. The Ig fraction of the antiserum was partially purified by precipita-tion with 33% saturated ammonium sulfate and dialyzed against 0.1 M phosphate buffer pH 7.5. Protein concentration was determined spectrophotometrically and diluted to 50 mg protein per ml with 0.1 M phosphate. The iodination method was carried out by the chloramine T method (56). 100 pi of protein solution (5 mg) was added to a shell v i a l and transferred to a fume hood at room temperature. One to 2.0 mCi of carrier-free Na 1 2 5I in 0.1 N NaOH (New England Nuclear) was added with a 20 y l capillary pipette fitted on a disposable 1.0 cc syringe. 200 yg (20 yl) of chloramine T (Eastman) freshly dissolved in 0.1 M phosphate buffer pH 7.5 was added and the solution was mixed by bubbling air through i t . The reaction was terminated after 3.0 min. by the addition of 200 yg (20 yl) of sodium metabisulfite (Fisher). 50 y l of 1% KI was added and the solution was applied to a 1.5 x 8 cm Sephadex G-25 column to separate the iodinated protein from the iodine. The column had been pre-treated with 5.0 mis of FCS and rinsed with PBS to minimize non-specific binding of the 1 2 5 I to the column. The sample was washed through the column with PBS and 8 drop fractions were collected in disposable plastic tubes. The radioactivity in each fraction was measured with a Geiger-Muller counter (Fig. 6), the samples in the voided peak pooled, and the volume measured. Triplicate 10 y l samples were dried on glass fiber f i l t e r discs (Reeve Angel), which were suspended in 8.0 ml of s c i n t i l l a t i o n f l u i d . Gamma radi-ation was counted with a Isocap 300 s c i n t i l l a t i o n counter for 0.1 min. The specific activity was calculated as follows: 6 8 The average counts per min. was 1.29 x 10 for 10 yl= 1.29 x 10 cpm/ml. 1.29 x 10 8 cpm x 1.25 = 1.615 x 10 8 DPM 1.615 x 10 8 DPM x yCi = 62.1 yCi = 0.062 mCi/ml 2.6 x 10 8 DPM 60 ^ i ,— i— . i — _ 5 10 15 FRACTION NUMBER ( 8 drops ) Fig. 6: The removal of free I from 5.0 mg of Ig conjugated with P-25j by a 1.5 x 8 cm Sephadex G-25 column. 61 If no protein was lost, we have 5.0 mg/2.2.mls „ 2.27 mg protein/ml ... 0.062 mCi n n ^ specific activity = ; = 0.027. 2.27 mg protein XX. S t a t i s t i c a l Analyses Gel s t a t i s t i c a l analyses were performed with a programmable Hewlett Packard 9810 A calculator. For most results, the significance was determined with a programme for the Student's t test. When the means of several groups were being compared, such as in the indirect immunoautoradiography surveys (Section XVIII.3.), significance was determined by the method of 'least significant difference' for a probability of 0.05 and 0.01 according to L i (71). I would like to thank Dr. J. Yensen for advice on s t a t i s t i c a l analysis. XXI. Skin Testing of Cancer Patients Whole extracts and DEAE cellulose fractions were sterilized by f i l t r a -tion through a 0.22 um membrane f i l t e r (Millipore) and were stored at -20°C. A l l skin testing was done by intradermal injection of 0.1 ml of material. Several extracts and fractions were skin tested in non-immune guinea pigs for the presence of skin reactive factors. I would like to thank Dr. P. Coy, B.C.C.I. for arranging patient cooperation and performing the tests. Patients were injected with extracts and fractions of their own tumours, either diluted or undiluted. The diameter of erythema (but not always of induration) was read by Dr. Coy i f possible, otherwise by the patients themselves at home. Most patients were tested for immunological energy by the a b i l i t y to respond to r e c a l l antigens such as a 1:100 dilution of Candida (Hollister), 5 T.U. of 62 tuberculin (Connaught), and Mumps Skin Test Antigen ( E l i L i l l y ) . XXII. Analysis of Extracts for CEA CEA analysis was performed by the Hansen method (73) (Hoffman-La Roche) on undiluted and on a 1:5 dilution of the extracts. I would like to thank Dr. E. Mincey and Ms. B. L. Archibald at the Division of Nuclear Medicine, V.G.H. for performing these assays. I. RESULTS AND DISCUSSION  Neonatal Tolerization of Rabbits to Antigens of Normal Lung Extracts The rationale of this technique i s to render a neonatal rabbit tolerant to the antigenic components of the malignant tissue, so that when the rabbit i s subsequently immunized with tumour extract, the predominant response w i l l be directed towards tumour specific antigens (38). A pool of six normal lung extracts was used as the tolerogen in order to minimize any histocompatibility differences. Normal lung from the cancer patient was not used because i t was suspected that microscopic foci of metastases might be present or the TAA produced by lung tumours might be free to circulate as does CEA from colonic tumours. Six rabbits were used for induction of neo-natal tolerance and a l l survived. Sera from these rabbits one week after immunization with alum precipitated tumour extract, when tested by immuno-diffusion against both normal and tumour extracts, produced a very faint precipitin band against tumour extract but not normal extract. It could not be determined whether the difference detected was qualitative or quan-titat i v e . To increase the t i t r e of these antisera so that these questions could be answered, the rabbits were given booster injections of the same alum precipitated tumour extracts. Subsequent testing of the antisera showed multiple heavy precipitin bands against both normal and tumour extracts. Apparently the tolerant state is not long lasting and i s easily reversed. This appears to be a common problem since no successes with this technique have recently been reported. Although the 3.0 M KC1 method of extraction was originally used for single c e l l suspensions (108), i t was shown to be efficient even when finely ground tissue was used. Attempts to further extract tissue pellets from 64 KCl extracts by treatment in a Waring blender with saline indicated that very l i t t l e material could be liberated this way. Disc gel electrophoresis of these samples showed that no new protein bands could be liberated from the tissue pellets by this method. Other workers (18,72,83) have reported successful tumour antigen solubilization for several tumours using hypertonic KCl. Studies on guinea pig tumours (84) have shown that i t is possible to recover 15 to 40% of the tumour antigen activity from tumours extracted in this manner. Immunologic specificity was maintained and the antigens were stable at -70°C for several months. While i t is probable that antigens may be extracted by other agents such as detergents, i t was decided to use KCl exclusively in this study since other laboratories were starting to use this method so extensively. It was fe l t that standardization of a single pro-cedure would simplify data analysis at later stages. II. Migration Inhibition of Allogeneic Human Leukocytes by Extracts of  Patients' Tumours Tests for the production of factors by sensitized lymphocytes upon reaction with specific antigen are among the best in vitro correlates of cell-mediated immunity (16). Tests for the production of migration inhibition factor (MIF) are possibly the most sensitive of these techniques and are certainly the most intensively studied. Since cell-mediated immunity may have a major role in tumour rejection, antigens detected by the MIF assay with cancer patients' leukocytes would li k e l y correlate with tumour specific transplantation antigens (TSTA). This method has been used successfully to detect solubilized guinea pig tumour antigens (72,124) and was found to be more sensitive than delayed cutaneous hypersensitivity by requiring 1/4 to 1/25 of the antigen for detection. Tumour immunity was specific for the 65 p a r t i c u l a r tumour being studied and stronger i n animals immunized with tumour than i n tumour bearers. This method has also been used i n hamster (15) and virus-induced mouse tumours (15, 131). MIF experiments were performed using allogeneic leukocytes and extracts to standardize the technique while waiting f o r s u r g i c a l specimens so that an autochthorous system could be tested. Migration index (MI) can be defined as: area of migration i n test culture . : ' x 100% = MI area of migration i n control (no antigen) culture The r e s u l t s (Fig.7 ) show a. non-tumour s p e c i f i c i n h i b i t i o n of migration f o r a l l of the leukocytes tested against some of the tumours and normal t i s s u e extracts tested. The a b i l i t y of the extracts to e l i c i t d i f f e r e n t responses with d i f f e r e n t leukocyte populations also made i t impossible to determine the protein concentration which minimizes non-specific reactions. The pr o t e i n concentrations used ranged from 47 ug/ml to 200 ug/ml. Other workers (115) used a protein concentration of human tumour extract of 100 ug/ml and claimed that non-specific i n h i b i t i o n may occur at concentrations greater than 200 Ug/ml. For KC1 extracts of breast carcinoma (75) , concentrations greater than 1000 yg/ ml were t o x i c to leukocytes whereas i n h i b i t i o n of migration occurred i n the narrow range of 500 to 750 yg protein/ml. Maluish and Hal l i d a y (78) report a method analogous to the MIF technique for the detection of cell-mediated immunity to human tumours: the leukocyte adherence i n h i b i t i o n t e s t . Leukocytes from patients with a wide v a r i e t y of tumours were tested against d i f f e r e n t tumour extracts. I n h i b i t i o n of leuko-cyte adherence was induced by extracts of tumours of the same type as the c e l l donor, even at protein concentrations from 50 yg to 4000 yg/ml. Extracts of 100 75 50 100 75 50 100 75 50 z o < BC O 100 75 50 a. n 1 1 r i i J.L. i'i i-i H.W. c. i l.i P.N. d. r i R.W. f. "T \ i i I i r ALL. J A M . h . MUR. I. V A N . e. 100 75 50 i i -i - i D.G. MIL. J L J I I I I 1 L N-pool C-2 C-5 C-24 N-25 C-26 C~30 E X T R A C T S N-pool C-2 C-5 C-24 N-25 C-26 C-30 The effects of normal lung extracts (N-pool and N-25) and tumour extracts (C-2, C-5, C-24, C-26 and C-30) on the migration of leukocytes (including the standard deviation) from healthy donors (a-e) and heterologous lung cancer patients ( f - j ) . 67 other tumour types did not cross react. The data i n Fig.7 suggest that a normal extract control should be included i n such studies. The r e s u l t s of the normal extract controls i n Fig.7 have been supported by the r e s u l t s of Mclllmurray et a l (76). They report that extracts of colonic tumours i n h i b i t the migration of leukocytes from cancer patients s l i g h t l y but s i g n i f i c a n t l y more than leukocytes from sex and age matched con t r o l donors. However, there was a considerable amount of overlap, i n responses, and extract of normal mucosa from the same s u r g i c a l specimen also produces s i m i l a r r e s u l t s . Since t h e i r extracts were used at a f i n a l concentration of 1.0 to 2.0 ug</protein/ml, i t i s possible that the r e s u l t s obtained were due to non-specific c y t o t o x i c i t y i f healthy donor leukocytes are h e a l t h i e r and better able to handle the s t r e s s . Tumour antigens could be released from the tumour and trapped by the nearby normal t i s s u e , although t h i s i s an u n l i k e l y explanation since migration i n h i b i t i o n s induced by both extracts were minor. The occasional enhancement of leukocyte migration which has been observed (Fig. 7 ) has also been reported by others (23). Their r e s u l t s also frequently showed migration i n h i b i t i o n of healthy donor leukocytes by several extracts, and migration i n h i b i t i o n induced by normal t i s s u e extracts. Wolberg (141) suggests that at l e a s t part of the migration i n h i b i t i o n may be due to k i l l i n g of the migratory c e l l s by factors i n the tumour ex-t r a c t s . This does not explain the r e s u l t s of F i g . 7 because: normal lung extracts also could i n h i b i t migration, and tumour extracts did not induce migration i n h i b i t i o n with a l l of the leukocyte preparations. I t has been suggested (75) that the unexplained r e s u l t s may be due to h i s t o c o m p a t i b i l i t y d i f f e r e n c e s , but t h i s i s not l i k e l y the case with the r e s u l t s i n F i g . 7 since the c e l l cultures were incubated for only 20 hr which should exclude any i n v i t r o s e n s i t i z a t i o n to h i s t o c o m p a t i b i l i t y antigens (75). 68 MIF tests using homologous tumour extracts and leukocytes were not performed because we did not receive any.surgical specimens during this time. Even though s t a t i s t i c a l analysis might demonstrate significantly greater reactivity with tumour extracts and cancer patients' leukocytes than with normal controls, the frequent reactivity by the normal controls suggests that at least some of the antigens detected were not tumour specific. Until such antigens are eliminated from the extracts, MIF tests are not considered suitable as an immunochemical assay for TAA. Further discussion of the reactivity by the controls i s included with the skin test results (section VIII ). III. Disc Gel Electrophoresis of the Extracts and their Fractions The i n i t i a l disc gel electrophoresis studies were performed on one of the tumour extracts (C-26) and fractions separated by DEAE cellulose chroma-tography with gradient elution (Fig. 8). The control extract (N-pool) was separated into two fractions by step DEAE cellulose chromatography. One of the fractions of C-26 (C-26-I) was further fractionated by chromatography on Sephadex G-75 (Fig. 9 ) . By careful examination of the protein banding patterns, a band with an Rf of 0.28 to 0.30 could be located i n the tumour extract (C-26) which was not present in the normal extract (N-pool) (Fig.10). This band was found in the f i r s t fraction collected from DEAE cellulose (C-26-I) and the second peak from Sephadex G-75 (C-26-Ib). This column was calibrated with Blue Dextran, ovalbumin, chymotrypsinogen A, and ribonuclease, and the molecular weight of C-26-lb was determined to be between 21,000 and 60,000. Further evidence that this component was tumour specific was obtained by hyperimmunizing a rabbit with C-26-I. The antiserum was absorbed free of 69 Fig- 3: C-26 extract (40.0 ml) in 0.01 M phosphate buffer pH 7.5 was fractionated on a 2.5 x 20 cm DEAE cellulose column by a linear NaCl gradient. 70 FRACTION NUMBER ( 5 . 0 m l ) Fig. 9: 5.0 ml of C-26-I (10.0 mg/ml) fractionated on a 2.5 x 80 cm Sephadex G-75 column. N-25 C-26 C-26-I (kOO) (*»50) (200) C-26-fa C-26-lb C-26-lc C-26-II C-26-IM N -poo l N - p - l N - p - l l (230) (150) (125) (360) (350) (390) (200) (650) Fig. 10: Disc gel electrophoresis of fractions of C-26 tumour extract and normal extracts. The DEAE cellulose fractions of C-26 (I, II and III) were prepared by gradient elution and the fractions of C-26-I (a, b and c) were prepared by chromatography on Sephadex G-75. The fractions of N-pool (I and II) were prepared by step elution from DEAE cellulose. The figures in parentheses are the amounts of protein added to the gels (yg). The arrows point to bands not present in the normal extracts or fractions but present in C-26 and fractions. 72 anti (normal antigen) antibodies with an immunoadsorbent prepared by co-valently coupling normal lung extract to Sepharose 4B. When tested by immuno-diffusion, this absorbed antiserum did not react to give a precipitin band against normal lung extract, but did against C-26, C-26-I and C-26-Ib (Fig. 11). This antigen was also precipitated by 50% saturated (NH^^SO^. The properties of this TAA differ from the TAA detected in broncho-genic carcinoma extracts by other workers. Both antigens detected by Yachi et a l (142) were larger than IgG and both antigens detected by Okada and Ikeda (93) are not eluted from DEAE cellulose u n t i l the phosphate concentration is greater than 0.05 M at pH 7.5. Tumour antigens from experimental animal tumours have been reported with a wide range of properties. For example, Baldwin et a l (9 ) report an antigen with a molecular weight of 55,000, and Meltzer et^  a l (84) report an antigen of 75,000 to 150,000 molecular weight. In order to test whether the TAA detected here was an embryonic anti-gen, extracts of embryonic lungs were analyzed by disc gel electrophoresis (Fig.12b). The tumour associated material detected in C-26 was not detected in the embryonic extracts F-48 or F-49. However, this could be due to enzymatic digestion in vivo or by previous extraction by the salt used to induce the abortions. Because the i n i t i a l disc gel electrophoresis results looked so promis-ing, several more extracts and fractions were also analyzed (Fig.12) i n the hope of finding a common TAA or other 'unique' antigens. From these photo-graphs, i t i s obvious that the extracts display a wide variety of electro-phoretic patterns. Similar results have been reported for guinea pig tumour extracts (59). The d i f f i c u l t y of comparing grossly dissimilar electrophoretic patterns, and the in a b i l i t y to detect the TAA of C-26 in other extracts, limit the usefulness of the technique. (The antigen in C-26 was detected in a few other tumour extracts, but only after a large number were tested). The Fig.11: Immunodiffusion results using abosrbed antiserum to C-26-I against extracts and fractions designated as follows: 1. C-26-lb 2. C-26-I 3. C-26 4. N-pool 5. C-26 fraction insoluble in 50% saturated (NH^SO^ 6. C-26 fraction soluble in 50% saturated (NH.).SO Note that the precipitin band which i s obvious against C-26 and C-26-I can also be seen near the C-26-Ib well. F i g . 12a: C-24 C-24-1 C-24-11 C-24-III C-30 C-30-1 C-30-11 C-30-II1 C-46 C-46-I C-46-III (425) (175) (200) (325) (600) (340) (400) (750) (460) (475) (700) Fig.12: 75 + CM H ti •H 76 minor differences between the extracts make i t d i f f i c u l t to decide which band to focus attention upon in determining antigenic properties. Neverthe-less, on the basis of these results i t is clear that TAA were present in some tumour extracts. .Using doubling dilutions of bovine serum albumin (Sigma), i t was determined that 2.5 yg of protein could be detected by the disc gel electro-phoresis conditions used. Although the sensitivity of disc gel electro-phoresis can be increased by staining with Coomassie Blue instead of amido black (77), sensitivity was generally not the main problem. The large quantity of other materials present could obscure tumour specific bands, making comparison d i f f i c u l t . Disc gel electrophoresis would be of more use at a later stage of the studies as a step in purification or a test for purity, but at this time was not pursued. Further studies on the biochemistry of the TAA detected in C-26 were not done because the apparent uniqueness of this antigen limited i t s usefulness. IV. Immunodiffusion Studies 1. The Detection of TAA in Extracts Using Absorbed Rabbit Antisera One of the more common methods of demonstrating the presence of human TAA is with the use of antisera from animals hyperimmunized with tumour extract or tissue. The animals must be hyperimmunized in order to induce a detectable antibody response against the minor antigens present. Antigenic competition by the large quantities and number of normal antigens could inhibit the antibody response to the very low concentration of TAA. It has been suggested (55) that HL-A antigens comprise at most 1% of the total 77 membrane protein extracted by 3.0 M KC1. TSTA i s probably much less than 1% of the tumour extract because there i s probably much less TSTA than HL-A antigen. For a tumour extract with 10.0 yg'protein/ml, the maximum amount of TSTA would be less than 100 ug/ml. This quantity would be within the limit of sensitivity for the immunodiffusion technique of 1.0 yg protein/ml (35) only under optimum conditions. Since i t is unlikely that a high t i t r e anti (TSTA) serum would be produced by immunization with a crude extract, any TAA detected by this technique would probably not be TSTA. A. The Use of Immunoadsorbents The large quantities of antibodies to normal antigens must be removed from the antisera. This i s usually done by absorbing the antisera with normal cells and tissues. Large volumes of tissue and antisera would be required to absorb these antibodies. It can also be d i f f i c u l t to reproduce the absorption when studying human tumours, as the tissues do not store well and different tissues can vary considerably in their expression of antigenic components. Another disadvantage to the use of cells for absorbing antisera i s that internal c e l l components, against which antibodies may be developed, are not necessarily present on the c e l l surface, and w i l l therefore not be removed by such a procedure. Extracts, however, store well as measured by the reproducibility of immunodiffusion and disc gel electrophoresis results. A technique was developed whereby anti (normal) antibodies in the rabbit antisera were removed on a solid phase immunoadsorbent prepared by the coupling of normal lung extract proteins to cyanogen bromide activated Sepharose 4B. The antisera were then concentrated and tested for antibodies against tumour and normal extracts by semi-micro immunodiffusion. Examples of whole antiserum compared to absorbed antiserum are shown in Fig. 13a and' 79 1 4 Fig. 13d: Fig. 13: Immunodiffusion and Immunoelectrophoresis results using rabbit anti-C-26. a. Immunodiffusion of whole anti-C-26 against: 1. C-26 2. N-pool 3. C-30 4. C-40 5. C-24 6. C-41 b. Immunodiffusion of absorbed anti-C-26 against: 1. C-26 2. N-50 3. C-26-I (DEAE cellulose fractions) 4. C-40 5. C-24 6. N-pool c. Immunoelectrophoresis of absorbed anti-C-26 against: 1. N-pool and 2. C-26 Note that this absorbed antiserum appears to be thoroughly absorbed and that only one tumour antigen is resolved. d. Immunodiffusion of eluted 'anti-normal' antibodies from anti-C-26 serum against: 1. C-26 2. C-41 3. C-24 4. C-40 5. C-30 6. N-pool 13b respectively. The antiserum in Fig.Bbwas absorbed by one pass through the column. This can also be demonstrated by Immunoelectrophoresis (Fig.13c). That these results were not s t r i c t l y due to inactivation of antibodies was demonstrated by eluting the adsorbed antibodies and testing by immuno-diffusion as in Fig. 13d.. By eluting the antibody and re-equilibrating in starting buffer, the columns were used for up to 10 times. Most of the antisera could not be absorbed as completely as that in Fig. 13b,c. More representative results are demonstrated in Fig. 14. With many of the antisera, none of the methods tried could remove a l l of the anti (normal) antibodies. A range of volumes (0.5, 1.0; 2.0 and 4.0 ml) of 4 antisera were absorbed by a 25.0 ml immunoadsorbent column. Up to 2.0 ml could usually be absorbed with equal effectiveness, but even the small volume (0.5 ml) of some antisera were not completely absorbed. Larger immuno-adsorbent columns (70.0 ml) could handle 4.0 ml of antiserum but 0.5 ml of some antisera were s t i l l not completely absorbed. Pre-absorbing the anti-sera with normal lung extract by precipitation of immune complexes, and pre-absorbing the antisera with normal lung tissue were also not effective at eliminating a l l of the anti (normal) antibodies, (see Materials and Methods, section XIV)• The best results were obtained by a combination of the Sepharose immunoadsorbent followed by a batch immunoadsorbent using normal extract insolubilized by polymerization with glutaraldehyde (Fig. 15). Although the anti (normal) antibodies were often not completely eliminated even by this combination of methods, the diminished intensity of the reaction made visualization of tumour-specific precipitin bands clearer and the interpretation easier. If tumour associated bands could not be detected or i f the normal component bands were prevalent the antisera were re-absorbed a third or more times. The adsorption capacity of the glutar-aldehyde conjugated immunoadsorbents was considerably decreased after use, 81 5 4 3 Fig. 14a: + Fig. 14b,: Fig. 14: Immunodiffusion results using absorbed antiserum to C-40 against: a. extracts designated as follows: 1. C-40 4. C-6 2. N-50 5. C-26 3. C-24 6. PBS and Immunoelectrophoresis results against: b. 1. N-pool 2. C-40 Note that a tumour associated precipitin band can be resolved by counting the bands. 82 1 Fig. 15a: 6 4 Fig. 15b: 1 T Immunodiffusion results using absorbed antiserum to C-67 against six extracts designated as follows: 1. C-67 4. N-pool 2. N-pool 5. C-67 diluted 1:9 3. C-67 diluted 1:1 6. N-pool diluted 1:9 The antiserum in part a, was absorbed once through an immunoadsorbent of Sepharose 4B insolubilized N-pool. The antiserum in part b was re-absorbed with glutaraldehyde insolubilized N-pool. Note that the number and intensity of precipitin bands is less after re-absorption of the antiserum. 83 so were used only once. B. The Detection of Tumour Associated Antigens The r e s u l t s of a survey of 15 absorbed a n t i s e r a tested against a panel of extracts by immunodiffusion are presented i n Table I, according to tumour pathology. Each s e r i e s of 6 extracts included one sample of homologous extract and at l e a s t one normal or f o e t a l lung extract. The normal lung extracts tested were N-pool, N-25, N-50 and N-83. The extracts were used at the protein concentration attained by the o r i g i n a l e x t r a c t i o n . The majority of the extracts had protein concentrations between 5.0 and 15.0 yg/ml with the exception of normal extracts which were used at higher protein concentra-tions (see Appendix A ). The reason for using normal extracts at protein concentrations higher than the tumour extracts i n t h i s study was that i t was hoped that simple quantitative differences i n antigenic components between normal and tumour extracts might be resolved by t h i s approach. I t i s recog-nized that such a precaution would only eliminate f a l s e p o s i t i v e s of t h i s nature i f quantitative differences were i n the range of 5 f o l d (the d i f f e r e n c e i n p rotein concentration between normal and tumour e x t r a c t s ) . Greater differences would not be detected. However, i f consistant quantitative differences of t h i s magnitude were found and could be shown to be tumour rel a t e d , such an observation would s t i l l have p o t e n t i a l diagnostic value, even though the component might not be unique to the tumour. Several extracts and antisera were also tested at d i l u t i o n s ranging from 1:1 to 1:4, but i n no case could previously undetectable bands be seen, although some bands became sharper. The r e s u l t s from t h i s extensive study (Table I ) were not easy to i n t e r p r e t i n a l l instances. A f t e r a l l of the absorbed a n t i s e r a were tested, Table I: Immunodiffusion results using absorbed antisera to extracts from tumours of 5 main types of pathology tested against individual extracts of tumours of a variety of pathologies, a l l thought to arise from the lung. A positive result means that the precipitin band was not detected in any of the normal lung extracts (N-pool, N-25, N-50 and N-83). A '±' was used i f there was any reason to doubt a positive result. A ' i ' indicated that the precipitin band was closer to the central than the outer wells. a. Absorbed antisera vs. squamous c e l l carcinoma extracts b. Absorbed antisera vs. adenocarcinoma and anaplastic carcinoma extracts c. Absorbed antisera vs. oat c e l l and alveolar c e l l carcinoma, a variety of less common lung tumour, and foetal lung extracts 85 Table l a : Absorbed A n t i gen Extract Antisera Squamous C e l l Carcinoma Squamous C e l l Carcinoma C-l C-6 C-53 C-60 C-61 C-63 C-66 C-69 C-71 C-74 C-75 C-76 C-79 C-87 C-90 C-93 C-94 C-6 + + + ± • + _ + + • + + C-53 - + + + + + + + + + + _ + + + + C-57 - + +i +i +i +i -H-i +i +i +i +i +i .+i +i +i +i -l- i C-63 +i + +i +i +i +i +i _ +i C-66 - - - - - + - - - • _ _ C-71 + + - - + Adeno-carcinoma C-24 + _ C-26 - - - - - + - - - + - - - - -Anaplastic Carcinoma C-41 +i +i +i +i +i ' +i'' • +i +i +i +i +i +i +i +i +i +i C-46 C-62 - + +i +i +i + +i +i _ + + _ + C-67 — — +i - +i +i ' . ± + - - -Oat C e l l Carcinoma C-40 + + - + + + + C-78 + + + - - - + -H-i - - + + + - +i + Alveolar C e l l Carcinoma 86 Table l b : Absorbed Anti s e r a Antigen Extract Adenocarcinoma Anaplastic Carcinoma Squamous C e l l Carcinoma C-6 C-53 C-57 C-63 C-66 C-71 C-3 C-5 C-24 C-26 C-36 C-45 C-58 C-85 C-88 C-41 C-46 C-56 C-62 C-67 C-92 + + +i +i + +i +i + +i + + + + + + + + + + -+i +i +i +i +i +i + - +i +i -- - - - + Adeno-carcinoma C-24 C-26 Anaplastic Carcinoma C-41 C-46 C-62 C-67 +i +i +i +i + +i +i +i +i +i +i + H-H +i +i + +i + +i +i +i -H-i + +i Oat C e l l Carcinoma C-40 C-78 Alveolar C e l l Carcinoma C-30 87 Table It: Absorbed A n t i s e r a Oat C e l l Carcinoma Antigen Extract Alveolar C e l l Carcinoma a i\j 3 a 0 o 0 a a o o •a •H 3 'rH 3 O • H 4-1 O r-I CJ* M O a j a • a ) rjl r d c 4J O •H C/J 3 •U TJ O O ' t o c r o a ) a U 4J t o « a ) CJ 0) •H 13 o S s <: « 0) 60 Squamous C e l l Carcinoma C-40 C-6 4 C-65 C-70 C-72 C-73 C-30 C-81 C-4 C-54 C-32 C-77 F-48 C-6 _ + + + + . C-53 + • + - + + + + - _ _ + C-57 - - +i +i +i +i - f i +i +i -H-i _ +i +i C-63 - +i +i +i - + - _ +i +i _ C-66 - - ' - •- .' + - + _ C-71 - - - - - - - - - - - -Adeno-carcinoma C-24 _ + C-26 - - + - - - + - + - - + -Anaplast i c Carcinoma C-41 +i +i +i +i _ _ _ +i +i +i +i +i +i C-46 - - - - - - ± - - _ C-62 +i - + +i +i - + - _ _ _ + _ C-67 - - + - - + - +1 +1 + -Oat C e l l Carcinoma C-40 + _ _ + + + + _ _ _ C-78 - - - • - - +i - - - - + -A l v e o l a r C e l l Carcinoma C-30 - + - + + - + • - - - - -* metastasized from the lung to the mouth 88 the immunodiffusion slides were evaluated independently by myself, Anajane Smith and Professor Julia Levy. Positive results which were not definite were labelled "±" in order to minimize the possibility of including false positives in the study. It should be emphasized that false negative results are expected because of the limited sensitivity of the technique. (The pathology of the tumours was not known at this time). There was considerable variation from one antiserum to another in terms of the degree of cross-reactivity, the presence of any tumour specific bands and the number of apparently tumour specific antigens detected. Because of the marked differences between individual antisera in Table I each one w i l l be discussed briefly here. C-6. This antiserum was absorbed only by the column procedure and did not present any major problem in terms of anti-normal activity. It was a relatively weak antiserum and anti-tumour bands were faint (Fig. 16). A considerable degree of cross-reactivity with other squamous c e l l carcinoma and anaplastic carcinoma extracts was noted (Table I ). C-53. This antiserum retained considerable anti-normal antibody even after multiple absorptions, as seen in Fig.17. The immunodiffusion results were d i f f i c u l t to interpret, but anti-tumour bands which cross-reacted extensively with other types of tumour pathology were found (Table I). . C-57. Even after both column and batch absorption, this antiserum s t i l l had considerable anti-normal activity (as did most of the antisera). Further absorption decreased the intensity of a l l of the bands, not just the anti-normal bands. A tumour associated inner band ("i") could be observed towards the centre well (Fig.18). This antibody was very cross-reactive with a l l tumour types except adenocarcinomas, against which there was only moderate(3 of 9) cross-reactivity (Table I).'.Another tumour associated band was observed occasionally (C-57 and C-69 in Fig.18). Immunodiffusion results using absorbed antiserum to C-6 against six extracts designated as follows: 1. C-6 2. C-l 3. C-2 4. N-pool 5. C-3 6. C-5 Note the weak positive reaction against C-6 and C-l. Fig. 17; Immunodiffusion results using absorbed antiserum to C-53 against six extracts designated as follows: 1. C-53 2. C-57 3. C-58 4. N-pool 5. C-60 6. C-61 Immunodiffusion results using absorbed antiserum to C-57 against six extracts designated as follows: 1. C-57 2. C-66 3. C-67 4. N-pool 5. C-53 6. C-69 Note the inner band which shows a line of identity with a l l of the extracts but N-pool. 92 C-63. This antiserum was similar to anti-C-57, only there were more anti-normal bands and weaker tumour bands, making analysis more d i f f i c u l t (Fig.19). TAA were not detected as often with this antiserum as with anti-C-57 (Table I ) . C-66. This antiserum was very weak (in spite of hyperimmunization of the rabbit). A single absorption could remove almost a l l of the antibody. However, antigen was detected in one extract (C-45) and suspected in 5 others (Table I ). C-71. Anti-C-71 was stronger than anti-C-66, but was s t i l l too weak for a good analysis of the extracts. When the precipitin'bands are faint and not long enough to meet the bands of the adjacent wells, i t i s d i f f i c u l t to determine identity or non-identity of the components, and the bands can-not be compared (Fig.20). Nevertheless, TAA were detected in 3 of the squamous c e l l carcinoma extracts (Table I ). A possible explanation for the ina b i l i t y to detect TAA i n the homologous extracts i s given in the discussion of anti-C-62. C-24. This was a very weak antiserum from a rabbit not as intensively hyperimmunized as later rabbits. Absorption was easy and efficient (Fig.21), but TAA were detected i n only 6 of the extracts tested (Table I ) . C-26. This antiserum could be easily and ef f i c i e n t l y absorbed, and was much stronger than anti-C-24. More than one TAA could be detected with homologous extract (Fig.22), but these could only be detected with certainty in 4 of the extracts (Table I ), which limits the usefulness of the TAA. The infrequent cross-reactivity was in agreement with the disc gel electro-phoresis results (section I I I ) , where the tumour associated materials could not be detected in any of the other extracts tested. The antigen(s) was stable for several months whether stored at -20°C and freeze-thawed often, or stored at 4°C (sterile). 93 Fig. 19: Immunodiffusion r e s u l t s using absorbed antiserum to C-63 against s i x extracts designated as follows: 1. C-63 2. C-l 3. C-2 4. N-pool 5. C-3 6. C-4 Note the presence of p r e c i p i t i n band with C-63 and C - l , s l i g h t l y i n s i d e ( i ) the heavier outer bands. 94 Fig. 21: Immunodiffusion results using absorbed antiserum to C-24 against six extracts designated as follows: 1. C-24 2. C-26 3. F-48 4. N-50 5. C-2 6. C-5 This was a particularly weak antiserum which could be effectively absorbed, resulting in a single, faint precipitin band with C-24. Fig. 22: Immunodiffusion results using absorbed antiserum to C-26 against six extracts designated as follows: 1. C-26 2. N-pool 3. C-6 4. C-24 5. C-40 6. C-30 Note the presence of more than one precipitin band with C-26, one of which i s also found with C-30. 97 C-41. This antiserum was very similar to anti-C-57 in terms of the wide cross-reactivity of an inner precipitin band (Table I ). This anti-serum detected the presence of an antigen i n foetal lung extract which showed partial identity (by spur formation) with an antigen in C-63 (Fig.23). No other antisera showed any tumour associated reactivity with foetal extracts. Tests were done later to determine i f the TAA was carcinoembryonic antigen (CEA) or a.i-foetoprotein (AFP). The results (section V and-IV.2.) demon-strate clearly that the antigen detected by anti-C-41 was neither of these antigens. C-46. This was a f a i r l y strong antiserum (Fig.24), but i t could not irrevocably detect TAA. Frequent reactions of C-46 extract with other anti-sera (Table I ) suggest that a TAA was present in the extract. Several different immunoabsorbents (N-50, N-pool and a pool of a l l of the normal extracts prepared with both Sepharose 4B and glutaraldehyde) were used without success. These factors suggest that properties of the immunological system of the individual rabbits may be important in the production of anti-TAA. C-62. This was one of the more interesting antisera studied. Anti-normal reactivity could be removed effectively i f the antisera were absorbed more than twice. Inner bands were detected in 12 of the tumour extracts, but the strong outer band (Fig.25) was of far more interest. (Similar precipitin bands were observed with-anti-C-78). Finding such an antigen in an appreciable number (ie. 11, Table I ) of extracts suggests that this antigen might be of use for diagnostic purposes. Recent results have shown that the antigen of Fig.25 was not detectable i n extracts which had been freeze-thawed repeatedly, even though i t was readily detectable in extracts which had been stored at -20°C (Fig.26). Therefore, i t i s probable that this antigen i s even more common than indicated by Table I . (Thermal denaturation or enzymatic degradation could be responsible for the loss of 98 F i g . 23: Immunodiffusion r e s u l t s using absorbed antiserum to C-41 against s i x extracts designated as follows: 1. C-41 2. C-65 3. C-70 4. F-48 5. C-63 6. C-85 The p r e c i p i t i n band against C-63 shows p a r t i a l i d e n t i t y with an antigen i n F-48. None of the normal lung extracts demonstrated such a reaction. 99 Fig. 24: Immunodiffusion results using absorbed antiserum to C-46 against six extracts designated as follows: 1. C-46 2. C-l 3. C-2 4. N-pool 5. C-3 6. C-5 Immunodiffusion results using absorbed antiserum to C-62 against six extracts designated as follows: 1. C-62 2. C-77 3. C-78 4. N-pool 5. C-93 6. C-94 While strong precipitin bands developed against C-77 and C-94, no bands formed against homologous extract. 101 Fig. 26: Immunodiffusion results using absorbed antiserum to C-62 against six extracts disignated as follows: 1. N-pool 2. repeatedly freeze-thawed C-94 3. stock preparation of C-94, stored at -20°C 4. N-pool 5. stock preparation of C-77, stored at -20°C 6. repeatedly freeze-thawed C-77 Note the complete absence of detectable antigen in the freeze-thawed extracts. 102 antigen activity). The loss of antigen activity by freeze thawing could also explain the frequent non-reactivity of antisera with homologous extracts (Fig. 25), because they were freeze-thawed at least 5 times for the hyperimmuniza-tion of the rabbits. (The bulk of each extract was stored at -20°C and thawed only when a 10 ml aliquot had been used. This small sample was stored at -20°C but was frequently freeze-thawed). Because of the possibility of a change i n antigenicity upon storage, fresh normal lung extracts were regularly added to this study. C-67. Anti-C-67 was another antiserum which was d i f f i c u l t to absorb. Anti-normal activity persisted and anti-tumour activity was very weak, making analysis d i f f i c u l t (Fig.27). Both inner and outer bands were detected, and they cross reacted widely (Table I ). C-40. This antiserum was much like anti-C-67 in terms of the moderate cross reactivity, but inner bands were not observed (Table I ). Fig.28, which shows the tumour associated band for C-40, also clearly demonstrates the importance of using different normal lung extracts for controls. Some apparently lack antigens found in other normal extracts. C-78. This antiserum was very much like anti-C-62 except that inner bands were only detected in 3 extracts (Table I ). The loss of activity of antigens in the outer precipitin bands detected by this antiserum (as des-cribed for anti-C-62) could explain the ina b i l i t y to detect TAA in the homo-logous extract (Fig.29a), and the infrequent occurrence of outer band antigens in other extracts (Table I ). However, when the absorbed anti-C-78 was tested against the stock solutions of C-78 and C-62, antigenic activity was only detected in C-62. From the two sharp antigen bands in C-94, i t is apparent that two similar antigens were present (Fig.29a). A later test of absorbed anti-C-78 against doubling dilutions of C-94 extract from the stock solution (Fig.29b) detected antigen at a 1/4 dilution of C-94, although only 4. N-pool 5. C-90 6. C-92 104 Fig. 28: Immunodiffusion results using absorbed antiserum to C-40 against six extracts designated as follows: 1. C-40 2. C-53 3. C-54 4. N-pool 5. C-56 6. N-50 The arrow points to the tumour associated band-105 Fig, 29b: Fig. 29: Immunodiffusion results using abosrbed antiserum to C-78 against: a. six extracts designated as follows: 1. C-78 2. C-77 3. C-78 4. N-pool 5. C-93 6. C-94 b. six dilutions of C-94 in saline as follows: 1. C-94 undiluted 2. diluted 1/2 3. diluted 1/4 4. diluted 1/8 5. diluted 1/16 6. diluted 1/32 In part a. note the occurrence of two heavy precipitin bands against C-94. 106 one antigen was detected. Since this antigen was not detected in any normal extract even at f u l l strength (including the stock solution of N-83, the most recent normal lung extract), C-94 contains at least 4 times as much of this antigen as the normal extracts. C-30. Anti-C-30 demonstrated a strong precipitin band with homologous extract and a faint reaction with 15 other tumour extracts as in Fig.30a. Again, analysis was d i f f i c u l t . Further absorption could clear the antiserum of a l l detectable anti-normal activity, but this antiserum could not detect antigen in any extracts but the homologous (Fig.30b. The results of Table I suggest not only that TAA exist and are common to many tumour extracts, but also that at least two TAA may be present in several extracts. The percentage of positive results from Table I i s summarized in Table II according to tumour pathology. It should be noticed that these results can only be compared within each antisera column because of the wide variations in the antisera. There appears to be a correlation between tumour pathology and the degree of cross-reactivity as demonstrated by this technique, and that this correlation was better after the questionable positives were removed (Table II ). The results with the antisera against the squamous c e l l , anaplastic and oat c e l l carcinoma extracts were generally similar; the percentage of positive reactions was highest for extracts of these pathologies and lowest for adenocarcinoma extracts (Table II). The antisera against the adeno-carcinoma (and similarly the alveolar c e l l carcinoma although the number of samples was small) extracts reacted maximally (27%) with extracts of similar pathology and lower (0,5,and 13%) with extracts of squamous c e l l , anaplastic and oat c e l l carcinomas. The differentiation between these 2 groups of lung tumour pathology i s of particular interest because there i s l i t t l e or no relationship between the incidence of alveolar c e l l carcinoma and adeno-107 Fig. 3Qa: Immunodiffusion results using antiserum to C-30 absorbed with N-pool insolubilized by both Sepharose 4B and glutar-aldehyde and tested against six extracts as follows: 1. C-30 2. C-53 3. C-54 4. N-pool 5. C-55 6. C-56 Note the outer band with C-30 which shows a line of identity with a faint band near the C-53 well. 108 F i g . 30b: Immunodiffusion r e s u l t s using antiserum to C-30 absorbed twice as i n part a. and re-absorbed with N-83 i n s o l u b i l i z e d by Sepharose 4B, and tested against s i x extracts as follows: 1. C-30 4. N-83 2. C-79 5. C-82 3. C-81 6. C-85 Absorbed EXTRACTS Antisera (number) squamous c e l l carcinoma (17) adenocarcinoma (9) anaplastic carcinoma (6) oat c e l l carcinoma (6) alveolar carcinoma c e l l (2) . total only total only total only total only total + only anti-squamous c e l l 55 47 23 13 59 44 46 34 55 27 carcinoma (6) anti-adenocarcinoma 14 5 27 27 25 13 14 0 67 33 (2) anti-anaplastic 50 46 42 33 63 54 39 39 50 38 carcinoma (4) anti-oat c e l l 53 34 17 0 55 9 33 33 25 25 carcinoma (2) anti-alveolar c e l l carcinoma 50 19 33 33 17 0 50 33 50 50 (1) • Table II: A summary of the data presented in Table I showing the percentage of positive results (+) and positive plus equivocal (±) results. Because of the wide variations in the antisera, these results should only be compared within each row of antisera results. 110 carcinoma and known carcinogens (ie. as in tobacco), while as much as a 25 fold increase in the incidence of squamous c e l l , oat c e l l and anaplastic carcinomas (although this group may be heterogeneous) has been shown for smokers (66). Thus there would appear to be a relationship between TAA and aetiological factors i n lung cancer. On other factors which may be associated with the aetiology of lung cancer -. Watson (135) has suggested that viruses may be related to alveolar c e l l carcinoma on the basis of an increased incidence of v i r a l lung infections in the history of these patients. Further, Mohr et a l (57), using an antiserum against a pellet containing virus-like particles from human alveolar c e l l carcinomas, could detect (by immuno-diffusion) common TAA in the serum of patients with both alveolar c e l l carcinoma and primary adenocarcinoma of the lung. Because of the detection method used, negative results do not necessarily mean that TAA were not present in the extracts. The vast quan-t i t i e s of normal antigens could have impaired the anti-tumour response of the rabbits or the normal bands could have obscured the TAA precipitin bands. Also, the TAA concentration i n the extract could have been too low (either i n t r i n s i c a l l y or by degradation) to form a visible immune precipitation or to stimulate antibody production. In order to resolve the negative results and verify the questionable positive results, two approaches need to be taken. One approach requires the development of a more sensitive .detection method and the other i s either eliminating some of the normal antigen load in the tumour extract or minimizing the antibody response to normal antigens. The latter approaches should decrease the antigenic competition and allow a better antibody response to the TAA. The concommitant decrease in the amount of anti (normal antigen) antibodies should allow the immunoadsorbent tech-niques to absorb the antisera more efficiently. Poor sensitivity i s indicated by the lack of reactivity of some of I l l of the absorbed antisera and the general weakness of the anti-TAA reactions. This i s because the rabbits, the immunoadsorbents and/or the immunodiffusion techniques were probably being taxed to near their limits. Many of the anti-TAA reactions were diminished i f not eliminated even by a 1:1 dilution of either component. The faintness of the precipitin bands and the interference by normal components made i t d i f f i c u l t to determine whether the antigens we have called TAA are a qualitative or quantitative difference between tumour and normal extracts. If the antigens are common, a significantly different quantity between non-malignant and malignant diseases could be useful c l i n i c a l l y in a manner described in part VI of the introduction. If the TAA are unique or only rarely cross-reacting, their c l i n i c a l use i s not nearly as great. Immunodiffusion studies have been used to show the presence of tumour antigens in a variety of tumours. Baldwin <at a l (9 ) have used immuno-diffusion to confirm other assays for TAA in rat hepatomas. Bhattacharya et a l (13) have shown the presence of at least two TAA with some cross-reactivity and common antigens in carcinoma of the ovary (13). Lewis et a l (68) claim to be able to detect cancer in general by a simple immunodiffusion technique, although 'some serum factors which occur in abnormal but non-neoplastic conditions may have molecular spatial configurations stereoiso-merically similar to the cancer antigen'. Several workers have used immunodiffusion techniques to assay human lung cancer antigens with a variety of results. Okada and Ikeda (93) reported the detection of two TAA, one of which could also be detected at lower levels in normal tissue extracts. These antigens were chemically different from the two lung cancer associated antigens detected by Yachi et a l (142). The latter antigens were found widespread in extracts of tumours of different pathology and tissue of origin. One of the antigens (which was more frequently 112 associated with lung cancer than the other) gave a reaction of partial identity with an antigen in foetal tissue extracts which i s comparable to the results with anti-C-41 (Fig.23). A l l three of these reports used pools of tumour extracts for antiserum preparation, which has a great advantage in the ease of preparation and absorption of the antiserum. This study was not done with antiserum against pooled tumour extracts because i t was f e l t that a more complete study could be done with individual antisera against extracts of different tumour types. It was also f e l t that the length of time i t took to receive less common types (ie. alveolar carcinoma) of lung cancer (and their pathology reports) might have allowed considerable anti-genic changes in the f i r s t extracts collected. Mohr et a l (87), in an immunodiffusion study using absorbed antiserum against alveolar cell-culture supernatant, could detect antigen in the supernatants of cultures of tumour cells but not normal lung cel l s . 2. cti-f oetoprotein The finding that a TAA detected by absorbed anti-C-41 showed partial identity (Fig. 31) with an antigen in a foetal extract suggested that one (or more) of the TAA might be foetal associated. One of the possi b i l i t i e s was oti-foetoprotein (AFP). Extracts were tested against 3 dilutions of absorbed goat anti (AFP) by micro immunodiffusion. Large quantities of AFP were detected in the foetal extract but none in the tumour or normal lung extracts. These extracts were tested further for AFP by semi-micro immunodiffusion against a 1:2 dilution of the anti-AFP. The results in Fig. 31 show that AFP could not be detected in the tumour extracts by this method and on the basis of the large quantity of AFP in the foetal extract, the TAA detected in the tumour extracts with the absorbed antiserum were not AFP. 113 Fig. 31: Immunodiffusion results using purified goat antiserum to ai-foetoprotein (diluted 1:2) against extracts designated as follows: 1. C-53 2. N-pool 4. C-40 5. C-3 3. F-48 6. C-57 114 3. S t u d i e s on P a t i e n t s ' S e r a f o r D e t e c t i o n o f TAA U s i n g A b s o r b e d  R a b b i t A n t i s e r u m S i x o f t h e a b s o r b e d a n t i s e r a were t e s t e d a g a i n s t serum samples f r o m t e n l u n g c a n c e r p a t i e n t s and two h e a l t h y d o n o r s . P a t i e n t s ' s e r a i n c l u d e d p r e - o p e r a t i v e , p o s t - o p e r a t i v e and post-mortem samples. I t was hoped t h a t c i r c u l a t i n g TAA might be d e t e c t e d i n t h i s way, i f i t s c o n c e n t r a t i o n were h i g h enough. No TAA were d e t e c t e d i n any o f t h e s e r a . T h i s o b s e r v a t i o n was n o t s u r p r i s i n g s i n c e t h e i m m u n o d i f f u s i o n t e c h n i q u e used w o u l d be u n l i k e l y t o d e t e c t a n t i g e n l e v e l s l o w e r t h a n about 2.0 ug/ml ( s e c t i o n IV.5 £ ) . 4. S t u d i e s on P a t i e n t s ' S e r a f o r D e t e c t i o n o f A n t i b o d i e s t o TAA Oth e r i m m u n o d i f f u s i o n e x p e r i m e n t s t e s t e d human serum as a p o s s i b l e s o u r c e o f a n t i - t u m o u r a n t i b o d y . A n t i b o d i e s i n serum w h i c h can p r e c i p i t a t e a n t i g e n f r o m homologous tumour e x t r a c t w o u l d n o t be e x p e c t e d t o be a g a i n s t s e l f a n t i g e n s , a l t h o u g h t h e e x i s t e n c e o f a n t i - s e l f a n t i b o d i e s h a s b e e n s u g g e s t e d (110)to be r e q u i r e d f o r t h e mai n t e n a n c e o f immuno l o g i c t o l e r a n c e t o s e l f a n t i g e n s . I f such a n t i b o d i e s were p r e s e n t i n human serum, d e t e c t i o n w o u l d be u n l i k e l y b ecause o f t h e i r e x p e c t e d l o w c o n c e n t r a t i o n s . H a l l (40) s u g g e s t s t h a t a u t o i m m u n i t y may be a s s o c i a t e d w i t h n e o p l a s t i c d i s e a s e s . Such a n t i b o d i e s a g a i n s t s e l f w o u l d be o f g r e a t i n t e r e s t b o t h c l i n i c a l l y and i n t h e s t u d y o f n e o p l a s t i c d i s e a s e p r o c e s s e s . I f a n t i b o d i e s were d e t e c t e d , i t s h o u l d n o t have been d i f f i c u l t t o d i f f e r e n t i a t e between a u t o i m m u n e - a n t i -b o d i e s and T A A - a n t i b o d i e s by t e s t i n g a g a i n s t a u t o l o g o u s n o r m a l l u n g e x t r a c t . Serum samples from 14 c a n c e r p a t i e n t s ( a p p e n d i x c) and 2 h e a l t h y d onors were each t e s t e d a g a i n s t a l l o f t h e homologous tumour e x t r a c t s and 4 n o r m a l l u n g e x t r a c t c o n t r o l s by m i c r o - i m m u n o d i f f u s i o n . No p r e c i p i t i n bands were d e t e c t e d . 115 Anti-TAA antibodies in serum can be either freely circulating (6 ) or in the form of antibody-antigen complexes (10). In order to dissociate complexes which might be present and to separate the antibodies from the tumour antigens, the patients' sera (or the (NH^^SO^ precipitate of) were acidified and chromatographed on a Sephadex G-150 column equilibrated in 0.1 M HOAc, 0.15 M NaCl pH 2.8 (Fig. 4). Separation depends upon the antigen being included i n the column volume. Although Fig. 4 demonstrates that included and excluded fractions were not well separated, testing them against anti-human immunoglobulin (Ig) (Fig. 5) shows that the included fractions contained only small amounts of Ig. The excluded and included fractions were immediately neutralized and concentrated to the starting volume by ul t r a f i l t r a t i o n . Seven cancer patients' sera were fractionated in this way. Semi-micro immunodiffusion tests were done with included vs. excluded fractions, excluded fractions vs. tissue extracts, included fractions vs. absorbent-tumour-sera, and included and excluded fractions vs. whole sera according to Fig. 32 . No tumour specific reactions were observed. 5. Studies on Radioimmunodiffusion for Improvement of Resolution In an attempt to improve the sensitivity of immunodiffusion, a radio-immunodif fusion technique was developed. Instead of detecting immune pre-cipitates by a protein stain, which also stains protein not removed by the 125 washing procedure, the micro-immunodiffusion plates were reacted with I-conjugated anti-Ig after the original immunodiffusion had been run. Purified human Ig at seria l 1:4 dilutions was^.used as the test antigen and was run against purified rabbit anti (human Ig) antibody. The rabbit antibody had been previously purified by elution from a human Ig immunoadsorbent (pre-pared by insolubilization of human Ig with glutaraldehyde). Dilutions of 116 Fig. 32: Testing patient whole serum, serum fractions, tumour extract and absorbed antisera for antibody and antigen by immunodiffusion. Centre well - patient serum. 1. homologous tumour extract 2. S-a; high mw serum fraction from Sephadex G-150 in HOAc 3. S-b; low mw serum fraction from Sephadex G-150 in HOAc 4. absorbed antiserum (homologous for C-66 and C-71, anti-C-57 for others) 5. normal human serum 6. heterologous cancer patient serum The sera and fractions tested in this way were: S-53, S-66, S-68, S-71, S-89, S-90, and S-92 (see Appendix C). 117 the rabbit antibody were also tested. After thoroughly washing the plates 125 to remove unreacted components, a dilution of I-conjugated sheep anti (rabbit Ig) globulin was added to the central well only or a l l 7 wells, and incubated. After the washed and dried plates were exposed to X-ray film, they were stained with amido black for comparison (Fig. 33). Using purified components, this radioimmunodiffusion technique was not able to detect any precipitin bands not detected by the protein stain. In fact, the 125 addition of the I-conjugate made analysis more d i f f i c u l t because of the 125 heavy background from the remaining I-conjugate and the formation of new 125 precipitin bands with residual rabbit antibody and the I-conj.ugated sheep anti (rabbit Ig) (Fig. 33a ). The latter finding suggests that this method 125 could be very sensitive i f the whole plates were soaked in I-conjugated anti-Ig. However, this would require the preparation and handling of large 125 volumes of I-conjugate, which we were not prepared to do at the time. When the results of Fig. 33 were compared with identical dilutions of anti-125 body and antigen by semi-micro immunodiffusion (without reaction with I-conjugate), the semi-micro technique (Fig.34) was able to detect 2 yg protein per ml and the micro technique (Fig. 33b), only 50 yg/ml. Thus, the semi-micro technique was more sensitive both qualitatively and quantitatively, and was used for a l l immunodiffusion experiments unless otherwise indicated. The micro technique was frequently used for pre-testing dilutions of antigens and antisera. V".. Carcinoembryonic Antigen Assay Although early reports almost exclusively found carcinoembryonic antigen (CEA) in the serum of patients with digestive tract cancers, later reports showed that CEA could be detected in the serum of patients with 118 Fig. 33: Micro-fadioimmunodiffusion using the following serial 1:4 dilutions of human Ig as antigen in the outer wells: a. 6.0 mg Ig/ml b. 1.2 mg Ig/ml c. 0.25 mg Ig/ml d. 0.05 mg Ig/ml e. 0.01 mg Ig/ml f. 0.002 mg Ig/ml The rabbit anti-human Ig was added to the central wells of the patterns as follows: Patterns 1 and 4, undiluted; 2 and 5, 1:9 diluted; 3 and 6 1:100 diluted. 125 The I conjugated sheep anti-rabbit Ig was diluted 1:4 and 5.0 ul (20,000 DPM) was added to a l l of the wells in patterns 1, 2, and 3. A 1:19 dilution (5,000 DPM) was added to the wells in patterns 4, 5, and 6. Photograph a. The resulting X-ray film shows a very high background and human Ig was detected only in wells a, b, and c of pattern 1 and well a of pattern 4. Photograph b. After staining the original slide the backgrounds were low and human Ig was detected down to 0.05 mg/ml. The bands dejected near the antigen wells are probably due to a reaction between the I-conjugate and rabbit antibody which was not removed by the washing. 119 F i g . 33b: 120 Fig. 34: Immunodiffusion results using rabbit antibodies to human Ig against serial 1:4 dilutions of human Ig as follows: 1. 6.0 mg Ig/ml 2. 1.2 mg Ig/ml 3. 0.25 mg Ig/ml 4. 0.05 mg Ig/ml 5. 0.01 mg Ig/ml 6. 0.002 mg Ig/ml Note that a precipitin band can be seen adjacent to well 6. 121 other forms of malignancies and certain non-malignant diseases (1 ). Since many lung cancer patients have detectable circulating CEA (1 ), i t was decided to have the tumour extracts assayed for CEA by radioimmunoassay. The extracts f i r s t tested were of tumour and normal lung tissues from the same specimen. Normal lung tissues for extraction were taken as far as possible from the site of the tumour and were macroscopically free of tumour. The results of Table BI show high CEA levels in both tumour and normal extracts. (Normal plasma levels of CEA are 0 to 2.5 yg/ml (128)). Although this was originally interpreted to suggest that CEA was released from the tumour into ' the surrounding tissue (possibly via the serum), later experiments showed that high levels of CEA could also be detected in extracts of lung not associated with any malignant disease (Table IV)• Later CEA analyses were carried out to determine i f the TAA detected by other methods were CEA, and i f there was a relationship between the tumour pathology and CEA level. The CEA levels (Table IV) of the fractions of C-26 (described in section III) prove that the antigen(s) detected in C-26-I were not CEA. High levels of CEA in normal lung extracts (Table IV) are also reported by Tillack et_ a l (128). This finding, and the low levels of CEA in several tumour extracts prove that the common TAA(i) detected by immunodiffusion (Table I ) were not CEA. However, i t i s possible that some of the other TAA were CEA. The low levels of CEA in F-48 could be due to the method of induction of the abortions as discussed in section I I I . There was no apparent correlation between CEA level and lung tumour pathology (Table IV) which also i s in agreement with Tillack e_t a l (128). The osteogenic sarcoma extracts were included as negative controls because of the high CEA levels in normal extracts. Tumour E x t r a c t P r o t e i n C o n c e n t r a t i o n (mg/ml) CEA ng/ml Lung E x t r a c t P r o t e i n C o n c e n t r a t i o n (ng/ml) CEA ng/ml C - l 6.9 >25 N-1 22.4 21.8 C-2 17.6 >25 N-2 9.8 !>25 C-5 13.8 >25 N-5 17.2 >25 C-6 15.4 9.0 N-6 25.7 16.8 T a b l e I I I : Q u a n t i t a t i o n o f CEA i n u n d i l u t e d e x t r a c t s by r a d i o -immunoassay. The n o r m a l l u n g was f r o m t h e same i n d i v i d u a l as t h e tumour o f t h e same number, and was m i c r o s c o p i c a l l y f r e e o f m a l i g n a n t d i s e a s e . Normal p l a sma l e v e l s o f CEA a r e 0 t o 2.5 ng/ml. Squamous Cell Carcinoma Adeno-carcinoma Anaplastic Carcinoma Oat Cell Carcinoma Alveolar Cell Carcinoma Mixed Squamous Adeno-carcinoma Mesothelioma Rhabdomyo-sarcoma Squamous Metastasis Foetal Lung Normal Lung Osteogenic Sarcoma CEA CEA "CEA CEA CEA . CEA CEA CEA CEA CEA CEA CEA C-l 82 C-6 9 C-53 90 C-60 150 C-61 0 C-63 Al C-66 1 C-69 150 C-71 150 C-74 64 C-75 57 C-76 >150 C-79 150 C-87 17 C-90 53 C-93 40 C-94 15 C-3 57 C-5 >150 . C-24 >150 C-26 >150 C-56 120 C-45 42 C-85 58 C-88 58 C-41 38 C-46 24 C-56 150 C-62 150 C-67 18 C-92 1 C-40 50 C-64 1 C-65 90 C-70 150 C-72 120 C-78 150 C-30 >150 C-81 43 C-82 >150 C-54 >150 C-77 26 C-57 11 F-48 3 H-pool 120 N-25 44 N-50 98 N-83 36 C-68 0 C-73 1 C-91 0 C-96 0 Table IV; CEA content of extracts according to tumour pathology. CEA was measured by radioimmunoassay of undiluted and 1:5 diluted extracts. The CEA values are given in ng/ml and values greater than 100 ng/ml are approximations. Protein concentrations of the extracts are in appendix A. N3 124 VI. Immuno fluorescence The studies reported previously on the detection of TAA by immuno-diffusion were interesting and potentially important. However, several questions regarding the nature of these observations must be considered. In the f i r s t place, these antigens were detected in tissue extracts, and there was no information on their location on or in the tumour c e l l . While tumour specific materials within tumour cells may have significance i n diagnosis and/or treatment, those antigens located on the c e l l surface would appear to have greater potential i n terms of tumour immunology. Therefore, some probe into the location of these materials would be important. Secondly, these antigens were detected by immunization of rabbits with tumour extracts, and therefore no knowledge of their antigenicity in humans was achieved. Attempts to identify either these antigens, or antibodies to them in patients' sera by immunodiffusion, were unsuccessful: this i s not surprising because of the relative insensitivity of this technique and because of the low levels of either antibody or antigen one would expect to find in the patients' sera. For these reasons, further experiments were designed to develop techniques which would both probe the locations of these antigens and increase the sensitivity of the detection methods so that some conclusions could be drawn regarding the immunodiffusion data. The sections on immunofluorescence and autoradiography ( VII ) techniques address themselves to these issues. The rationale of the indirect immunofluorescence (IF) technique i s similar to that described for the indirect autoradiography technique in section VII. The indicator was anti-Ig conjugated with fluorescein instead 125 of I. Many problems were common to both techniques which were being used concurrently and they were similarly approached. When the absorbed antisera (anti-C-66, C-71 and C-78) were used, anti-125 body was bound to the normal lung and tumour tissue sections, but could not be shown to be tumour specific even i f the antisera were further re-absorbed. The poor quantitative nature of this technique meant that in order to detect differences in the binding of fluorescent label to the tissue sections, an antiserum with a higher t i t r e of anti-TAA and/or better absorbed would be required. Experiments using cancer patient and healthy donor sera as a possible source of antibody also did not allow the resolution of tumour associated differences in fluorescence. Two sources of background fluorescence were found to contribute to the problem of quantitation. There was back-ground from the binding of the conjugated-anti-Ig to the tissue and from autofluorescence of the tissue (possibly the connective tissue). The IF technique was pursued no further because the autoradiographic technique appeared more promising. IF techniques are commonly used for the demonstration of TAA. Baldwin and Glaves (7 ) and Borsos e^ t a l (17) report quantitation of the IF technique' by the inhibition of the fluorescent labelling of experimental animal tumour c e l l suspensions by the pre-incubation of antiserum with tumour extract. Studies on human tumours (89, 91) report the detection of TAA or c e l l smears or suspensions by patients' sera, although positive fluorescence by the normal controls implies that the results may be equivocable. Other workers (29) could not detect anti-TAA in patients' sera by IF techniques. Yachi e_t a l (142) reported preliminary results of positive fluorescent staining of lung tumour tissue sections (cryostat) and negative staining of normal tissue sections by absorbed xenogeneic anti-tumour serum. I have not been able to obtain this kind of result using a similar technique, although the incomplete absorption of the antisera was known to be a problem. Also, the work of Yachi e_t a l (142) was reported in 1968, arid they have not published any information on the subject since then. 126 VII. Indirect Autoradiography The indirect autoradiography (IA) technique was expected to be very sensitive, versatile and readily quantitated. Most studies of cellular antigens by indirect, labelled anti-Ig techniques (7, 17, 43 ) use c e l l suspensions, either fresh or from c e l l cultures. This was not considered practicable for the studies we wished to undertake for several reasons. Dead 125 cells and debris bind much higher levels of I-conjugate than viable ce l l s . This suggests that i f fresh tumour cells are used, they should be used immediately, as v i a b i l i t y decreases greatly with storage. This i s not suit-able for comparing samples over a period of time. Tumour c e l l cultures are not suitable because: i t i s d i f f i c u l t to maintain large numbers of different cultures, the cultures generally cannot be prolonged indefinitely, they are frequently overgrown by fibroblasts, and surface antigen properties can change during culture (89). Cells in culture may also contain various con-taminants, including mycoplasma and extraneous viruses (ie. from the foetal calf serum). Frozen sections were used because they could be stored easily, minimum antigen denaturation or loss was expected, and i t was hoped to be able to locate tumour antigens within the tissues and cel l s . Although i t was realized that the non-viability may lead to an increased background, i t was expected to be similar for normal control tissue sections and therefore could be eliminated in the quantitation. 1. Studies with Absorbed Rabbit Antiserum Tumour and normal lung tissue sections were incubated with a pre-determined dilution of absorbed rabbit anti (tumour extract) serum, then 125 tested for bound antibody with a pre-determined dilution of I-conjugated 127 sheep anti (rabbit Ig) globulin. After reacting with nuclear track emulsion and developing, the tissue sections were observed for grains. The grains were not only on the tissue sections, but also on the microscope slide glass i t s e l f (similar effects were also observed with plastic microscope slides). This was shown to be non-specific binding to the glass by the observation of grains when microscope slides with no tissue sections were incubated with 125 I-conjugate. So far i t has not been possible to demonstrate specific binding with any particular part of the tissues or cel l s . Welsh et^ al_ (138) have, recently reported a technique which may be of use for this type of study. 125 Instead of using radio-iodinated anti-Ig preparations, they used I-con-jugated protein A from Staphylococcus aureus, which has a high a f f i n i t y for the Fc portion of most mammalian IgG subclasses. In the attempts to minimize background grains, one of the absorbed antisera (anti-C-66) was re-absorbed u n t i l no more antibody was detectable by immunodiffusion. When this re-absorbed antiserum was tested undiluted by IA, the grains were far too numerous to count. A 1/50 dilution was required to lower the grain count to less than 10 grains/cell for both normal and tumour tissues. Thus the technique i s at least 50 times more sensitive in antibody detection than i s immunodiffusion. Also, the immunoadsorbents did not remove a l l of the antibodies against normal antigens. The i n i t i a l results with the dilutions of the absorbed antisera against C-66 usually showed significantly higher grain counts/cell on the tumour than on the normal lung sections. The specificity of the sheep anti (rabbit Ig) serum was demonstrated by immunoelectrophoresis against rabbit antisera and human tissue extracts as shown in Fig. 35 . To further test the 125 specificity of the binding of I-conjugate, experiments were carried out to test the inhibition of binding by pre-incubation of 10 ul absorbed anti-sera overnight at 4°C with 50 ul normal lung and tumour extracts. 128 Fig. 35: Immunoelectrophoresis results on the specificity of the sheep anti-rabbit Ig against: 1. N-pool 2. rabbit anti-C-71 Note that no antigen was detected in the human lung extracts. 129 From F i g u r e 36 i t c a n be seen t h a t when th e a b s o r b e d a n t i s e r a were p r e - i n c u b a t e d w i t h homologous e x t r a c t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t h e number o f g r a i n s / c e l l o f tumour and n o r m a l t i s s u e s e c t i o n s . P r e - i n c u b a t i o n w i t h PBS o r n o r m a l l u n g e x t r a c t s g e n e r a l l y r e s u l t e d i n s i g -n i f i c a n t l y more b i n d i n g t o t h e tumour s e c t i o n c e l l s t h a n t o n o r m a l s e c t i o n c e l l s ( F i g . 3 6 ) . These f i n d i n g s a r e c o n s i s t e n t w i t h t h e i d e a t h a t TAA were p r e s e n t on t h e tumour s e c t i o n s and t h a t t h e a n t i (TAA) a n t i b o d y a c t i v i t y c o u l d be a b s o r b e d f r o m t h e a n t i s e r a w i t h homologous e x t r a c t b u t n o t w i t h n o r m a l l u n g e x t r a c t s . When t h e t i s s u e s e c t i o n s were i n c u b a t e d w i t h d i l u t e d ( F i g . 36a,b) o r u n d i l u t e d ( F i g . 3 6 c ) n o r m a l r a b b i t serum, no s i g n i f i c a n t d i f f e r e n c e was d e t e c t e d between tumour and n o r m a l s e c t i o n s . On t h e b a s i s o f t h e p r o m i s i n g r e s u l t s o f F i g . 36 , a l a r g e r e x p e r i m e n t was c a r r i e d o u t t e s t i n g 4 d i l u t e d , a b s o r b e d a n t i s e r a and n o r m a l r a b b i t serum a g a i n s t 8 d i f f e r e n t t i s s u e s e c t i o n s . Data were a n a l y z e d by t h e method o f L e a s t S i g n i f i c a n t D i f f e r e n c e . No s i g n i f i c a n t b i n d i n g o f t h e l a b e l t o t h e tumour s e c t i o n c e l l s was o b s e r v e d . The g r a i n c o u n t s v a r i e d f r o m 1.5 t o 3.5 g r a i n s / c e l l compared t o 2.5 t o 9.5 g r a i n s / c e l l i n F i g . 36 . U s i n g a s i m i l a r t e c h n i q u e , Dr. I . B e r c z i ( p e r s o n a l communication) and h i s a s s o c i a t e s f ound t h a t t h e d i l u t i o n s o f the r e a g e n t s a r e c r i t i c a l f o r m a x i m i z i n g t h e tumour-n o r m a l d i f f e r e n c e s and t h a t many tumour c e l l s may have a n t i b o d y bound t o t h e i r s u r f a c e a n t i g e n s . They used a t e c h n i q u e ( f o r c o u n t i n g r a d i o a c t i v i t y bound t o a known number o f f i x e d c e l l s f r o m a c e l l s u s p e n s i o n ) w h i c h c o u l d be q u i c k l y and e a s i l y q u a n t i t a t e d . T h e i r t e c h n i q u e may be a p p l i c a b l e t o t h e s t u d i e s r e p o r t e d h e r e , a l t h o u g h two o b s e r v a t i o n s we have made w i t h t h e t i s s u e s e c t i o n s s u g g e s t t h a t s e r i o u s p roblems c o u l d a r i s e : c e r t a i n c e l l s ( p o s s i b l y macrophages o r mast c e l l s ) i n t h e t i s s u e s e c t i o n s bound v e r y h i g h 125 amounts o f l a b e l ( F i g . 37a) even i f o n l y t h e I - c o n j u g a t e was added, and p a t c h e s o f t h e t i s s u e s e c t i o n s o c c a s i o n a l l y had h i g h e r backgrounds t h a n 130 10.0 u \>5.0 Z < O a. .01 .05 C-66 PBS N-pool N-50 Extracts pre-incubated with ANTI-C-66 PBS NRS control .02 C-66 PBS C-67 Extracts pre-incubated with N-50 ANTI - C-66 01 N-pool .05 N-83 PBS NRS control 10.0 2 5.0 < U. O C. f + + + • + + + » 4 + + *•*** «- + + + *• + + • .01 .005 .005 .005 -71 PBS C-78 N-50 N-pool Extracts pre-incubated with A N T I - C - 7 1 N-83 + + + 4 + + + +• + + + • + + + + + + + + NRS control Fig. 36: Absorbed antisera tested for the binding of antibody to tumour and normal lung tissue sections by indirect autoradiography. The absorbed antisera were pre-incubated with PBS on the extracts indicated below each figure. The standard deviation (S.D.) for each grain count i s given and the probability (p) i s given where the difference in grain counts between tumour and normal tissue sections was significant by the Student's t test. • N-86 tissue section C-71 tissue section C-66 tissue section a,b. Absorbed anti-C-66 and normal rabbit serum (NRS) diluted 1:55 Absorbed anti-C-71 diluted 1:100 and NRS undiluted. 131 Fig.37a Fig.37b Fig. 37: Photographs of uneven indirect autoradiography results. a. This photograph was from a N-86 normal tissue section incubated with a 1/100 dilution of absorbed anti-C-78, but similar heavily grained cells were also found in some tumour sections and whether T O C I conjugated anti-rabbit Ig or anti-human Ig were used. b. This photograph was from a C-74 tumour tissue section incubated with PBS and ^ I conjugated anti-rabbit Ig. Note the higher grain counts in the top half. The cells in such areas were not counted. 132 surrounding areas (Fig. 37b). It was not practicable to determine the optimum experimental conditions and the experiment was not repeated because with the technique we used i t was very time-consuming and d i f f i c u l t to do accurate quantitation. It would appear that antiserum raised in rabbits to human tumour ex-tracts, no matter how carefully and thoroughly i t i s absorbed, w i l l continue to have levels of anti-normal components detectable by this technique and any experiments u t i l i z i n g this type of antiserum would be self defeating. However, because of i t s sensitivity, i t was. thought that the IA technique might be amenable to the detection of specific anti-tumour antibodies in the serum of patients. 2. Studies with Human Serum The tissue sections were incubated with lung cancer patients' sera 125 undiluted, followed by I-conjugated rabbit anti (human Ig) globulin, or 125 later, by purified anti (human Ig) antibody conjugated with I. However, the background counts remained high and a dilution of the conjugate was ire-quired so that the grains could be counted. To test whether the high grain count was due to human antibody already present in the tissue sections, they were incubated with non-conjugated anti-Ig and washed, then incubated with 125 the I-conjugate. No difference was detected whether or not the non-con-jugated anti-Ig was added, suggesting that pre-existing antibodies were not responsible for the high backgrounds. A major difference was observed between the absorbed rabbit antisera experiments and the human sera experiments: the human sera could be incubated with the tissue sections undiluted and the grain counts remained within a countable range. If this result i s compared to the 1/50 dilution required of the re-absorbed anti C-66 serum which had a l l of the detectable precipi-133 tating antibody absorbed, human serum contains less than 1/50 of the auto-antibody required for detection by immunodiffusion against the extracts. This technique was further studied in spite of the serious problems encountered because of i t s potential sensitivity and the small amounts of materials required suggested that i t might be useful c l i n i c a l l y . Anti-tumour antibody may be circulating freely in the blood, in which case i t would be present in whole serum and the (NH^)2^°^ precipitated globulin fraction or i t might circulate as a complex with tumour antigen. To dissociated immune complexes, the serum was chromatographed on an acidified Sephadex G-150 column as discussed in section IV.4. It has been shown that blocking factors (immune complexes?) may be present in tumour bearer serum, but when the tumour was removed, anti-tumour antibodies could be detected (43,120). Thus, an assay for this antibody could be a prognostic aid by determining complete tumour removal. A series of experiments was carried out to test patient whole serum, the serum globulin fraction, the G-150 excluded fraction and normal human serum for antibody which would bind to tumour and normal tissue sections. Each serum sample and i t s components, and normal human serum were tested against homologous tumour, heterologous normal lung (N-86), and another tumour as in Fig. 38. (Not a l l of the other tumour sections were counted). The grain counts/cell for each serum sample were analyzed by the Student's t test for the significance of counts on the tumour sections, compared to the normal lung sections. The results of Fig. 38' suggest that anti-tumour antibody (or immune complexes i n antibody excess) were present in the serum samples S-71 and S-68, as well as in the fractions, although i t could not be determined i f more antibody were present in the fractions. The increase in label bound to normal tissue sections with the serum fractions (Fig. 38 ) could be due to the decreased protein solubility observed in immunodiffusion studies (Fig. 10.0 a. z < DC o 5.0 P 10.0 I I -5.0 I + + + + !*•+++ . NHS •*• + +• + + • + + + + + + + + + + + + + + + + + + + + + + ++ + + . .005 S-71 + + + + ++++ ++++ *•+++ ± ± ± ± . a a a\ a a i a a i • • al • • Q| a a i + + + + + + + + + + + + + +++ + > + • + + + + ++ + + *-+++ • +•+ + +++ + + + + + Itttt_ | a - . i • • I I a o • I • a a I o a a l S-71-1 .01 .05 S-71-1a b. < O a a o • o o • • • NHS .005 S-68 .005 S-68-10.0 • i • 5.0 P 10.0 C. z < O a a a • a a o • • a n a gay • * • • • • • • • • • • .005 .005 NHS S-90 .01 S-90-1 S-90-1a d. z < Of o 5.0 L • M • a • • o • a • • • • a n • • Q a • • • • a • • • P 10.0 N HS S-89 S-89-a e. o ^ 5 . 0 z < DC o P • a _ • a • • • • H O B o -L a • DO z> a a a a a 3 • n .025 .005 NHS .01 .05 .05 S-92 S-92-1 S-92-1a F i g . 38: Indirect autoradiography assay for the binding of antibody from normal human serum (NHS) and cancer patient serum against homologous tumour reactions and heterologous normal lung and tumour tissue sections. Serum fractions were also tested. S - : serum sample 1 : (NH,)„SO. precipitate fraction 4 2 4 a : high mw fraction eluted from Sephadex G-150 column in HOAc. SD i s given for a l l grain counts and the probability (p) i s given where the difference in grain counts between tumour and normal tissue sections was significant by the Student's t test. N-86 a. S-71 vs. C-71 + + + • + + + C-71 b. S-68 vs. C-68 C-68 c. S-90 vs. C-90 * • • 1 * * a a a C-90 d. S-89 vs. C-89 a a • • o a C-89 e. S-92 vs. C-92 • • • • • mlm-d C-92 136 '5 ), or the presence of immune complexes not composed of tumour antigen-antibody. The latter possibility could be due to an autoimmune process, perhaps related to the neoplastic disease (40) • S-71 (Fig. 38a ) was a post-., mortem specimen and S-68 (Fig. 38b) was a 3 month post-operative specimen from a patient with multiple metastases already present (Appendix C). (Tumour specimen C-68 was an osteogenic sarcoma variant (see Appendix B) included because of the ava i l a b i l i t y of materials and because post-operative serum samples were d i f f i c u l t to get). Immune complexes could be present in such sera. There was no specific binding to the tumour sections by normal serum for C-71 and C-68 (Fig. 38a,b). Specific binding of label to the tumour sections was not detected with sera S-90 or S-89 (Fig. 38c,d). The results with S-92 (Fig. 38e) suggest that antibody might be present in this serum, but the low grain counts on the normal lung tissue sections by normal human serum makes this equivocal. The unaccountable differences occasionally encountered severely limit the usefulness of this technique as an assay for biochemical studies of tumour antibody or antigens, but i t may s t i l l be applicable for surveying different serum samples for tumour antibody. Because antibody could not be detected more readily with the serum fractions, only whole serum was used for subse-quent experiments. The i n a b i l i t y to detect more antibody with serum fractions could be because: tumour immune complexes were not present in the serum, the higher backgrounds could have interfered with quantitation, the antigen was large enough that i t was not separated from the antibody, or some of the antibody activity could have been lost during the fractionation procedure. A survey was set up to test 16 sera against 15 different tissue sections. The experiment was to test for the presence of antibody in the sera and for relationships between the antigens in the' tissues. S t a t i s t i c a l analysis for significance was by the Least Significant Difference method, 137 comparing the mean grains/cell in the presence of patient serum with the mean of normal human serum. The tumours include osteogenic sarcomas (C-68 and C-91) and a rhabdomyosarcoma (C-77) (Appendix B) which were added for extra controls. For more information on tumour pathology and the sera, see Appendices B,C. The results of Table V and Fig.39 suggest that the serum of cancer patients contains antibodies reactive with tumour sections, and that in some cases there i s a high degree of cross-reactivity, even with tumours of different pathology. Because of the latter cross-reactivity and because there appeared to be clustering of positive results associated with certain tumours, the results appeared somewhat suspect. However, a l l the specimens were frozen and stored under the same conditions and only the time of storage was different. The tumours which had the most cross-reactivity (C-68 to C-77) were stored the longest, so the clustering of positive results was not due to loss of antigenic activity. Also, these results (Table V ) are in agreement with those presented in Fig.38 regarding positive results with S-68 and S-71 (and S-92?) and negative results with S-89, S-90 and the normal control tissue sections, N-86. The results of Table V are also comparable to those presented by Morton e_t a l (59) and Moore jat .al (91) , using an analogous IF technique, in the demonstration of wide cross-reactivity-within malignant melanomas and sarcomas. The latter report (91) also found some cross-reactivity between sarcomas and carcinomas. In the studies reported here, there would have been an advantage in using normal control and tumour tissue from the same patient. Then the possibility of interpreting histocompatibility differences (eg. A,B,0. blood groups) as tumour specific would be eliminated. However, such an approach would not have been suitable for comparing different sera and tumour sections. Ideally, both homologous and heterologous normal tissue should have been used, but the d i f f i c u l t y of quantitation precluded this for the present. Sera Tissue Sections N-86 C-68 C-71 C-74 C-77 C-79 C-85 C-87 C-88 C-89 C-90 C-91 C-92 C-93 C-94 Normal MG 1.3 2.0 1.7 2.0 1.2 2.7 1.5 1.5 1.6 2.0 1.9 1.2 0.9 1.4 1.3 HM 1.3 1.9 1.7 2.3 1.7 2.1 2.1 1.7 1.3 1.3 1.6 1.3 1.0 1.6 1.3 Cancer Patient S-68 1.1 3.6 2.7 3.8 2.1 2.5 1.6 1.1 1.4 1.2 1.2 1.1 1.0 1.6 1.2 S-71 1.2 2.2 3.3 3.7 2.0 1.9 1.5 1.4 1.6 2.1 1.2 1.2 1.0 1.8 1.2 S-74 1.1 2.9 2.7 3.2 2.4 3.4 1.5 1.5 1.1 0.8 1.9 1.1 0.8 1.7 1.5 S-77 1.0 2.7 1.8 3.7 1.7 1.8 1.0 1.5 1.7 1.4 1.6 1.5 0.9 1.5 1.1 S-79 1.3 2.2 2.2 2.6 1.8 1.7 1.3 1.3 1.4 1.5 1.6 .9 1.1 1.4 1.8 S-85 1.1 2.3 2.3 1.8 1.3 2.0 1.2 1.2 1.4 2.1 2.1 1.8 1.8 2.1 1.6 S-87 1.4 5.1 3.1 4.4 3.0 2.3 1.9 1.6 1.9 2.2 1.7 1.7 1.3 2.0 1.6 S-88 1.1 2.3 "277 1.8 T77 2.6 1.6 1.7 1.2 1.6 1.6 1.3 1.1 2.1 2.1 S-89 1.3 1.8 1.9 1.9 1.1 2.7 2.3 2.1 1.1 1.0 1.8 1-5 1.1 1.9 1.4 S-90 1.1 1.7 2.0 2.7 1.1 2.8 1.9 1.7 2.1 1.9 1.3 1.7 1.3 1.8 1.9 S-91 1.2 1.8 2.0 2.7 1.0 2.0 1.4 1.7 1.8 1.1 1.7 1.2 1.4 1.5 1.2 S-92 . 1.6 2.9 3.0 3.4 1.4 2.2 2.0 1.5 2.3 1.7 1.1 1.9 1.4 2.0 2.0 S-93 1.3 1.8 2.1 '1.9 1.7 2.0 1.5 1.9 3.0 1.6 1.7 1.4 1.5 1.8 2.0 S-94 1.6 2.6 3.2 2.7 1.8 2.7 1.9 1.3 1.7 1.6 1.4 1.4 1.1 1.5 1.5 Table V; Indirect autoradiography assay for the binding of antibody from normal human sera and cancer patient sera to various tumour and normal tissue sections. Tumour pathologies are presented in appendix B and information on the sera in appendix C. The results are in grains per c e l l . The significance of the grain counts with patient serum com-pared to normal serum for each tissue section was evaluated by the method of Least Significant Difference. 139 Fig. 39: Photographs of indirect autoradiography results with serum 68 tested against: a. C-68 tumour tissue section and b. N-86 normal tissue section. 140 The IA technique has severe limitations when used with tissue sections. The expected sensitivity was not achieved because of the high backgrounds and the necessity to use dilutions of the test antiserum. The d i f f i c u l t y i n quantitation precluded multiple testing and the inclusion of some controls. It was also d i f f i c u l t to define the experimental parameters which would allow specific reactions to be detected maximally and to keep backgrounds to a minimum. It was not possible to locate antigens within the tissue or ce l l s . It i s possible that i f viable single c e l l suspensions could be used from either fresh tumour or tissue cultured cells for these studies, many of these problems could be eliminated. Unfortunately, the use of normal lung cells as controls, as well as tumour preparations, would be exceedingly d i f f i c u l t at present. VIII. Skin Tests of Patients with Extracts of Their Own Tumours Perhaps the simplest, most direct assay for TAA i s to skin test cancer patients with extracts of their own or heterologous tumours. Although skin testing i s a less sensitive method of detecting cell-mediated immunity than some in vitro tests (72), i t more closely parallels the c l i n i c a l state of the patient (50,83). The latter r e s u l t - i s possibly due to components such as blocking factor and immunosuppressive factors produced by tumours, which may operate in vivo but would be washed from the effector cells or diluted in vitro, or to a possible requirement for tissue structure. Therefore, besides assaying for TAA, this technique could serve as a diagnostic and prognostic aid as well as monitoring the effects of chemotherapy and/or immuno therapy. For ethical reasons and because of the possibility of detecting histo-compatibility differences, we did not wish to inject patients with extracts 141 of tumour or normal tissue from other patients. Similarly, injection of healthy persons with any of the extracts was out of the question. In order to control the experiments, the patients were skin tested with extracts of their own tumour and DEAE cellulose fractions which were concentrated to the original volume of the extract. Autochthonous normal lung extracts were not available when these experiments were done, but future work should include them. The expected positive result would be a skin test reaction against the whole extract and an equal or smaller reaction to one (or possibly more) of the fractions. The f i r s t patients skin tested with extracts at DEAE cellulose fractions of their tumours were osteogenic sarcoma patients about to receive immuno-therapy (BCG plus acetoacetylated tumour extract). Patient 68 was tested with undiluted (Fig. 40a,b) and 1/10 diluted (Fig. 40c,d) materials to determine the effects of protein concentration on cutaneous reactivity. Induration and erythema was measured after different time intervals to aid in determining the types of response. There appears to be l i t t l e effect of protein concen-tration on skin test reactivity over the range tested (Fig. 40), except for the reaction to C-68-Ace (acetoacetylated extract) which was probably non-specific due to the high protein concentration of 21.0 mg/ml. Other workers (53) have reported non-specific reactivity to materials over 3.0 mg protein/ml, but C-68 at 8.6 mg/ml (Fig. 40) induced no delayed skin reaction at a l l . Since a classical delayed hypersensitivity reaction reaches a maximum at 24 to 48 hours, the reaction observed against C-68 is probably a residual immediate reaction instead (specific or non-specific), although i t i s possible that part of the reaction at 24 hours was cell-mediated. It. should be noticed that the reactivity to some of the fractions was greater than to the whole extract which contained much more protein. This finding was common to a l l of the patients tested. 09 -f>-O Pi E R Y T H E M A D I A M . ( cm ) o -INDURATION D I A M . ( c m ) o ro I 01 X ro A . I CO PROTEIN CONC. (mg / m l . ) -4>-i—i—O-O—i—1—L®L . ro on eo PROTEIN C O N C . ( m g / m l ) ro o E R Y T H E M A D I A M . ( cm ) _* ro o o -O-—D Q-- i ^ j — i — o-a— i — i — L Q i—//oi ro o oo -» ro P R O T E I N C O N C . ( m g / m l ) ° b 143 Hrs 6 Hrs 24 Hrs 48 Hrs Fig. 40: Skin tests of osteogenic sarcoma patient with whole extract (C-68), DEAE cellulose fractions (I, II and III), and aceto-acetylated (Ace) extract of autochthonous tumour. • C-68, whole • C-68-I • C-68-II O C-68-III ' . 9 C-68-Ace The dashed line represents a borderline reaction. The protein concentrations of the skin test materials are given on the right. a. Diameter of erythema with undiluted materials b. Diameter of induration with undiluted materials c. Diameter of erythema with 1/10 diluted materials d. Diameter of induration with 1/10 diluted materials 144 Similar results were obtained with another osteogenic sarcoma patient, 96. Patient 73 gave a maximal reaction to two of the fractions at 24 hrs. (Fig.41). While the complete lack of a reaction at 48 hrs i s not typical of the time course of delayed cutaneous hypersensitivity, the increased induration at 24 hrs (Fig.41b) shows that a residual immediate reaction was not respons-ible and suggests that the reaction was delayed hypersensitivity. Note that the protein concentration of C-73-III was only 1/5 that of C-73-Ace. (It may be of interest that this patient is c l i n i c a l l y free of disease over a year since surgery). The results of Figs.40 and 41 show a good correlation between erythema and induration, and appear in agreement with reports that a negative reaction can be defined as less than 1.0 cm diameter of erythema ( i f induration i s also present)'(123)or less than 0.5 cm diameter of induration (94,137). Three bronchogenic carcinoma patients were skin tested with dilutions of extract and fractions of their tumour. Although the results were not quantitated, they can be summarized as follows: No reactions were observed at 48 hrs and no reactions were observed against the 1/10 dilution after a few hrs. There were no reactions to any dilution of or undiluted C-67 materials after a few hrs. There were no reactions to the 1/5 dilution of C-57 materials but there was a positive reaction to the undiluted C-57-I and III at 24 hrs. There were positive reactions to 1/5 diluted and undiluted C-53-I and II at 24 hrs. These results clearly paralleled those of the osteogenic sarcoma patients (Figs. 40 and 41) and suggested that more work should be done to 145 a. 2 Hrs 6 Hrs 24 Hrs 48 Hrs b. 2 Hrs 6 Hrs 24 Hrs 48 Hrs Fig. 41: Skin tests of osteogenic sarcoma patient with whole extract (C-73),.DEAE cellulose fractions (I, II and III) and aceto-acetylated (Ace) extract of autochthonous tumour. • C-73, whole • "C-73-1 • C-73-II O C-73-III £ C-73-Ace The dashed line represents a borderline reaction. The protein concentrations of the skin test materials are given on the right. a. Diameter of erythema b. Diameter of induration 146 determine the cause of the delayed skin reactions to some of the f r a c t i o n s but not the whole extracts. To test the f r a c t i o n s for the presence of non-specific skin r e a c t i v e f a c t o r s , 4 extracts and t h e i r f r a c t i o n s were tested undiluted i n non-immune guinea pigs before t e s t i n g i n the patients. The r e s u l t s of 3 of the animals were s i m i l a r to Fig.43, and the fourth gave reactions to C-76 and f r a c t i o n s , which were a l l near 1.0 cm diameter. This i s not a good test for non-specific factors because a l l of the materials stimulated a reaction although some of them were negative i n the patients (Fig.43). However, i n 3 of 4 guinea p i g s i the r e a c t i o n was somewhat greater to f r a c t i o n I I I than the whole extract, although the whole extracts contained more protein (Fig.42). On t h i s basis, there could be non-specific skin r e a c t i v e factors i n f r a c t i o n III of the extracts, but the time course of the reactions (Fig.42) does not suggest a delayed h y p e r s e n s i t i v i t y reaction. The r e s u l t s of the patient skin tests with these materials are presented i n Fig.43. Patients 87 and 85 who f a i l e d to give a p o s i t i v e reaction (at 24 hrs) to t h e i r tumour materials (Fig.43a,b ), also did not respond to any of the r e c a l l antigens, thus demonstrating impaired immune responsiveness. Similar r e s u l t s have been reported by others (94,137). The time course of the r e a c t i o n of patient 72 to the f r a c t i o n s (Fig. 43d) suggest that i t i s a r e s i d u a l immediate reaction, possibly with no delayed h y p e r s e n s i t i v i t y at a l l . Again, however, there i s some reaction against the f r a c t i o n s and not the whole extract.-The time course of the r e a c t i o n of patient 76 to f r a c t i o n II and I I I (Fig. 43c) i s suggestive of delayed h y p e r s e n s i t i v i t y , while the reaction to C-76 i s smaller and p o s s i b l y not delayed h y p e r s e n s i t i v i t y . These r e s u l t s are d i f f e r e n t from studies reported on two transplantable methylcholanthrene-induced guinea pig sarcomas (55). The animals gave a Skin tests of a non-immune guinea pig with C-87 extract and DEAE cellulose fractions. The protein concentrations of the skin test materials are given on the right 0 C-87, whole • C-87-I A C-87-II O C-87-.HI 2 Hrs 6 Hrs 24 Hrs Fig. 43: Skin tests of bronchogenic carcinoma patients with extracts and DEAE cellulose fractions (I, II and III) of their own tumours. 9 whole extract • fraction I A fraction II O fraction III a. patient 87 b. patient 85 c. patient 76 d. patient 72 150 delayed hypersensitivity reaction to whole extracts and one of the fractions upon skin testing with tumour extract prepared by sonication of tumour cells and 5 DEAE cellulose fractions. The reactions were specific for the individual tumours and although the positive fractions were eluted under different con-ditions, both would be eluted from the DEAE cellulose under the conditions for the preparation of fraction III of Figs.40 to 44 . Other workers (83,132) using whole 3.CM KC1 extracts of human tumours, were able to detect positive delayed cutaneous hypersensitivity i n approximately 50% of the immunocompetent patients tested. Vanky et a l (132) also tested patients with3.0MKCl extracts of normal tissue and found a few weak reactions, suggesting the possibility that the reactions were not tumour specific. In a study by Wells et^ al (137), 7 of 14 immunocompetent lung cancer patients demonstrated delayed cutaneous hypersensitivity to autologous tumour c e l l membranes. However 2 of these 7 patients also reacted to autologous leukocyte membranes and 1 reacted to normal lung c e l l membranes. Hollinshead et^ a l (53) detected no delayed hypersensitivity to immuno-competent lung cancer patients skin tested with tumour c e l l membranes. How-ever, 50% of these patients demonstrated delayed cutaneous hypersensitivity (confirmed by histology) to Sephadex G-200 fractions or gradient polyacrylamide gel fractions of sonicated membrane extracts. (There was no indication (53) that any patients were tested with the sonicated membrane extract before i t was fractionated). Their results correlate very well with those presented-here in Figs. 40, 41, 43, 44 although the extraction and fractionation methods were different. They also compare their lung cancer results (53) with the results of their studies on different types of tumours under the same conditions. Membranes for intestinal cancer and leukaemia cells induced delayed hyper-sensitivity i n the respective patients, while the reactions to membranes from breast cancer and malignant melanoma cells were weaker and less frequent. 151 There i s no e x p l a n a t i o n g i v e n f o r the d i f f e r e n c e i n r e a c t i v i t i e s o f d i f f e r e n t t y p e s o f tumours, b u t i t i s s u g g e s t e d t h a t t h e poor r e a c t i v i t y t o t h e membranes i s due t o b l o c k i n g f a c t o r s w h i c h were removed from t h e a n t i g e n s i n t h e f r a c -t i o n s . ( B l o c k i n g f a c t o r s were p r o b a b l y n o t r e s p o n s i b l e f o r t h e l a c k o f r e a c t i v i t y o f the 3.OM K C l whole e x t r a c t s o f F i g s . 40,41,43,44 ) because t h e DEAE c e l l u l o s e f r a c t i o n a t i o n p r o c e d u r e would n o t be e x p e c t e d t o d i s s o c i a t e a n t i g e n - a n t i b o d y c o m p l e x e s ) . Some r e a c t i o n s were a l s o o b s e r v e d when p a t i e n t s were t e s t e d w i t h a u t o l o g o u s and h e t e r o l o g o u s n o r m a l l u n g e x t r a c t f r a c t i o n s (53), a l t h o u g h i t was p o s s i b l e t o d i f f e r e n t i a t e between l u n g s p e c i f i c and tumour s p e c i f i c a n t i g e n s . A v e r k i e v a and T r a k h t e n b e r g (3 ) a l s o r e p o r t f r e q u e n t d e l a y e d c u t a n e o u s h y p e r s e n s i t i v i t y o f l u n g c a n c e r p a t i e n t s t o p r o t e i n - c o n t a i n i n g tumour e x t r a c t f r a c t i o n s and l e s s f r e q u e n t r e a c t i v i t y t o n o n - m a l i g n a n t t i s s u e e x t r a c t f r a c -t i o n s . They d e t e c t e d r e a c t i o n s t o tumour e x t r a c t f r a c t i o n s by 2 o f t h e 130 n o r m a l s u b j e c t s t e s t e d . I n a r e c e n t s t u d y on b r e a s t c a r c i n o m a a n t i g e n s , H o l l i n s h e a d e t a l (51) r e p o r t poor d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s t o tumour c e l l membranes, and s o n i c a t e d membrane e x t r a c t s , and f r e q u e n t s t r o n g r e a c t i o n s t o some f r a c t i o n s o f t h e e x t r a c t s . Four o f 8 p a t i e n t s r e a c t i n g t o tumour e x t r a c t f r a c t i o n s a l s o r e a c t e d t o s i m i l a r f r a c t i o n s o f n o r m a l t i s s u e e x t r a c t . F u r t h e r f r a c t i o n a t i o n by g r a d i e n t p o l y a c r y l a m i d e e l e c t r o p h o r e s i s c o u l d d i f f e r e n t i a t e between a no r m a l t i s s u e a n t i g e n and a t u m o u r - a s s o c i a t e d a n t i g e n (51). R e a c t i v i t y t o n o r m a l e x t r a c t was a l s o d e t e c t e d i n t h e human p e r i p h e r a l b l o o d MIF s t u d i e s ( I I ) . Because some o f t h e r e c e n t s t u d i e s on d e l a y e d c u t a n e o u s h y p e r s e n s i t i v i t y t o tumour e x t r a c t s (51,53,132 ) c l e a r l y show t h a t some o f t h e r e a c t i v i t y was a g a i n s t n o r m a l a n t i g e n s , and because t h e p a t i e n t a c c e s s r e q u i r e d t o c l e a r l y e s t a b l i s h whether t h e s p e c i f i c i t y o f t h e r e a c t i o n s a r e due t o o r g a n , v i r u s , 152 or tumour antigens was not a v a i l a b l e here at the time, t h i s approach was deemed unsuitable as an assay for biochemical and immunochemical studies of tumour antigens. Nevertheless, I have t r i e d to demonstrate i f i t i s even poss i b l e that the r e a c t i v i t y seen against some of the extract f r a c t i o n s (Fig. 41,43c) i s against tumour antigens. I t i s possible that greater r e a c t i v i t y to some of the f r a c t i o n s than the whole extract was due to: Contamination of the fractions (ie. chemical or bacterial). Modification of existing materials (eg. by exposing an antigen or a non-specific skin reactive factor). An immune suppressor (specific or non-specific) present in the extract, which is capable of inhibiting skin reactions to tumour and/ or normal antigens. Such a suppressor would be separated from anti-gen or lost by the fractionation procedure. To test these p o s s i b i l i t i e s , patients were skin tested with 1:2 dilutions (in 0.15 M NaCl) of the fractions and extract of their tumour and a 1:1:1 dilution of the 3 DEAE cellulose fractions. A l l 5 patients tested were shown to be immunocompetent by reacting to at least one r e c a l l antigen. Erythema and induration were measured at 2 and 24 hrs, but only the diameter of^induration is presented in Fig.44 because the primary concern is the delayed hypersensitivity reaction. The results of Fig.44a,b show positive reactions only against the mixture of the fractions. This is not readily explainable, but does show that the fractionation procedure produced a change in these extracts which could not be reversed by reconstitution. The results of Fig. 44c,d,e are much more interesting. (It should be emphasized that the mixtures contained as much of each fraction as the dilution of the fractions used for skin testing). From Fig.44c i t can be seen that C-87-I and C-87-II INDURATION DIAM. ( cm ) HQ I 0 ) - 0 — O O - - a — PROTEIN C O N C . (mg / m l ) t—1 to CO IB o • • • to s! -P~ CT H M r-l kin M Hi Hi Ml H- r-l rt H l-i cr rt H" ** ro P> P) ft) o cr cn cn M n o n M P> rt H rt rt rt cn 3 CO H- H- H- P) l-i CL O O O M ro o H 3 3 3 H- CO H i-h M rt 3 ro H M r-l M M H ro 3 H cr • M H ^. P> • rt *. ' l-i H V • O ro «* o \a • I-1 1—1 rt CL ft) nc M ro l-> cr 3 cr •. to •. ID P. o ro ro H 09 ro £L o cr . ft) XJ X) XI ft) ft) P) ft). ft) rt rt rt rt rt H- H- H- H-ro ro ro ro ro 3 3 3 3 3 rt rt rt rt rt CT\ 0 0 0 0 VO l_n O oo LO rt ro cr 3 HI ro H-o cn n ri ro n ft) ri o 3 o 3 p> ro n ft) i-i ^ O • 3 CO XI H 9 ft) CT H> rt ro x H-ro ro CL 3 rt I—1 CO cr o ro (-1 si ro h-1 rt x • cr rt ri ro P O X n 3 rt rt M i-i cn ft) n ft) rt rt .3 CT cn a. ro -rt P . a cr H- w ro ft) ' S M I ro ri rt p> ft> ro n i-i h-1 rt M H- o c O Ml h-1 o cn ro > n H-3 CL C M M l-i ro ft) ft) rt O &. ro i-i o 3 o 3 rt ft) cn ro rt CL INDURATION DIAM. ( cm ) ro. o ro • ar - C 3 0 — L ro o PROTEIN C O N C . ( m g / m l ) INDURATION D I A M . ( c m ) CO o ->-C- @- -67 PROTEIN C O N C . ( m g / m | ) INDURATION DIAM. (cm ) - 0 -i -£—ao -1- a ' © — i PROTEIN C O N C . ( m g / m l ) INDURATION D I A M . ( c m ) (5 - r - ro o CD X X ) -PROTEIN C O N C . ( m g / m l ) 154 could eliminate the 2.0 cm reaction to C-87-III and that the mixture contains 3 times the protein concentration of C-87-III. C-88-II could reduce a 1.0 cm reaction against C-88-I and C-88-III to 0.5 cm (Fig. 44d) • A 1.0 cm reaction against C-60-III was eliminated by mixing with C-60-I and II (Fig.44e). Thus, a factor(s) in fraction I and/or II (II in the case of C-88) was shown to be capable of eliminating or diminishing a delayed skin reaction to autologous tumour extract antigens. The results presented here (Fig.43c,44d ) and the results of others (53) have shown delayed skin reactivity to more than one lung tumour extract fraction. This suggests that either more than one antigen was present or that the antigen was present in different forms, such as aggregated or in associa-tion with other materials. Although i t i s possible that normal tissue anti-gens were present, the detection of reactivity to more than one fraction (53) when no reactivity was detected against normal antigens suggests that in some lung tumours more than one tumour antigen may be present. This would support the results from the studies by immunodiffusion with absorbed anti (tumour extract) serum (IV.l) and indirect autoradiography with patient serum (VII.2.). Whether the antigens are tumour or normal tissue associated, the detection of a factor which can diminish a cell-mediated immune response in  vivo could have tremendous implications for the immunotherapy of patients with malignant diseases. Further studies on the nature of this factor(s) are presently under investigation. These studies are beyond the scope of this thesis and w i l l not be included. 155 GENERAL DISCUSSION The original goal of this project was to'definitively study the biochemical and immunochemical properties of antigens associated with human bronchogenic carcinoma. The absence or the presence of common or unique antigens could t e l l whether immunotherapy might be feasible and i t is possible that purified tumour antigens could be used for specific immunotherapy. Ultimately, immunization methods might be developed which would protect against malignant diseases. If the TAA are common or cross-reactive (but not necessarily antigenic in the patient), the detection and quantitation of antigen in the serum could have diagnostic and prognostic value and possibly allow the screening of large numbers of high risk individuals for cancer. This last factor would be of particular value for lung cancer because high risk patients are known and lung cancer diagnosis is often late in the course of the disease.. Such tests would require purified antigen and highly specific antibody. Studies on the biochemical and immunochemical relationships of TAA could supply information about the possible causative agents for malignant diseases, such as the studies on RNA virus antigens and human leukaemia (36,116) and information about the nature of oncogenesis, such as the relationship between histocompatibility antigens and TSTA. One of the chief problems in this study arose from misleading reports in the s c i e n t i f i c literature. The method of inducing neonatal tolerance in rabbits was shown to be ineffective in this study and by other workers (Dr. J. Berczi, Department of Immunology, University of Manitoba, personal communications). The significance of certain immunofluorescence techniques might also be questioned because they measure the percentage of fluorescent labelled cells in a population rather than the amount of fluorescence on 156 each c e l l . There are many reports of tumour antigen detection using cancer patients' immune responses, such as the MIF test and delayed cutaneous hyper-sensitivity. However, the MIF test results reported in this study and the skin test results of others (51) where normal control results were reported, demonstrated that the reactivity to tumour materials was not necessarily tumour specific (although careful analysis could resolve tumour specific reactivity (51)). Such non-tumour-specific reactions to tumour materials could be autoimmune phenomena, perhaps related to malignant diseases as suggested by Hall (40). The detection of immune responses of cancer patients to tumour antigens is a prerequisite for studies on TSTA. The skin test results reported here suggest that tumour antigens may be present (although tumour specificity was not ascertained) in some of the tumour extracts, but were not detectable u n t i l the extracts were fractionated on DEAE cellulose because of an immuno-logical inhibitor which could be separated from the antigen. The results of the indirect autoradiography studies (Table V ) where tissue sections were reacted with patients' sera,do not appear to be due to autoimmunity because the normal lung tissue sections and the two normal sera were not associated with significant binding of radioactivity. However, many more normal controls would need to be studied before the possibility of auto-immunity could be eliminated. An interesting observation from Table V i s the negative results with some homologous sera where heterologous sera gave a positive reaction. It i s certainly possible that false positive and nega-tive results can occur or that antibody might induce a change in phenotypic expression of immunogenicity (57), but the non-reactivity with homologous serum could also suggest that multiple antigen systems are present. This would be in agreement with the results of the immunodiffusion studies using absorbed antisera (section IV.1.) Non reactivity in the homologous system 157 when reactions were detected in a heterologous system was also reported by McCoy et a l (75), using an MIF technique to study extracts of human breast cancer. The large quantity of normal antigen and the low concentration of TAA in the tumour extracts were the main problems i n the detection of TAA by xeno-antisera. Although methods of inducing tolerance to normal antigens were unsuccessful, some good results were obtained by using solid phase immunoadsorbents to remove antibodies to normal antigens from the serum of rabbits hyperimmunized with tumour extract and testing these absorbed anti-sera by immunodiffusion against normal lung and tumour extracts. The system reported here probably demanded too much from the techniques because anti-bodies to normal antigens were often s t i l l present after multiple absorptions, but i t does indicate a direction for further tumour antigen studies. The results of this system should be greatly improved by minimizing the antibody response to the normal antigens during hyperimmunizatibn. (Immunoadsorbents would s t i l l be required!) This could be done a few different ways: Non-human primates instead of rabbits could be immunized with tumour extracts (85,86). Rabbits could be treated with normal tissue extracts neonatally or with cyclophosphamide before hyperimmunization with tumour extracts. The tumour extracts could be pre-absorbed with an immunoadsorbent of antibodies against normal antigens. This technique could also augment other studies on tumour antigens in extracts. If monospecific antibody were prepared, more sensitive antigen detection methods such as radioimmunoassay could be used. We can be confident that the antigens detected by the immunodiffusion 158 technique are tumour-associated since they were not detected in any of the 9 normal lung extracts or the 3 normal serum samples employed at higher.protein concentrations than the tumour extracts. (These antigens were also not detected in any of the cancer patients' sera). However, i t i s possible that the antigens detected are from infection by bacteria or opportunistic viruses. It i s unlikely that the antigens would be bacterial, since more would be expected in the normal lung extracts than the tumour extracts and the con-ditions of extraction and storage did not encourage bacterial growth. Studies with preparations of different viruses would be required to demonstrate a v i r a l origin of these antigens. If further studies with normal controls s t i l l showed tumour specificity of such v i r a l antigens, these antigens could be of value both c l i n i c a l l y or sc i e n t i f i c a l l y . The results of immunodiffusion studies presented here also suggest that more than one antigen may be present in the tumour extracts and that some of these antigens may be common to tumours of different pathology. A similar common antigen was detected by several of the antisera in a precipitin band which formed near the central (antiserum) well on the immunodiffusion plates. One of the antisera demonstrated a partial identity reaction with this antigen and an antigen in the foetal extracts. This antigen was shown to be neither carcinoembryonic antigen nor oii-foetoprotein. Other TAA (in C-77 and C-94) were also detected, but not as often as the TAA in the inner precipitin band. At least one of these antigens could be detected in larger quantities than others as evidenced by a heavy pre-c i p i t i n band, and i t was shown to be inactivated by repeated freeze-thawing. Another TAA (C-26) was detected in only a few tumour extracts. Although the common TAA were frequently detected in extracts of tumours of different pathologies, when the overall results for each antiserum and tumour extract were compared according to tumour pathology, correlations 159 were observed. Squamous c e l l , anaplastic and oat c e l l carcinomas a l l showed a similar level of cross-reactivity with each other and lower cross-reactivity with adenocarcinomas (and possibly alveolar c e l l carcinomas). Adenocarcinomas and alveolar c e l l carcinomas cross-reactions were similar with each other and lower with squamous c e l l , anaplastic and oat c e l l carcinomas. The differentiation between these two groups of lung cancer pathology i s of particular interest because most tumours in the latter group are carcinogen induced while the adenocarcinomas and alveolar c e l l carcinomas are not (66). There is some evidence of v i r a l association with the latter two types of tumours (87,135). 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Immunological diagnosis and prognosis of digestive tract cancer: some c l i n i c a l considerations. Ann. Immunol. (Inst. Pasteur) 124: 635. APPENDIX A Protein concentration of extracts Extract mg protein/ml Extract mg: protein/ml N-pool 29 C-62 6.7 N-25 40 C-63 8.1 N-50 20 C-64 4.0 Norm 30 C-65 6.5 N-83 33 C-66 5-7 F-48 14.5 C-67 6.9 C-68 8.6 C-l .6.9 C-69 11.6 C-2 17.6 C-70 5.7 C-3 6.3 C-71 4.4 C-4 4.7 C-72 3.2 C-5 13.8 C-73 6.7 C-6 15.4 C-74 11.1 C-24 9.5 C-75 8-2 C-26 18.0 C-76 8.2 C-30 27 C-77 7.9 C-36 1.9 C-78 6.6 C-40 19 C-79 6.5 C-41 20.1 C-81 10.4 C-45 13.7 C-82 7.9 C-46 18.5 C-85 5.5 C-53 11.0 C-87 12.1 C-54 7.5 C-88 5.0 C-55 7.2 C-90 7.9 C-56 5.1 C-91 6.0 C-57 8.6 C-92 7.0 C-5 8 4.9 C-93 8.2 C-60 4.6 C-94 6.3 C-61 6.0 C-96 18.3 A'PI'KNMX H A l l t h i s I n f o r m a t i o n t s from p a t h o l o g y o r a u t o p s y reports Reference Patient Age /Sex An top sy or Cell. Type Size Extent of Number Surgical Metastases A S C-l VER 76 M / Squamous c e l l 1 large' none C-3 LAN 61 M / Adenocarcinoma mediastinal lymph nodes C-4 1I0L 33 M / Carcinoid peribronchial nodes C-5 PHI M / Ad cno c a rc i no ma h i l a r node3 C-6 NYS 63 M / Squamous c e l l 7 x 6 cm h i l a r nodes C-24 . DIC 62 M / Adenocarcinoma brain, l i v e r C-26 ROB 62 H . / Adenocarcinoma 10 x 6 cm h i l a r nodes and widespread* C-30 CON 49 M / Alveolar c e l l carcinoma 'huge' h i l a r nodes and brain C-36 STE 72 M / Adenocarcinoma 'huge1 l o c a l extension and l i v e r C-38 MIK 68 M / Anaplastic 9 cm diam widespread C-40 CHA 44 M / Oat c e l l 2.5 cm l o c a l nodes and , l i v e r , bones C-41 MOO 54 F / Anaplastic (some squamous elements) 8 cm diara widespread C-4 5 LEH 54 M / Adenocarcinoma medium widespread C-46 DRY 60 F / Anaplastic 1 large' widespread C-53 JOH 67 M / Squamous (recurrence of b i l a t e r a l h i l a r primary resected 3 yrs ago) nodes C-54 MOT 50 F / Anaplastic (viewed by some extensive chest wall as a mesothelioma) invasion C-56 ALL 71 M / V Anaplastic (some sarco-matous elements) 4 cm diam residual disease C-57 HOP 62 M / Squamous (biopsy of distant s i t e (floor metastatic l e s i o n , of mouth) lung primary) C-58 FIS M- / Adenocarcinoma (biopsy distant s i t e from metastatic nodule (abdominal mass) in abdomen from lung primary) C-60 AND 72 M / Squamous c e l l 3 cm diara C-61 TUC M / Squamous c e l l 123 gm h i l a r nodes C-62 CHAL M / Anaplastic 96 gm C-63 BUR M / Squamous c e l l 133 gm mediastinium and brain C-64 LeJ M / Oat c e l l 35 gm widespread C-65 AND 48 M / Oat c e l l C-66 TRE M / Squamous c e l l 105 gm widespread C-67 DAV F /. Anaplastic (some sarco-matous elements) 52 gm lung and l o c a l lymph nodes C-68 McP 54 M / t A t y p i c a l osteogenic sarcoma 11 cm none C-69 CLI F / Squamous c e l l >.1.4 gm pleural extension C-70 LeB F / Oat c e l l >15 gm h i l a r nodes and residual disease C-7.1 BAR 83 M / Squamous 5x4.8 cm lymph nodes C-72 CAR F / Oat c e l l >12 gm l o c a l nodes C-73 FLI 9 M • Osteogenic sarcoma none * Widespread: numerous organs involved with metastatic disease t Tumour tissue o r i g i n - bone APPENDIX B (Continued) Reference Number Patient Age/Sex Autopsy or Surgical A S C e l l Type Size C-74 HAL M Squamous >10 gm C-75 SHU M / Squamous 35 gm C-76 BAL M / Squamous 56 gm C-77 McC M / Rhabdomyosarcoma§ 46 gm C-78 HAR F / Oat c e l l >13 gm C-79 FOL 73 M / Squamous c e l l 'large' C-8.1 MON 65 M / Alveolar c e l l both (2 primaries) 'small' C-82 MEL 63 M Mixed (squamous and 5 cm diam adenocarcinoma elements) C-85 WAL 60 F / Adenocarcinoma 5 x 3 cm Extent of Metastases none h i l a r sub pleural extensio pulmonary lymph node lo c a l extension and distant sites l o c a l nodes and s a t e l l i t e nodules C-87 POB 68 M Squamous 'large' 1 l o c a l nodes C-88 GOR 69 F / Adenocarcinoma 3 cm diam none C-89 FOS 53 F / C-90 CHA .62 F / Squamous 5 x 3 cm extensive l o c a l C-91 DIX 12 F / Osteogenic sarcoma C-92 BAL 60 M / Anaplastic 6 x 6 cm l o c a l nodes C-93 SMI 58 M / Squamous C-94 MOR 64 M / Squamous 6 x 5 era l o c a l nodes C-96. LAN 15 M / Osteogenic sarcoma § Tissue of o r i g i n - (Chest wall) s t r i a t e d muscle 174 APPENDIX C Serum sample Condition of donor Metastases detected within when serum taken 2 months of serum collection S-53 5 mo. post-op. S-66 autopsy S-68 3 mo. post-op. + S-71 autopsy S-74 pre-op. S-77 3 wk. post-op. + S-79 autopsy S-85 pre-op. + S-87 pre-op. + S-88 pre-op. S-89 pre-op. + S-90 pre-op. S-91 pre-op. + S-92 pre-op. + S-93 pre-op. -S-94 pre-op. + 

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