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Clinical and in vitro observations in cancer patients during immunotherapy Clements, Donna V. M. 1975

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CLINICAL AND IN VITRO OBSERVATIONS IN CANCER PATIENTS DURING IMMUNOTHERAPY by DONNA V. M. CLEMENTS B.Sc, University of B r i t i s h Columbia, 1966 M.Sc., University of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Pathology We accept t h i s thesis as conforming to the required ^taneferd THE UNIVERSITY OF BRITISH COLUMBIA August, 1975 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f i nanc ia l gain sha l l not be allowed without my writ ten pe rm i ss i on . Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date dterf&C* / ABSTRACT The value of immunotherapy, i n conjunction with radiotherapy, chemotherapy and surgery, i n c o n t r o l l i n g cancer i s slowly becoming established. Reported here are c l i n i c a l and i n v i t r o observations on the e f f e c t of BCG immunization i n a preliminary study of patients with lymphomas and of BCG, PPD, and v a c c i n i a virus i n the management of malignant melanoma. As o r a l BCG was used i n these patients to stimulate the maxi-mum amount of lymphoid t i s s u e , i t was deemed necessary to determine whether o r a l BCG was an e f f e c t i v e means of inducing s e n s i t i v i t y . Con-sequently, normal healthy volunteer student nurses were divided i n t o two groups; one group received o r a l BCG while the second group were immunized with intradermal BCG. Follow-up skin t e s t i n g showed a much lower degree of s e n s i t i v i t y i n those nurses who had received o r a l BCG. This difference, however, was not apparent using an i n v i t r o assay to determine s e n s i t i v i t y . Maintenance of remission s o l e l y by repeated BCG vaccinations i n seven patients with non-Hodgkin's lymphoma who had achieved a com-ple t e c l i n i c a l remission with i n i t i a l standard therapy has provided s u f f i c i e n t encouragement to begin a randomized c l i n i c a l t r i a l . In  v i t r o lymphocyte responses to mitogens and PPD used as parameters of cell-mediated immunity have not proved to be of value i n p r e d i c t i n g early or l a t e recurrence i n p r e - t r i a l and t r i a l patients. i i i i i Eight out of twenty-one patients with malignant melanoma have shown a s a t i s f a c t o r y c l i n i c a l response to immunotherapy. Those who respond must show immunological r e a c t i v i t y to the stimulating agent, however the best c l i n i c a l responses were not associated with the highest degrees of i n vivo and i n v i t r o s e n s i t i z a t i o n . The s k i n r e a c t i v i t y and the i n v i t r o lymphocyte response to PPD as w e l l as a 2 - 3 f o l d increase i n the appearance of colony-forming units i n the per i p h e r a l blood following the intratumour i n j e c t i o n of BCG or PPD are h e l p f u l i n prognosis and management of these p a t i e n t s . In general patients with malignant melanoma who presented with a PHA response less than 40% of normal made a poor response to immunotherapy. Autopsies performed on seven patients dying with extensive melanocarcinomatous disease f a i l e d to show any serious adverse t o x i c reactions or i n f e c t i o n s from o r a l and intratumour i n j e c t i o n s of BCG. TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i INTRODUCTION 1 1. C l i n i c a l Management of Cancer 1 2. Immune Surveillance 2 (2-a) Antigenic Make-Up of Cancer Tissue 3 (2-b) Recognition and Reaction 4 3. Suggestive Evidence Supporting the Concept of Immune Surveillance 4 (3-a) Age Incidence 4 (3-b) Spontaneous Regression 5 (3-c) Histology 6 (3-d) Immunosuppression 7 (3-e) Neonatal Thymectomy 8 4. Immune Escape Mechanisms 8 (4-a) Mechanisms at the Level of the Host C e l l 9 (4-b) Mechanisms at the Level of the Tumour C e l l 9 (4-c) Mechanisms at the Level of the Host-Tumour Interaction 10 i v V Page 5. Tumour K i l l Mechanisms 14 (5-a) Cytotoxic Antibody 14 (5-b) Cytotoxic E f f e c t o r C e l l s 16 6. General Aims of Immunotherapy 18 (6-a) Cancer Antigen A l t e r a t i o n 19 (6-b) Passive Immunization 20 (6-c) Active Immune Stimulation 21 7. Successful Immunotherapy 23 8. Evaluation of the Success of Immunotherapy 26 9. Object and Scope 33 MATERIALS AND METHODS 35 1. Subjects Studied 35 (1-a) Healthy Volunteers 35 (1-b) Patients 36 2. Leukocyte Culture 39 3. Serum Immunoglobulin Levels 41 4. Culture of Colony-Forming Units 41 5. Photography of Colony-Forming Units 42 6. Spontaneous DNA Synthesis (Baseline Transformation) . . . 42 7. Autoradiographs 43 (7-a) Preparation of Blood Films 43 (7-b) Autoradiography . . 44 (7-c) Development of Prepared Autoradiographs 44 (7-d) Staining 44 8. Preparation of Neuraminidase Treated Tumour C e l l s . . . . 45 v i Page RESULTS 46 1. Healthy Volunteers 46 2. Student Nurses 46 3. C l i n i c a l Observations i n Lymphoma Patients 51 4. C l i n i c a l Observations i n Melanoma Patients 57 5. Laboratory Observations 68 (5-a) Lymphocyte Stimulation i n Lymphoma Patients . . . . 68 (5-b) Lymphocyte Stimulation i n Melanoma Patients . . . . 73 (5-c) Immunoglobulin Levels i n Lymphoma and Melanoma Patients 84 (5-d) Spontaneous DNA Synthesis (Baseline Transformation). 84 (5-e) Autoradiographs 93 (5-f) Colony Forming Units 9 3 6. C l i n i c a l Status of the Patients Studied Nine Months Following Completion of In V i t r o Studies 108 DISCUSSION 112 CONCLUSIONS 132 REFERENCES . . 134 LIST OF TABLES Table Page I Results of tuberculin t e s t i n g two groups of student nurses 10 - 13 weeks post-BCG vaccination showing per cent of group tested according to reaction s i z e . . . 47 II Results of tuberculin t e s t i n g two groups of student nurses 10 - 13 weeks post-BCG vaccination giving average s i z e of reaction i n mm 48 III Results of tuberculin t e s t i n g two groups of student nurses 6 months post-BCG giving average s i z e of reaction i n mm 49 IV PHA response i n group "A" and "B" student nurses pre-and post-BCG vaccination 50 V PPD responses i n group "A" and "B" student nurses pre- and post-BCG vaccination 52 VI Lymphoma - non-randomized t r i a l 53 VII Lymphoma - randomized t r i a l 54 VIII Melanoma patients - treatment and c l i n i c a l responses . 58 IX ' Milligrams of BCG taken o r a l l y by melanoma patients during immunotherapy treatment 60 X Milligrams of BCG d i r e c t l y i n j e c t e d i n t o melanomatous nodules during immunotherapy treatment ., 61 XI In v i t r o responses i n lymphoma patients 69 XII In v i t r o responses i n lymphoma groups 72 XIII Peripheral white blood c e l l counts on lymphoma patients 78 XIV In v i t r o responses i n melanoma patients 79 XV In v i t r o responses of melanoma patients according to group and stage of disease 81 v i i v i i i Table Page XVI In v i t r o responses i n two groups of melanoma patients divided according to sex 82 XVII Analysis of variance of jLn v i t r o responses of grouped and staged melanoma patients 83 XVIII E f f e c t of BCG treatment modality on baseline trans-formation and CFU-C lev e l s 92 XIX S e r i a l spontaneous baseline transformation and auto-radiograph l a b e l l e d c e l l s i n two melanoma patients . . 94 XX CFU-C i n male and female patients 107 LIST OF FIGURES Figure Page 1 C l i n i c a l improvement while on immunotherapy i n patient 9 65 2 Monthly fluctuations i n PHA and PPD responses i n patient 3 70 3 Recovery of i n v i t r o responses following chemotherapy. 74 4 Recovery of i n v i t r o responses following i r r a d i a t i o n and chemotherapy 75 5 Divergence of i n v i t r o responses following i r r a -d i a t i o n and chemotherapy 76 6 F a i l u r e of i n v i t r o responses to recover following i r r a d i a t i o n and chemotherapy i n patient 01 77 7 In v i t r o responses to PHA expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment 85 8 In v i t r o responses to Pokeweed expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment 86 9 In v i t r o responses to Concanavalin-A expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment 87 10 In v i t r o responses to PPD expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment 88 11 To t a l white c e l l counts during the f i r s t 11 months of immunotherapy treatment . . 89 12 Absolute lymphocyte counts per cu mm during the f i r s t 11 months of immunotherapy treatment 90 13 Absolute monocyte counts per cu mm during the f i r s t 11 months of immunotherapy treatment 91 i x X Figure Page 14 Spontaneous uptake of 3H-thymidine i n the p e r i p h e r a l blood of melanoma patients expressed as a percent of normal 95 15 Autoradiograph on p e r i p h e r a l blood buffy coat showing a l a b e l l e d large transformed lymphocyte 96 16 Autoradiograph on peripheral blood buffy coat showing a l a b e l l e d 'Turk' c e l l 97 17 Phase microscopy of t y p i c a l macrophages showing two c e l l s i n mitosis 4 99 18 Phase microscopy of t y p i c a l macrophages , 100 19 Phase microscopy of early e o s i n o p h i l i c c e l l s ; one i n mitosis 101 20 Phase microscopy of eosinophils showing development of granules 102 21 Phase microscopy of a 'mixed' type colony showing one eosinophil ( l e f t ) and one neutrophil (right) . . . 103 22 Phase microscopy of an eosinophil colony showing an agranular c e l l i n the centre 104 23 Phase microscopy of a second eosinophil colony . . . . 105 24 Phase microscopy of a macrophage colony showing ce n t r a l c e l l i n telephase 106 25 E f f e c t of BCG vaccination on two i n v i t r o parameters measured d a i l y f or one week following immunotherapy . 109 26 E f f e c t of intratumour i n j e c t i o n of BCG on peripheral blood CFU-C 110 ACKNOWLEDGEMENTS I would l i k e to take this opportunity to express my thanks and appreciation for the unreserved co-operation extended to me by a l l the patients studied during the l a s t f i v e years. The continuous encouragement and guidance from my supervisor, Dr. J . W. Thomas has meant a great deal to me. I would l i k e to thank Dr. D. M. Whitelaw and h i s technicians, Mrs. P. Aldred and Miss M. B e l l f o r t h e i r invaluable assistance i n se t t i n g up both the colony-forming and autoradiographic assays. Without the help and co-operation of Dr. S. Grzybowski, Miss E. Dorkin, and Mrs. R. Burge the study on the student nurses would not have been possible. Dr. D. E. Hay gave h e l p f u l c r i t i c i s m s and advice. Last, but by no means l e a s t , I express a great deal of thanks to the g i r l s of laboratory 1 at the B.C. Cancer I n s t i t u t e not only f o r a l l the help they have given throughout t h i s study but also f o r making the laboratory such a nice place to have spent the l a s t s i x years: Mrs. P. Ballem, Mrs. G. Forrest, Miss N. Hasham, Mrs. J . McGee, Mrs. R. McMullan, and Mrs. A. Yuen. xxi INTRODUCTION 1. CLINICAL MANAGEMENT OF CANCER Cancer denotes a disease i n which the c e l l and i t s descendents lose t h e i r normal growth con t r o l and acquire the most fearsome properties of invasiveness, permeation and metastases. Surgery "cures one out of every three patients with cancer. De-f i n i t i v e s u r g i c a l treatment i s based on the p r i n c i p l e that cancer begins as a l o c a l i z e d disease and that excision of a l l malignant c e l l s w i l l r e s u l t i n a cure . . . and when cure i s no longer p o s s i b l e , surgery can p a l l i a t e symptoms and prevent some l i f e - t h r e a t e n i n g complications of cancer" (1). Radiation therapy involves the k i l l i n g of tumour c e l l s with X or y rays which act by disrupting such b i o l o g i c a l l y important molecules as DNA, proteins or enzymes. However, ra d i a t i o n has the d i s t i n c t d i s -advantage i n the lack of s p e c i f i c i t y of i t s destruction, and therapy i s thereby l i m i t e d by the need to maintain the i n t e g r i t y of the v i t a l tissues through which the damaging rays must t r a v e l . The varying degrees of r a d i o s e n s i t i v i t y of d i f f e r e n t tumours i s a further l i m i t i n g factor i n the usefulness of r a d i a t i o n therapy. Chemotherapy i s the f i n a l conventional form of therapy which i s now the "key factor responsible for long-term s u r v i v a l i n at l e a s t ten types of widespread cancer" (2). The various agents now i n use may 1 2 act by modifying n u c l e i c acid biosynthesis (6-mercaptopurine); modifying DNA function ( a l k y l a t i n g agents); i n h i b i t i n g protein synthesis (1-aspara-ginase); i n h i b i t i n g mitosis (vinca a l k a l o i d s ) ; or a l t e r i n g hormonal balance ( s t e r o i d s ) . The usefulness of chemotherapy i s l i m i t e d by the tolerance of the indivudual, the s e n s i t i v i t y of the malignant c e l l , and the de-velopment of tumour c e l l resistance to the drug. Immunotherapy as a form of treatment i n cancer c o n t r o l has de-veloped due to experimental evidence that cancer antigens e x i s t and that the i n d i v i d u a l may recognize such antigens as being foreign and mobilize i t s immune resources against the neoplastic c e l l s . By i n i -t i a t i n g or augmenting these immune responses, immunotherapy hopefully w i l l eradicate the tumour c e l l s completely or at l e a s t ensure a longer remission time. 2. IMMUNE SURVEILLANCE Recently there has been a resurgence of i n t e r e s t i n the immuno-l o g i c a l r o l e that the tumour host may play i n c o n t r o l l i n g the development of h i s own cancer. As early as 1909 Paul E h r l i c h stated "I am convinced that during development and growth malignant c e l l s a r i s e extremely f r e -quently, but that i n the majority of people they remain latent due to the protective action of the host. I am also convinced that t h i s n a t u r a l immunity i s not due to the presence of a n t i - m i c r o b i a l bodies but i s deter-mined purely by c e l l u l a r f a c t o r s . These may be weakened i n the older age groups i n which cancer i s more prevalent" (3). 3 I t was Burnett, however, who i n the 1960's coined the term 'immu-n o l o g i c a l s u r v e i l l a n c e ' (4,5) for the host defence mechanism whose role i t was to search out and k i l l cancer c e l l s ; the c o r o l l a r y being, that f a i l u r e of th i s system re s u l t s i n the development of c l i n i c a l cancer. Although this concept has yet to be firml y proved, i t has allowed f o r a r a t i o n a l approach to' a l t e r i n g the r e l a t i o n s h i p i n the host between the immunological system and the cancer i n an e f f o r t to overcome the malignant process. The concept of immune su r v e i l l a n c e implies three main conditions: (1) the antigenic make-up of cancer ti s s u e i s d i f f e r e n t from normal t i s s u e ; (:(2) the body i s capable of recognizing the di f f e r e n c e ; (3) the body i s capable of responding to and attempting to re j e c t t h i s ' f o r -eign' but autologous t i s s u e . (2-a) Antigenic Make-Up of Cancer Tissue There i s now good evidence for the existence of tumour-specific (or tumour-associated) transplantation antigens (TSTA). This evidence stems from studies (6,7) i n which g e n e t i c a l l y i d e n t i c a l (syngeneic) animals were used to overcome the d i f f i c u l t i e s of di s t i n g u i s h i n g immuno-l o g i c a l responses due to h i s t o i n c o m p a t i b i l i t y from those associated with tumour-specific antigens. I t has thus been shown that s p e c i f i c tumour immunity develops i n animals pretreated by: (1) allowing a trans-planted tumour to reach a c e r t a i n s i z e before l i g a t i n g or s u r g i c a l l y removing i t ; (2) i n j e c t i n g i r r a d i a t e d n o n - p r o l i f e r a t i n g tumour c e l l s ; or (3) i n j e c t i n g tumour c e l l s i n a dose too low to cause frank tumour. 4 Animals not pretreated by one of these methods develop tumours when challenged with an inoculum of tumour c e l l s whereas pretreated animals r e s i s t tumour development. Such TSTA antigens have been found i n chemically (8) and v i r a l l y (7,9) induced tumours as w e l l as a v a r i e t y of human neoplasms including Burkitt's lymphoma, neuroblastoma, colon cancer, breast cancer, s k e l e t a l and s o f t tissue sarcomas and malignant melanomas (10,11). (2-b) Recognition and Reaction The capacity of the body to respond to the cancer has now been c l e a r l y shown, not only by microscopic observation of immune c e l l i n -f i l t r a t i o n i n t o the cancer but also by immunological evidence (6,12) showing (1) the presence of c i r c u l a t i n g antibodies directed against the cancer, (2) a lymphocyte response against the cancer when the two are incubated i n a test tube, and (3) a delayed h y p e r s e n s i t i v i t y reaction i n cancer patients to the intracutaneous i n j e c t i o n of autologous cancer c e l l s , a reaction which i s analogous to the tuberculin reaction. Not only has this reaction been shown against the patient's own cancer, but there i s c l e a r l y c r o s s - r e a c t i v i t y of the immune response to homo-logous cancers of s i m i l a r type. 3. SUGGESTIVE EVIDENCE SUPPORTING THE CONCEPT OF IMMUNE SURVEILLANCE (3-a) Age Incidence The age incidence of malignant disease r e f l e c t s a greater emer-gence of malignant change at ages of r e l a t i v e immunological i n e f f i c i e n c y , 5 that i s , the a n t i - and p e r i - n a t a l period, and old age (5). In o l d age, t h i s would be due to the regular accumulation of carcinogenic noxae or spontaneous mutational events, and/or due to the decline of the immune sur v e i l l a n c e system, for i t i s w e l l established that, with aging, immuno-l o g i c a l reactions of various kinds become depressed (7). (3-b) Spontaneous Regression The occurrence, though rare, of spontaneous regression implies the existence of a tumour-associated antigen with a r e j e c t i o n inducing p o t e n t i a l i n the autochthonous host. In animals, t h i s i s exemplified by the Shope papilloma of rabbits and the MSV-induced sarcoma i n mice (7). Everson and Cole (13) have reviewed 176 cases of human neoplasms showing what they consider to be regression of cancer, e i t h e r t o t a l l y spontaneously or with minimal 'therapy which would not normally have had any influence on the cancer. More than f i f t y per cent were i n examples of four types of tumour; hypernephroma (31 cases), neuroblastoma (29 cases), melanoma (19 cases), and choriocarcinoma (19 cases). More recently, evidence (7) has indicated the i n c l u s i o n of B u r k i t t ' s lymphoma i n t h i s group. Although the l a t t e r responds remarkably w e l l to chemotherapy i n the majority of cases thereby d i f f e r i n g from other malignancies, p a r t i -c u l a r l y the melanomas, the proportion of long term survivors i n B u r k i t t ' s lymphoma i s approximately the same despite a large v a r i e t y of chemo-therapeutic agents and dosages. This suggests the importance of the host response as the decisive f a c t o r i n s u r v i v a l , rather than the primary s e n s i t i v i t y of the tumour c e l l to chemotherapy. 6 I t i s of i n t e r e s t that a l l the above tumours have a peak of age incidence much younger than the common cancers. This i s what would be expected i f , as has been suggested, there are two factors which are responsible for the t y p i c a l age incidence of most cancers, namely: the accumulation of sequential somatic mutations and the progressive weakening of the immune system with age. "Those tumours, which for genetic or other reasons are l i a b l e to reach the l e v e l of overt malignancy early i n l i f e , w i l l meet a mature immune system not yet weakened by aging" (14). Furthermore, blood from melanoma patients having undergone spon-taneous regression can sometimes induce regression i n other melanoma patients (15,16) implying the occurrence of an immunological phenomenon which i s probably associated with the existence of a common cross-reactding antigen. (3-c) Histology Large scale routine h i s t o l o g i c a l studies of common s i t e s of cancer show a higher proportion of h i s t o l o g i c a l l y diagnosable cancer than can be expected to emerge c l i n i c a l l y (5). Other evidence suggesting the r o l e of the hosts' immune system i n the defence against cancer i s the h y p e r p l a s t i c reaction often seen i n lymph nodes associated with non-metastasizing tumours; these share a s i m i l a r h i s t o l o g i c a l p i c ture to the one seen i n nodes responding to an immunological stimulus. The i n t e n s i t y of responses to tumours i s r e f l e c t e d morphologically i n the c e l l u l a r expression of active immune responses resembling those of 7 delayed h y p e r s e n s i t i v i t y or g r a f t r e j e c t i o n , not only i n the immediate area of the tumour, but i n the draining lymph nodes and, i n rodent tumours, i n the thymus and spleen as w e l l (17) . Recently, Zbar (18) and Hanna et al. (19) have performed compara-t i v e h l s t o p a t h o l o g i c a l studies on inbred guinea pigs at the s i t e of intradermally transplanted hepatocarcinoma and i n the draining lymph nodes, both i n the presence and absence of b a c i l l u s Calmette Guerin (BCG). BCG was e f f e c t i v e i n causing tumour regression and elimination of regional lymph node.metastases. Their r e s u l t s suggest that the conventional lymphoproliferative response of the regional node i s inadequate to cause regression i n the absence of what they f e e l are the major e f f e c t o r c e l l s — h i s t i o c y t e s . (3-d) Immunosuppression Immunosuppressive agents f a c i l i t a t e the spontaneous appearance or experimental transfer of tumour c e l l s . There i s a high incidence of reticulum c e l l sarcomas and other tumours i n patients receiving these agents following renal transplantation. McKhann (20)states the incidence of malignancy to be 1% as compared to a normal incidence of 0.58%. Support for s u r v e i l l a n c e has also come from the accidental transplan-t a t i o n of malignant tumours along with the renal homografts which have become autonomous and invasive. However, r e j e c t i o n of the metastases has occurred following removal of the graft (or primary tumour) and withdrawal of immunosuppressive drugs (6). 8 (3-e) Neonatal Thymectomy Neonatal thymectomy f a c i l i t a t e s the increased incidence of v i r a l l y and chemically induced tumours such that neonatally thymectomized adult mice are rendered susceptible to the oncogenic e f f e c t of polyoma virus upon room i n f e c t i o n which i s quite harmless to immunologically i n t a c t mice (21). Children with inborn errors of the immune system, e s p e c i a l l y those with defects of cell~mediated immunity, shown an increased ten-dency to develop malignancy, often of the lymphoreticular systems (20,22). There i s also evidence of a r e l a t i o n s h i p between autoimmune and malignant disease both i n man and i n animals (6). For example, i n chronic lympho-c y t i c leukemia, and lymphosarcoma, autoimmune hemolytic anemia not i n -frequently occurs, and, i n some cases, the hemolysis has preceded the apparent onset of the malignant disease. I t i s i n t e r e s t i n g that animals of the NZB s t r a i n of mice which spontaneously develop autoimmune hemo-l y t i c anemia also develop both malignant thymomas and malignant p r o l i -f e r a t i o n of reticulum c e l l s leading to death from reticulum c e l l sarcoma. With other s o - c a l l e d autoimmune diseases, i n p a r t i c u l a r the rheumatic diseases, there also i s an increased incidence of lymphomas. 4. IMMUNE ESCAPE MECHANISMS In s p i t e of t h i s evidence for an ongoing immune response, i n a great v a r i e t y of malignancies there i s the undisputed fact that i n the vast majority of cases tumours grow progressively and show no obvious signs of a host response. Experimentally, various escape routes have been demonstrated by which tumour c e l l s can evade known forms of immune i n h i b i t i o n (7,21,23,24). 9 (4-a) Mechanisms at the Level of the Host C e l l - Immunological Tolerance. A slow growing tumour may produce tolerance as can v e r t i c a l transmission of the e t i o l o g i c agent. This phenomenon i s seen with both the Gross leukemia v i r u s i n AKR mice and the mammary tumour agent transmitted v i a the milk of C3H mice. Newborn animals that receive the agent are e i t h e r incapable of developing virus n e u t r a l i z i n g antibodies or antibodies directed against new c e l l u l a r antigens determined by the same v i r u s , or do so only a f t e r some delay, the delay being decisive for tumourogenesis. - Immunosuppression. This can be brought about by drugs, i r r a -d i a t i o n , inborn errors and increasing age. (4-b) Mechanisms at the Level of the Tumour C e l l - Immunoresistance. "The s e n s i t i v i t y of c e l l s to immune attack by humoral or cell-bound factors i s p a r t l y determined by the density of antigen s i t e s on the c e l l surface. Based on quantitative absorption studies, antigen density was found to be correlated with c e l l s e n s i -t i v i t y to the k i l l i n g action of antibodies or c e l l s . Selection of immuno-re s i s t a n t c e l l s with low antigen density from an immunosensitive popu-l a t i o n has been demonstrated i n experimental systems. . . . Cytotoxic s e n s i t i v i t y has been found also to f l u c t u a t e during the c e l l cycle i n s p i t e of the f a c t that complement a c t i v a t i o n on antibody coated c e l l s was found to remain e s s e n t i a l l y constant" (24). - Antigenic Masking. "The presence of surface l o c a l i z e d sub-stances which may mask the antigenic determinants has also been postulated. 10 In a s e r i e s of hybrids between E h r l i c h ascites tumour c e l l s and various partners, the isoantigens introduced by the partners were found to be e i t h e r undetectable or considerably decreased. In p a r a l l e l with the progressive chromosome loss occurring during prolonged passage of hybrid c e l l s i n v i t r o or i n vivo, the i n i t i a l antigenic suppression was released i n several sublines, and the missing antigenic phenotype was reacquired f u l l or i n part" (24) . (4-c) Mechanisms at the Level of the Host-Tumour Interaction - Rapid Tumour Growth. This may produce excessive antigen load and thus e f f e c t a high dose tolerance which would f i t with the obser-vations of Nairn et a l . (25) who found an absence of cytotoxic lympho-cytes both i n regional nodes and from the tumour i t s e l f i n patients with malignant melanoma, despite the concurrent presence of demonstrable c y t o t o x i c i t y by the patients' c i r c u l a t i n g blood lymphocytes. This was f e l t to be due to prolonged l o c a l exposure to excessive antigen. These observations are i n agreement with those of Alexander and H a l l (26) who demonstrated that the lymph nodes draining a tumour, and thus under constant bombardment by tumour antigen, do not release .tumour-specific immunoblasts. Only a f t e r e x cision of the tumour or autografting at d i f f e r e n t s i t e s are s p e c i f i c a l l y activated immunoblasts disseminated i n t o the c i r c u l a t i o n and a systemic immune response developed. - Tumour Sponges. The large tumour mass may 'sponge up' any anti-tumour antibody produced without s i g n i f i c a n t l y e f f e c t i n g tumour growth. "The immune s u r v e i l l a n c e system i s more l i k e l y to act as a 11 preventive mechanism designed to r e j e c t emerging small neoplastic f o c i rather than to eliminate tumours of c l i n i c a l s i z e which may overwhelm the system which cannot keep pace" (4). - S i t e of Tumour P r o l i f e r a t i o n . Tumour p r o l i f e r a t i o n may occur i n s i t e s i n a c c e s s i b l e to immune c e l l s or antibody. The presence of the blood-brain b a r r i e r suggests that the c e n t r a l nervous system (CNS) may be an immunologically p r i v i l e g e d s i t e . Several observations, however, suggest the presence of some host defence system against CNS neoplasia (27). (1) Brain tumours are not unduly common, a fact inconsistent with the absence of immunological s u r v e i l l a n c e although possibly ex-plained by the low m i t o t i c a c t i v i t y i n the CNS. (2) Brain tumours, even when characterized by an extremely anaplastic h i s t o l o g i c a l appearance, ra r e l y metastasize. On the other hand, systemic tumours frequently invade the CNS. (3) Spontaneous regression of experimentally induced br a i n tumours has been observed. (4) Brain tumours are more exposed to the host's vasculature than the b r a i n i t s e l f . Thus, arguments f o r immunological p r i v i l e g e do not hold f or CNS neoplasms. Studies by Levy je_t a l . (27) on gliomas, astrocytomas, and meningiomas i l l u s t r a t e the a b i l i t y of i n t r a c r a n i a l neoplasms to induce s e n s i t i z a t i o n of the p e r i -pheral immunological system. On the other hand, immunological i s o l a t i o n of the CNS i s suggested by the ease of induction and transplantation of tumours i n the b r a i n and the observation that the recurrence and ultimately f a t a l course of Bu r k i t t ' s lymphoma, a tumour i n which immunological defenses are thought to play an important role i n containing tumour progression, are often due to cerebral involvement. 12 - Depressed Immune Responses i n Cancer Patients. Humoral a n t i -body formation to known antigenic substances has been studied by many in v e s t i g a t o r s . The evidence c o l l e c t e d suggests that i n most patients with e i t h e r s o l i d or lymphoreticular neoplasms, the a b i l i t y to form humoral antibodies to a va r i e t y of antigenic substances, even i n the presence of advanced disease, i s not impaired. So there seems to be no evidence to implicate a defect i n humoral antibody production i n most cancer patients (28). However, i n association with o l d age, i t has been shown that the number of antibody forming c e l l s , detectable by the hemolytic plaque assay, decreases markedly i n aging mice (7). I t would be l i k e l y , then, to f i n d a s i m i l a r s i t u a t i o n i n human beings. Cell-mediated immune reactions i n cancer patients have been measured by t h e i r a b i l i t y to manifest a delayed h y p e r s e n s i t i v i t y response to a v a r i e t y of common skin test antigens (mumps, tuber c u l i n , strepto-kinase-streptodornase) to which normal i n d i v i d u a l s , due to previous exposure, are reactive. In addition, the a b i l i t y of cancer patients to manifest a primary immune response a f t e r exposure to an antigen f o r the f i r s t time, has been evaluated by studying the s u r v i v a l of skin homografts i n these patients or by determining t h e i r a b i l i t y to develop contact s e n s i t i v i t y to an antigen such as dinitrochlorobenzene (DNCB) (22,28). Such studies show the cell-mediated immune reaction to be s i g n i f i c a n t l y impaired i n patients with lymphoreticular neoplasia, a fact which i s not s u r p r i s i n g , sinee lymphoreticular malignancies are usually d i f f u s e throughout the immune e f f e c t o r system. A s i m i l a r im-pairment, however, was also found i n patients with l o c a l i z e d or f a r advanced s o l i d neoplasms i n which the immune system was not d i r e c t l y 13 implicated. These observations suggest the presence of a defect i n cell-mediated immune reactions i n patients with neoplasia which may unfavorably influence the course of the patients' malignant disease and i n p a r t i c u l a r h i s response to immunotherapy. Studies by Johnson et a l . (29) , however, in d i c a t e that f a i l u r e to respond to DNCB may be caused by a defective inflammatory response r e s u l t i n g from impaired l o c a l i z a t i o n of macrophages at the s i t e of antigen deposition, rather than by f a u l t y c e l l u l a r immune recognition or responsiveness. During progressive tumour growth i n mice, a sub-population of lymphocytes having B - c e l l c h a r a c t e r i s t i c s has been observed (30,31) which has the a b i l i t y to depress T-lymphocyte function; measured by the a b i l i t y of c e l l s to respond to the mitogen, PHA. In addition, K i l b u r n e_t a l . (31) f e e l there are also serum factors present i n such animals which have a s i m i l a r suppressor a c t i v i t y . I t i s not impossible that such active suppression could be responsible f o r depressed c e l l u l a r immune responses i n cancer patients. - Enhancement. The presence of enhancing or blocking 'antibody' i n tumour-bearing hosts has been demonstrated i n the experimental system of the Hellstrbms (32,33,34,35). In th i s system, sera from tumour-•I bearing hosts can be shown to block the i n v i t r o a c t i v i t y of immune lymphocytes normally capable of i n h i b i t i n g growth of tumour c e l l s i n the presence of normal sera. With the f i n d i n g that these blocking factors disappeared sh o r t l y following tumour regression or s u r g i c a l tumour removal, i t i s now believed that t h i s blocking a c t i v i t y i s the r e s u l t of antigen-antibody complexes ( i n antigen excess) as w e l l as free antigen (36) 14 which act by binding with TSTA thus blocking t h e i r immunogenicity and preventing t h e i r i n t e r a c t i o n with cytotoxic lymphoid c e l l s , and/or by attaching to e f f e c t o r c e l l s rendering them i n e f f e c t i v e ; the l a t t e r be-l i e v e d to be the more important mechanism. That such blocking a c t i v i t y may be very c r u c i a l i n abrogating anti-tumour immunity i s suggested by the detection of blocking serum a c t i v i t y before c l i n i c a l l y overt tumour recurrence and the poorer prognosis i n women with choriocarcinoma who have c i r c u l a t i n g antibody as compared to those who lack c i r c u l a t i n g antibody (37). More fasc i n a t i n g i s the fi n d i n g of a deblocking factor i n sera from patients and animals i n remission or tumour-free which can cancel the i n v i t r o e f f e c t of blocking sera. This deblocking factor i s believed to be immunoglobulin which can be s e l e c t i v e l y removed from sera by absorp-t i o n with the respective c e l l s (38). I t has been suggested that such an unblocking 'antibody' may be responsible f o r the occurrence of spon-taneous regressions and for the b e n e f i c i a l therapeutic e f f e c t s of such sera on cancer patients (15,16,39). 5. TUMOUR KILL MECHANISMS Evidence suggests that there i s a vari e t y of means by which tumour k i l l i s accomplished, one method not necessar i l y precluding another. (5-a) Cytotoxic Antibody (40) "In contrast to leukemia and lymphoma c e l l s , s o l i d tumour c e l l s 15 generally r e s i s t complement-mediated c y t o t o x i c i t y . Important exceptions to t h i s generalization should be emphasized, however, since they suggest that the apparent ineffectiveness of antibody i s neither absolute nor l i m i t i n g . In the Molony sarcoma virus system, cytotoxic antibody i s regularly found i n the serum of animals that have rejected t h e i r tumours. Among human tumours, melanomas, sarcomas, and neuroblastomas are apparently s i m i l a r exceptions since antibody that i s cytotoxic i n v i t r o i s found i n the c i r c u l a t i o n of patients a f t e r tumour removal or regression. Cyto-t o x i c anti-tumour antibody, although probably not an e f f e c t i v e f a c t o r i n the steady-state tumour-host r e l a t i o n s h i p , may thus become apparent a f t e r removal of the source of c i r c u l a t i n g tumour antigens and may act e f f e c t i v e l y i n c o n t r o l l i n g micrometastases" (17). Cytotoxic antibody present i n the serum of sarcoma and melanoma patients i n remission de-creases or disappears altogether at the time of recurrence of tumour growth. The p o s s i b i l i t y that d e c l i n i n g antibody t i t r e s were the r e s u l t of i n a b i l i t y to form antibody to any antigen as a consequence of wide-spread metastases was investigated by assaying the same serum samples for antibodies to mumps and influenza (41). Variations i n antibody to these antigens were not re l a t e d to var i a t i o n s i n anti-sarcoma antibody; and at i n t e r v a l s when no antibody to the sarcoma antigen was detected, complement-fixing antibodies to mumps and influenza were demonstrable. Thus, the decline of the anti-tumour antibody appears to be a s p e c i f i c phenomenon and not the r e s u l t of a non-specific loss of the capacity to form antibody to any antigen. Lewis et a l . (40) have suggested that the co-existence of tumour and cytotoxic antibody could be explained on 16 the basis of a c c e s s i b i l i t y ; antibody, p a r t i c u l a r l y i f i t i s of the IgM type, does not re a d i l y pass vascular b a r r i e r s or penetrate i n t r a c e l l u l a r spaces and probably does not reach much of the tumour. (5-b) Cytotoxic E f f e c t o r C e l l s Cell-mediated immune responses are believed to play a major r o l e i n tumour destruction. The highest y i e l d of new information on s p e c i f i c cell-mediated immunity to human and animal tumours has come through e x p l o i t i n g the capacity of lymphocytes f o r d i r e c t c y t o t o x i c i t y or for reduction of i n v i t r o colony formation by the tumour c e l l s to which the lymphocytotoxicity i s directed. Three important points have emerged from these studies (17). (1) In the absence of autologous serum, s p e c i f i c cell-mediated iri v i t r o i n h i b i t i o n of tumours can occur regard-less of the c l i n i c a l state of the patient. (2) In contrast to most carcinogen-induced animal tumours, s p e c i f i c c e l l u l a r recognition and colony i n h i b i t i o n are cross-reactive among human tumours of s i m i l a r o r i g i n and h i s t o l o g i c a l type. (3) In v i t r o and presumptive i n vivo manifestations of cell-mediated immunity do not correlate i n many s i t u a t i o n s . When exposed to antigen i n v i t r o , immune lymphocytes respond with the release of chemical mediators which not only amplify the onco-c i d a l e f f e c t s of previously committed immune c e l l s but also stimulate the recruitment of other uncommitted lymphocytes. I t i s probable that such agents play a v i t a l r o l e i n vivo, f o r the cytotoxic capacity of immune responses i s l i m i t e d by the number of ava i l a b l e antigen-sensitive 17 and antigen-reactive lymphocytes that can s p e c i f i c a l l y i n t e r a c t with the a n t i g e n i c a l l y offending c e l l s . Soluble mediators (42) that have so f a r been demonstrated i n v i t r o include the following: Lymphotoxin. This substance apparently exerts a d i r e c t cytotoxic e f f e c t on tumour c e l l s by destroying t h e i r membranes. Lymphotoxin has the decided advantage that i t can di f f u s e into s o l i d tumours and destroy c e l l s not r e a d i l y accessible to immune or r e c r u i t e d lymphocytes. Experimental evidence that lymphotoxin may be e f f e c t i v e has been shown by Kikuchi et_ a l . (43) who observed the destruction of sarcoma c e l l s separated from immune lymphocytes by a m i l l i p o r e f i l t e r . Inflammatory Factor. By increasing vascular permeability at the reaction s i t e , t h i s f actor allows the accumulation of c e l l s susceptible to the action of other mediators. Macrophage I n h i b i t i o n Factor (MIF). The immobilization of macro-phages by MIF contributes to t h e i r accumulation at the reaction s i t e . Macrophages provide a means of removing and processing a n t i -genic tumour-cell debris; this immunogenic material may then induce secondary immune responses by converting immunocompetent lympho-cytes i n t o immune c e l l s capable of cell-mediated or humor.al".re-sponses . In contrast to the frequently non-specific a n t i - m i c r o b i a l a c t i -v i t y of macrophages that occurs i n animals i n f e c t e d with l i v i n g microorganisms, Evans and Alexander (44) have shown that a n t i -tumour a c t i v i t y of macrophages from immunized animals i s made up 18 of an immunologically s p e c i f i c i n t e r a c t i o n followed by a non-s p e c i f i c l e t h a l reaction. Normal macrophages, incubated with e i t h e r hyperimmune spleen c e l l s or supernatants of cultures of immune lymphocytes with s p e c i f i c antigen, give r i s e to 'armed' macrophages which are cytotoxic to s p e c i f i c target c e l l s only. I f these i n v i t r o or i n vivo armed macrophages are then incubated with s p e c i f i c antigen they undergo a transformation or a c t i v a t i o n which renders them capable of k i l l i n g a l l lymphoma or sarcoma c e l l s . Mitogenic Factors. By n o n - s p e c i f i c a l l y transforming previously quiescent normal lymphocytes, mitogenic factors continually amp-l i f y the supply of activated c e l l s thereby increasing non-specific cytotoxic a c t i v i t y at the reaction s i t e . Transfer Factor. This small molecular weight material provides a rapid mechanism to expand the number of immune lymphocytes at the reaction s i t e and, i n contrast to other soluble mediators, i n t e r a c t i o n of transf e r f a c t o r with non-sensitized lymphocytes induces a n t i g e n - s p e c i f i c recruitment. 6. GENERAL AIMS OF IMMUNOTHERAPY It can be seen that the ce n t r a l problem i n tumour immunotherapy i s not that of inducing the patient to become immune to the products of h i s tumour but i s rather to make e x i s t i n g immune responses more e f f e c -t i v e i n c o n t r o l l i n g tumour growth. At the present time, there are three main areas under i n v e s t i g a t i o n . 19 (6-a) Cancer Antigen A l t e r a t i o n E f f o r t s to a l t e r cancer c e l l s i n such a way that they w i l l be more e a s i l y recognized and more immunogenic has been attempted by t r e a t i n g i r r a d i a t e d tumour c e l l s with V i b r i o cholerae neuraminidase (VCN) (45,46) which cleaves the 2-3' and 2-6' g l y c o s i d i c linkages between s i a l i c a c i d residues and the mucopolysaccharides on the c e l l surface. I t has been hypothesized by Currie and Bagshawe (47,48) that the sialomucins on c e l l s , by v i r t u e of t h e i r high negative charge, act to i n t e r f e r e with the perception of c e l l u l a r immunogens by lymphocytes. The fa c t that normal, as w e l l as malignant c e l l s can be rendered immunogenic by treatment with VCN suggests that VCN does not act to unmask s p e c i f i c antigenic determinants but rather acts n o n - s p e c i f i c a l l y to render c e l l s more susceptible to immunologic processing by the r e c i -pient. Increased immunogenicity can be r e l a t e d to several properties of c e l l s a f t e r treatment with VCN. (1) VCN removes s i a l i c a c i d from the c e l l surface which could s t e r i c a l l y hinder the perception of antigen i t s e l f or i n t e r f e r e with contact between the antigen-bearing and antigen-processing c e l l s . (2) VCN reduction of the negative charge on the c e l l surface may cause the negatively charged antigen-processing or antigen-responsive c e l l to be more attracted to the le s s s h i g h l y charged e e l l s . (3) Treatment of c e l l s with VCN renders them more e a s i l y phago-cytosed and may enhance c e l l aggregation. I t i s w e l l established that phagocytosis and the aggregation of antigen with antigen-responsive c e l l s seems to f a c i l i t a t e antigen processing and the development of immunity. (4) VCN treatment renders lymphoid c e l l s highly susceptible 20 to the c y t o l y t i c e f f e c t s of alloantibody and complement i n v i t r o (49). There i s no reason to suppose that VCN could not have a s i m i l a r e f f e c t on tumour c e l l s . Experimentally, i n h i b i t i o n of tumour growth i s not as great when the challenging VCN treated c e l l s are in j e c t e d simultaneously with the primary untreated i&noculum. Complete i n h i b i t i o n of primary tumour growth can be achieved i f a delay of 5-10 days i s allowed between primary i&noculum and the challenge; suggesting that a low degree of immunity induced by the tumour growth i s necessary to obtain the maximum immunity from VCN treated tumour challenge. (6-b) Passive Immunization Augmentation of immunity i s being attempted by passive t r a n s f e r of e i t h e r c e l l s or serum from normal i n d i v i d u a l s or from patients who have s u c c e s s f u l l y dealt with t h e i r cancer. This concept i s based on experimental proof that a growing tumour does not induce a maximum immune response (28). Programs have developed using cross-immunization with tumour tissue followed by cross-transfusions of either lymphocytes, alone or combined with serum. When using v i a b l e tumour c e l l s f o r immu-n i z a t i o n , close monitoring of the i n j e c t i o n s i t e i s necessary to prevent tumour growth. Another drawback to t h i s form of therapy occurs i n the form of immune elimination i n the immunocompetent r e c i p i e n t . Objec-t i v e c l i n i c a l responses have been seen i n 54/277 patients thus treated. Differences i n h i s t o c o m p a t i b i l i t y are undoubtedly accounting for some of the p o s i t i v e responses. 21 The adoptive trans f e r of tumour immunity with xenogeneic lympho-cytes rendered incapable of p r o l i f e r a t i o n by i r r a d i a t i o n has been demon-stra t e d by Alexander and co-workers (50). These experiments led to speculation (51,52) that tumour immunity mediated by xenogeneic lympho-cytes was due to the transfe r of an immunoreactive s u b c e l l u l a r f r a c t i o n of these c e l l s — p e r h a p s comparable to transfe r f a c t o r . The therapeutic use of such an agent would eliminate any graft versus host reaction. The l i m i t i n g factor i n cancer patients would be the deficiency of an adequate number of c e l l s able to respond. Five hundred and seventy-three documented cases of patients receiving autologous tumour vaccines have been reported; s i x t y showing objective responses (28). However, a major t h e o r e t i c a l objection usually raised to a l l forms of intervention that involve immunization with autochthonous tumour, or i t s products, i s the p o s s i b i l i t y of antibody-associated enhancement rather than i n h i b i t i o n . (6-c) Active Immune Stimulation One modality of active immune stimulation used s u c c e s s f u l l y by K l e i n involves the addition of new antigens to a tumour i n order to render i t more immunogenic (53). Following patient s e n s i t i z a t i o n , he has demonstrated r e s o l u t i o n of malignant and premalignant skin lesions with such agents as DNCB and p u r i f i e d protein d e r i v a t i v e (PPD) from Mycobacterium tuberculosis. I n t e r e s t i n g l y , the i n t e n s i t y of the i n -flammatory reaction c o r r e l a t e d d i r e c t l y with the degree of malignancy of the les i o n s . 22 Immune stimulation may also involve non-specific immunotherapy. In 1912, DePace (54,55) aroused i n t e r e s t i n the c l i n i c a l use of viruses for cancer treatment with h i s des c r i p t i o n of a woman who gained re-mission of her c e r v i c a l carcinoma following rabies vaccination f o r a dog b i t e . In the 1950's, attempts were made to treat melanoma patients with rabies vaccine; about 10% of the patients showed subsequent improve-ment. In the early 1960's, p a r t l y as a r e s u l t of these observations and also p a r t l y due to the fac t that tumour c e l l s were known to be lysed by viruses i n the te s t tube, v a c c i n i a v i r u s i n j e c t i o n s into meta-s t a t i c melanoma were c a r r i e d out with regression of the l o c a l lesions and also of a mediastinal l e s i o n (56). I t seems l i k e l y now that the action oftlthe virus i s the r e s u l t of non-specific stimulation of the r e t i c u l o e n d o t h e l i a l system. More commonly known stimulators of the r e t i c u l o e n d o t h e l i a l system such as BCG or various species of Corynebacterium are also being used f or non-specific immune stimulation. The mechanism of BCG therapy i s believed to involve a m p l i f i c a t i o n of macrophage or r e t i c u l o e n d o t h e l i a l function. In mouse systems, BCG causes a change i n the rate of a c t i -vation, lysosomal content and b a c t e r i o l y t i c power of macrophages (17). In addition to t h i s general e f f e c t , the d i r e c t i n j e c t i o n of BCG i n t o tumour nodules to induce a delayed h y p e r s e n s i t i v i t y reaction i n the nodule r e s u l t s i n a non-specific aggregation of immunologically competent c e l l s whose reaction to the tumour s p e c i f i c antigens would hopefully be enhanced, leading to d i f f u s e systemic immunization. This i s based on the observation that the regression of melanoma nodules following 23 t h e i r d i r e c t i n j e c t i o n with BCG occurred only i n those patients p o s i t i v e to tuberculin, suggesting that a large portion of the anti-tumour e f f e c t was non-specific and resulted from the delayed h y p e r s e n s i t i v i t y reaction to BCG (10). 7. SUCCESSFUL IMMUNOTHERAPY Successful immunotherapy requires of the patient a fun c t i o n a l immune system. During embryonic development, pluripotent stem c e l l s i n the embryonic yolk sac and p r i m i t i v e blood islands migrate to f e t a l l i v e r and bone marrow. From these areas c e l l s are seeded to the thymus and other lymphoid organs where, under the inductive influence of the microenvironment, the stem c e l l s p r o l i f e r a t e , and t h e i r progeny, having acquired t h e i r s p e c i f i c class properties, migrate to the peripheral lymphoid organs (57,58). Evidence f o r concurrent c e l l u l a r and humoral immunity i n i t i a l l y arose i n chickens (59,60) where neonatal thymectomy was shown to markedly impair T - c e l l cell-mediated immunity while leaving B - c e l l humoral immu-n i t y i n t a c t . Conversely, removal of the bursa of F a b r i c i u s , a c l o a c a l lymphoid organ unique to b i r d s , impaired only humoral immunity. Although no comparable bu r s a l equivalent i s evident i n human beings, i t has been postulated that the bone marrow or Peyer's patches of the gut subserve t h i s function. Nature's experiments have supported t h i s d i v i -sion of the immune response i n human beings. Children presenting with congenitally hypoplastic thymus (DiGeorge's syndrome) have l i t t l e or no cell-mediated immunity yet maintain an unimpaired humoral system (61). 24 Conversely, children affected with Bruton's agammaglobulinemia are d e f i c i e n t only i n humoral immunity ( 6 2 ) . The primary immune r e s p o n s i b i l i t y of B-lymphocytes involves most b a c t e r i a l i n f e c t i o n s and r e j e c t i o n of c i r c u l a t i n g c e l l s such as those introduced by blood transfusions. T-lymphocytes, on the other hand, appear to be responsible f o r primary immunity to v i r a l and fungal i n f e c t i o n s , tuberculin-type delayed h y p e r s e n s i t i v i t y reactions, a c t i -vation of macrophages to r e s i s t i n f e c t i o n , and r e j e c t i o n of f i x e d t i s s u e and neoplasms. Evidence has accumulated that the two c e l l types are not e n t i r e l y independent of one another; T - c e l l s can co-operate with precursors of the antibody-forming c e l l s (B-cells) thereby serving to increase antibody formation ( 6 3 ) . C e l l s other than lymphocytes have also been implicated i n the body's defence against foreign invaders. The ti s s u e macrophage or blood monocyte functions to phagocytose foreign antigenic m a t e r i a l — e i t h e r destroying i t completely or processing i t i n some way so that i t becomes more antigenic for the T- or B - c e l l . C e l l s of the macro-phage/monocyte series are believed by Grant ejt a_L. (64) to be the cyto-t o x i c e f f e c t o r c e l l s i n t h e i r animal model; c y t o t o x i c i t y being conferred by a population of s e n s i t i z e d lymphocytes. I t now seems w e l l established that BCG i s an.effective agent i n delaying the appearance of malignancies i n animals, as w e l l as i n t r e a t i n g animals that have already developed malignancies ( 6 5 , 6 6 , 6 7 , 6 8 ) . When BCG was used following treatment with the carcinogen methylcholan-threne, Old et a l . (69) observed a delay i n the appearance of the 25 chemically-induced sarcomas. Later, i t was reported (66,67) that BCG-treated animals had a reduced incidence of malignancy and a longer s u r i v a l time than untreated animals following i n f e c t i o n with the car-cinogenic polyoma v i r u s . Furthermore, i t has been demonstrated that animals can be s u c c e s s f u l l y protected against t r a n s f e r of l i v i n g tumour c e l l s by previous vaccination with BCG (70,71). Lemonde (72) has shown that tumour recurrence following s u r g i c a l removal of the primary tumour can be greatly delayed or even prevented by the use of BCG. Zbar et a l . used BCG immunotherapy to treat e f f e c t i v e l y both a s c i t i c (73) and s o l i d animal tumours (74,75) with the following conclusions. (1) L i v i n g BCG must be i n d i r e c t contact with the tumour for maximum effectiveness. (2) The f i n a l outcome i s c r i t i c a l l y dependent on tumour s i z e , with tumours greater than two centimeters giving r e l a t i v e l y poor r e s u l t s . (3) BCG therapy can be e f f e c t i v e i n s p i t e of metastatic disease i n the regional lymph nodes. Further studies along the same l i n e have been c a r r i e d out by Weiss (76) i n I s r a e l with a methanol extractable residue (MER) of BCG which s i m i l a r l y offered protection against tumour i s o g r a f t s . Human studies have also supported the use of BCG i n cancer treatment. Two studies (77,78) have reported a decreased incidence of acute leukemia i n BCG-vaccinated children when compared to a noh-vaccinated control group. The f i r s t immunotherapeutic use of BCG to prevent recurrence was made by Mathe e_t a l . (79) who took a s e r i e s of acute lymphoblastic leukemia patients and, following induction of re-mission by chemotherapy, repeatedly vaccinated these patients e i t h e r 26 with BCG alone or simultaneously with i r r a d i a t e d b l a s t c e l l s . Although others (80,81) have been unable to confirm t h i s , Mathe (82) has recently reported continuing good r e s u l t s i n h i s o r i g i n a l s e r i e s of p a t i e n t s ; seven of the o r i g i n a l group of twenty patients receiving active immuno-therapy were s t i l l i n primary remission a f t e r seven to ten years. Schinitsky e_t a l . (83) f e e l that these " c o n f l i c t i n g c l i n i c a l reports concerning the e f f i c a c y of BCG vaccination for cancer immuno-prophylaxsis and immunotherapy i n humans may be resolved i f , as (their) experiments ( i n mice) suggest, BCG i s an e f f e c t i v e immunoprophylactic against cancer only as long as the host i s able to mount a strong, cell-mediated response to PPD and the tumour i s immunogenic." More recently, Powles et a l . (84,85) reported on a group of patients with acute myeloblastic leukemia. Following remission induc-t i o n with standard chemotherapy, one group of patients received repeated BCG vaccinations accompanied by i r r a d i a t e d b l a s t c e l l s as maintenance therapy while the c o n t r o l group received standard chemotherapy main-tenance. A s i g n i f i c a n t l y b e t t e r s u r v i v a l was evident i n the former group. 8. EVALUATION OF THE SUCCESS OF IMMUNOTHERAPY Due to the m u l t i p l i c i t y of BCG s t r a i n s and t h e i r d i f f e r e n t methods of preparation, problems a r i s e when comparing the success of immunotherapy. MacKaness et_ a l . (86) have recently studied the immune response to d i f f e r e n t s t r a i n s and preparations and concluded that "the therapeutic usefulness of BCG may be undone, by the use of s t r a i n s of 27 excessive virulence or doses which can disorganize lymphoid tissues and make them f u n c t i o n a l l y i n a c t i v e . . . . " I t i s u n l i k e l y that t h i s problem of the various vaccines used throughout the d i f f e r e n t labora-t o r i e s w i l l be overcome i n the near future. One source of BCG was used throughout t h i s study. The second concern i n a l l laboratories involved i n immunotherapy i s the development of a laboratory test with which to evaluate the patients' response. The f i r s t l i n e of attack i n v o l v e s s s p e c i f i e assays. The prime concern i n any form of cancer therapy i s tumour destruction. In the case of immunotherapy this implies an i n t a c t afferent and efferent immune system. I t i s possible i n v i t r o , using a mixed leukocyte culture (MLC) t e s t , to demonstrate a lymphocyte blastogenic response against autologous tumour c e l l s (or tumour-antigen e x t r a c t ) ; b l a s t t r a n s f o r -mation i n d i c a t i n g an i n t a c t afferent immune response (87,88,89,90). I t i s also p o s s i b l e , using various c y t o t o x i c i t y assays, to show that host lymphocytes have the capacity to k i l l autologous (or homologous) tumour c e l l s (91,92,93,94). Such systems measure both the afferent and efferent arms of the immune response. Tumour extract or i r r a d i a t e d tumour c e l l s have also been used i n an i n vivo delayed h y p e r s e n s i t i v i t y assay of the patients' response to h i s tumour (95,96,97) with the decided advantage that i t i s occurring within the 'tumour m i l i e u . ' There are, however, problems associated with such assay systems. A source of tumour tissue i s necessitated. In the lymphoma patients studied at the B.C. Cancer I n s t i t u t e only one out of fourteen patients had accessible tumour t i s s u e . The problem i s not quite so acute when 28 the patients have malignant melanoma as most patients present with s u r g i c a l l y removable tumour. Strong evidence for the presence of cross-reactive antigens i n melanoma ti s s u e (41) permits the s u b s t i t u t i o n of homologous tumour tiss u e as antigen source i f autologous tumour tissue i s unavailable. Host blocking factors (93) could prevent recog-n i t i o n of the tumour antigen and subsequent c y t o t o x i c i t y . Furthermore, i n v i t r o c y t o t o x i c i t y assay systems require an enormous r a t i o r o f lympho-cytes (and macrophages) to tumour c e l l s to demonstrate k i l l . In the i n vivo s i t u a t i o n , lack of delayed h y p e r s e n s i t i v i t y could be a r e s u l t of a lack of such soluble factors as MIF or inflammatory f a c t o r thereby preventing an adequate number of e f f e c t o r c e l l s from accumulating at the reaction s i t e . To circumvent a lack of tumour t i s s u e , other antigens have been used for assaying both i n vivo delayed h y p e r s e n s i t i v i t y and i n v i t r o blastogenesis as a measure of the patients' general immune com-petence and as a prognostic a i d . The test agents include r e c a l l antigens (PPD, streptokinase-streptodornase—SKSD, and Candida) as w e l l as agents used f o r primary s e n s i t i z a t i o n and challenge (DNCB, and Keyhole Limpet Hemocyanin—KLH). C o n f l i c t i n g reports abound as to the value of these agents. Lee e_t a_l. (98) found the rate of progression of disease within s i x months of t e s t i n g lower i n patients with advanced s o l i d neoplasms (melanoma, sarcoma, squamous c e l l carcinoma and adenocarcinoma) who had a p o s i t i v e i n vivo response to challenge with DNCB. Re a c t i v i t y to r e c a l l antigens was of prognostic value only i n patients with 29 adenocarcinoma. A s i m i l a r s u r v i v a l advantage has been reported (99) i n bronchogenic cancer patients able to be s e n s i t i z e d to DNCB. In thi s type of cancer, however, longer s u r v i v a l times have also been reported i n patients able to respond to the r e c a l l antigen, PPD (100, 101) . Morton e_t a l . (10) have found delayed h y p e r s e n s i t i v i t y studies to DNCB and common skin test antigens to show a good c o r r e l a t i o n between immunological r e a c t i v i t y to those antigens and extent of disease i n i -t i a l l y as w e l l as the patients' response to immunotherapy; melanoma nodule regression being observed only i n tuberculin p o s i t i v e patients. Studies by E i l b e r e_t j i l . (102) on 116 malignant melanoma patients and 40 sarcoma patients have shown s i m i l a r c o r r e l a t i o n between delayed h y p e r s e n s i t i v i t y to DNCB and the c l i n i c a l extent of malignancy (stage I vs. I I I ) . They f e e l that the malignant disease state e f f e c t s the immune response to neoantigens more than the response to r e c a l l antigens as an analysis of the immune response to the common skin t e s t battery showed no c o r r e l a t i o n with e i t h e r the i n i t i a l extent of disease, or subsequently, with progressive disease. A l l malignant melanoma patients i n remission r e c e i v i n g BCG immunotherapy studied by Gutterman eit a l . (103) developed delayed hyper-s e n s i t i v i t y and a majority developed primary antibody response to KLH. Relapse pa t i e n t s , however, f a i l e d to do so. Pre-therapy evaluation i n these patients was a poor prognostic i n d i c a t o r as t h e i r two anergic patients were s t i l l i n remission. I t appears, therefore, that BCG immunotherapy potentiates delayed h y p e r s e n s i t i v i t y responses. Bluming 30 et al. (104) also found a non-specific potentiating e f f e c t on c e l l u l a r r e a c t i v i t y to both primary and r e c a l l antigens i n a group of melanoma patients c l i n i c a l l y free of apparent tumour receiving prophylactic BCG by dermal s c a r i f i c a t i o n . No such e f f e c t was noted, however, i n a s i m i l a r group of patients receiving intradermal BCG vaccination. Other studies (102) have suggested that t h i s p o t entiating e f f e c t de-pended very much on the stage of the disease as the administration of BCG as a stimulus to systemic immunity was not e f f e c t i v e where recurrent disease was noted. BCG therapy was not able to maintain immunologic r e a c t i v i t y to DNCB i n patients developing progressive or recurrent disease. In tumour-free patients with h i s t o r i e s of frequent bladder cancer recurrences, a s i g n i f i c a n t decrease i n the numbers of patients with recurrences and the number of new tumours has been observed during a study period of two years following KLH immunization (105). S i m i l a r l y , as with the other assay systems, the value of i n v i t r o mitogen and antigen responses varies according to the patient population studied and the laboratory doing the study. Butterworth et al. (90) reported PHA and PPD responses poor tests of malignant melanoma patients' a b i l i t y to recognize foreign t i s s u e antigens. Nearly a l l t h e i r patients, regardless of c l i n i c a l staging of disease, s i t e of o r i g i n a l primary, age, or sex responded to PHA. The response was subnormal only i n two patients both of whom died the following week. Ei g h t y - s i x percent had normal responses to PPD. P r i o r i to i n i t i a t i o n of BCG immunotherapy i n a group of melanoma patients with recurrent disease, S e i g l e r e_t al. (106) observed normal responses to PHA and abnormal responses to PW and PPD. However, following immunotherapy 31 lymphocyte unresponsiveness was reversed i n those patients who became skin t e s t p o s i t i v e and developed cell-mediated immunity. Malignant melanoma patients without detectable disease i n another study (103) did not show t h i s change i n lymphocyte blastogenic response to mitogens and antigens during BCG therapy and were s i m i l a r i n remission and re-lapse patients. Lieberman ejt a l . (89) f e e l that " i n v i t r o parameters of c e l l u l a r immunity inc l u d i n g MIF production, i n h i b i t i o n of leukocyte migration, are a f f e c t e d by i n t r a l e s i o n a l BCG; some, p a r t i c u l a r l y lymphocyte stimu-l a t i o n tests and rosettes seem to be correlated with c l i n i c a l response of the p a t i e n t s . " During i n t r a l e s i o n a l BCG i n j e c t i o n 4/4 responders had marked increases to PHA whereas non-responders had no increase i n lymphocyte stimulation e i t h e r to melanoma antigen or to PHA. They f e e l the increase i n PHA responsiveness r e f l e c t s stimulation of non-s p e c i f i c immunity. Similar controversy as to the value of lymphocyte transformation responses has been found i n patients with other types of cancer; for example, Hodgkin's disease, lymphomas and uro l o g i c cancers (107,108,109,110). I t has also been suggested (111) that only dose response curves are of value as some patients show normal responses only to maximal PHA stimulation. Serum factors have been implicated i n those patients showing abnormally low responses (112). In our laboratory, we have not been impressed by the a b i l i t y of homologous serum from normal i n d i -viduals to increase lymphocyte responses i i i patients showing low re-a c t i v i t y (113). 32 In v i t r o mitogen responses d i f f e r from antigen responses i n that (1) they involve a l a r g e r proportion of c e l l s and (2) they are independent of any immunogenicity possessed by the stimulant. "These features of p o l y c l o n a l responses provide the ra t i o n a l e f o r the use of such systems both as a model for the study of a c t i v a t i o n processes i n lymphocytes and c l i n i c a l l y f o r the assessment of p r o l i f e r a t i v e poten-t i a l of blood-borne lymphocytes" (114). I t would be expected that those i n d i v i d u a l s presenting with, or acquiring, a decreased or n e g l i -g i b l e response i n v i t r o to such agents would be unable to respond s a t i s -f a c t o r i l y to the growing neoplasm. The three mitogens used i n t h i s study appear to activate d i f -ferent populations of c e l l s allowing a broad spectrum of responding lymphocyte populations to be measured. PHA i s known to ac t i v a t e p r i -marily T - c e l l s as w e l l as a small percentage of B - c e l l s (115). E v i -dence has accumulated that PW stimulates a larger proportion of B - c e l l s (116). Con-A stimulates a population of immature T - c e l l s (115). Measuring the In v i t r o response to the antigen, PPD, serves a th r e e - f o l d purpose. (1) I t has been reported (117) that BCG possesses antigenic determinants i n common with a b a s i c protein extracted from cancer t i s s u e . Because of the close association and c r o s s - r e a c t i v i t y between PPD and BCG, PPD could thus be measuring the response to a bas i c cancer antigen. (2) I t i s another means of assessing the patients' a b i l i t y to become s e n s i t i z e d during the course of BCG immunotherapy. (3) I t also serves to measure immunological memory or r e c a l l i n those patients known to have been tuberculin p o s i t i v e . Reactions involving 33 memory appear easier to a t t a i n than de_ novo s e n s i t i z a t i o n (102) . In  v i t r o b l a s t transformation may be more s e n s i t i v e than i n vivo delayed h y p e r s e n s i t i v i t y induced by the same antigen. Although correlations have been reported between skin t e s t and i n v i t r o responses (118,119), there are occasions where, i n the face of negative dermal reactions, p o s i t i v e i n v i t r o responses are present and vice versa (120,121). 9. OBJECT AND SCOPE With some of the information and evidence presented i n t h i s introduction a v a i l a b l e at the beginning of t h i s study i t was deemed worthwhile to i n i t i a t e a study to confirm the e f f i c a c y of immunotherapy. Consequently, a l i m i t e d immunotherapy program has been i n operation since 1969 at the B r i t i s h Columbia Cancer I n s t i t u t e i n the hope of determining whether immunotherapy could be s u c c e s s f u l l y used to main-t a i n remissions and so prolong s u r v i v a l times i n lymphoma patients following successful treatment with i r r a d i a t i o n and/or chemotherapy as w e l l as i n malignant melanoma patients with or without obvious disease. Immunological treatment has consisted p r i m a r i l y of repeated BCG s c a r i -f i c a t i o n or i n j e c t i o n s and more recently the use of o r a l BCG has been introduced. More l i m i t e d treatments have involved v a c c i n i a v i r u s , PPD or neuraminidase-treated tumour c e l l vaccines. In concurrence with t h i s program, studies have been conducted with the object of f i n d i n g a s a t i s f a c t o r y laboratory procedure which would a i d i n prognosticating or determining the p a t i e n t s ' c l i n i c a l response to immunotherapy; one that would perhaps d i f f e r e n t i a t e between 34 those that would respond to immunotherapy and those that would not and/or warn the physician of an impending recurrence so that more aggres-sive methods or a d i f f e r e n t modality of therapy could be i n s t i t u t e d . MATERIALS AND METHODS 1. SUBJECTS STUDIED (1-a) Healthy Volunteers Throughout the study, healthy volunteers from amongst the labo-ratory personnel were p e r i o d i c a l l y used as controls. Student nurses entering t r a i n i n g at the Vancouver General Hospi-t a l were studied before and a f t e r t h e i r i n i t i a l b a c i l l u s Calmette Guerin (BCG) vaccination to compare the e f f i c a c y of o r a l BCG versus i n t r a -dermal BCG as a method of primary s e n s i t i z a t i o n . An incoming student class of approximately eighty nurses, having been informed of the study being undertaken, were skin tested f o r r e a c t i v i t y to tuberculin. Those students showing a negative reaction were divided i n t o two groups. Individuals of group A (alphabetical surname A-L) received the standard intradermal i n j e c t i o n of 0.1 mg BCG. Tuberculin negative group B nurses (alphabetical surname Mc-Z) received 120 mg BCG o r a l l y once a week for four weeks. Skin t e s t i n g was performed by Miss E. Dorkin. A l l s k i n tests were made intradermally into the dorsal surface of the forearm. BCG immunization was ca r r i e d out three days post-skin t e s t i n g at which time blood was drawn for lymphocyte stimulation t e s t s . Ten to thi r t e e n Connaught Laboratories Ltd. 35 36 weeks following vaccination, blood was again drawn for culture. At the same time, repeat skin t e s t i n g was done and g i r l s f a i l i n g to con-vert i n i t i a l l y were revaccinated with intradermal BCG. (1-b) Patients A l l patients receiving immunotherapy treatment were attending the B r i t i s h Columbia Cancer I n s t i t u t e . C l i n i c a l and i n v i t r o obser-vations reported were completed by August, 19 74. Informed consent was obtained from a l l patients studied. Lymphoma Patients The diagnosis of malignant lymphoma, lymphocytic poorly d i f f e r e n t i a t e d , u n d i f f e r e n t i a t e d or h i s t i o c y t i c type, was confirmed i n a l l patients both by h i s t o l o g i c a l and c l i n i c a l c r i t e r i a . These patients represented the vanguard of investigations of possible t o x i c i t y and effectiveness of a c t i v e immunotherapy by BCG i n maintaining remission i n patients who had achieved complete remission following standard radio- and chemotherapy. A l l such patients had been previously untreated; and following i r r a d i a t i o n and chemotherapy (cyclophosphamide, v i n c r i s -tine and prednisone) had complete regression of a l l palpable and v i s i b l e disease, disappearance of X-ray evidence of tumour including lymph nodes seen on lymphangiogram, absence of fever, disappearance of any lymphoma c e l l s from the peripheral blood and marrow, and no evidence of l i v e r or s p l e n i c enlargement. Seven patients (Table VI), a f t e r achieving a remission as defined above, received BCG by multiple s c a r i f i c a t i o n and subsequently by mouth. 37 The i n i t i a l s c a r i f i c a t i o n dose was 40 mg and the o r a l dose 120 mg. The s c a r i f i c a t i o n s i t e s were rotated between the four extremities to stimulate the maximum number of lymph node areas, while o r a l BCG was used to stimulate the lymphoid areas i n the gut and mesentery. I n i -t i a l l y , one extremity was vaccinated each week and as an immune re-sponse was observed the cycles of BCG were a l t e r e d , with the next cycle c o n s i s t i n g of vaccinations every two weeks, then stretched to three weeks, four weeks, f i v e weeks and then maintained at every s i x weeks. A f t e r a cycle of four s c a r i f i c a t i o n s , the f i f t h dose of BCG was given o r a l l y . Where the violence of the reaction became too marked, 40 mg was reduced to 20 mg or 10 mg and the time i n t e r v a l extended. A further eight patients (Table VII) treated s i m i l a r l y are now i n a randomized t r i a l . - Method of BCG Immunization. Forty milligrams of BCG was dissolved i n 0.3 ml of s t e r i l e normal s a l i n e . Using a 1 ml p l a s t i c disposable syringe with a 25 gauge needle, the milky contents were spread on a l e v e l , 4 x 4 cm area of the appropriate limb, and s i x ver-t i c a l and s i x h o r i z o n t a l scratches made j u s t deep enough to draw blood. The area was then dried by fan without extra l i g h t or heat, and no dressing applied. For o r a l administration, 1 120 mg of BCG was added to one h a l f cup of clear f r u i t j u i c e . The mouth was then rinsed with a f u l l cup of f r u i t j u i c e . BCG was always stored i n darkness at 4°C and sol u -b i l i z e d immediately before use. 38 Melanoma Patients The diagnosis of melanoma was confirmed i n a l l patients by h i s t o l o g i c a l ! c r i t e r i a . Tables VIII, IX, and X outline the therapy and c l i n i c a l r e s u l t s . - Method of Vaccinia Virus Therapy. Immunity was established by routine vaccination with v a c c i n i a v i r u s . Three to s i x weeks l a t e r , tumour areas were i n j e c t e d with from 1 - 6 vaccinating doses of vac-c i n i a v i r u s . In patient 18, v a c c i n i a virus was i n j e c t e d into h i s a s c i t i c f l u i d . Patient 9 received weekly increasing subcutaneous doses to a maximum of 10 vaccinating doses subcutaneously at one time. - Method of BCG Therapy. Routine skin t e s t i n g to 5 and 250 tuberculin units (TU) was c a r r i e d out and patients who were negative to these two strengths were routinely immunized with 0.1 mg BCG i n t r a -cutaneously. I n t r a - l e s i o n a l i n j e c t i o n of BCG was c a r r i e d out using i n j e c t i o n s of from 0.05 to 3 mg BCG i n 0.1 - 0.3 ml depending on the s e n s i t i v i t y of the patient and the s i z e of the l e s i o n . Injections were c a r r i e d out e i t h e r once a week or every second week. Oral BCG was given i n doses of 80 mg once a week up to doses of 200 mg three times a week. Routine average dosage was 120 mg given e i t h e r once a week or every second week. Connaught Laboratories Ltd. 0.0001 and 0.005 mg PPD i n 0.1 ml s a l i n e diluent. 39 2. LEUKOCYTE CULTURE The technique of measuring the lymphocyte response to mitogens by the incorporation of 3H-thymidine was a modification of the procedure described by Bain and Lowenstein (122). S t e r i l e technique was used throughout. Blood was obtained by venipuncture and placed i n t o Falcon 3033 tubes containing 400 units of phenol-free heparin f o r every 15 ml of blood. The tubes were c e n t r i -fuged f o r 10 minutes at 1,800 rpm and plasma and buffy coat transferred to another tube, resuspended, and placed i n a 37°C a i r incubator on a 60° angle. A f t e r the red c e l l s had sedimented, the tubes were placed v e r t i c a l l y f o r a further 15 minutes to obtain a d i s t i n c t demarcation between red c e l l s and the w h i t e - c e l l r i c h plasma. The w h i t e - c e l l r i c h supernatant was then spun at 1,000 rpm f o r 10 minutes, the c e l l button resuspended i n Eagle's medium (MEM) con-t a i n i n g 100 units of p e n i c i l l i n per ml, 100 yg of streptomycin per ml, and 15% autologous plasma and an aliquot removed to determine the lympho-cyte count. For mitogen stimulation, the c e l l suspension was d i l u t e d to give a f i n a l concentration of 2 x 10 5 lymphocytes per ml and d i v i d i e d into 4 aliquots of 4 ml each to which was added one of the following: phyto-hemagglutinin (PHA) , concanavalin A 2X (Con-A), or pokeweed (PW). Burroughs-Wellcome (0.01 ml/4 ml) N u t r i t i o n a l Biochemicals (0.01 ml/4 ml) Grand Island B i o l o g i c a l Co. (0.04 ml/4 ml) 40 One tube was l e f t as a c o n t r o l . Each preparation was dispensed, i n t r i p l i c a t e 1 ml a l i q u o t s , i n t o 10 x 75 disposable glass culture tubes and incubated at "37°C for 3 days. The lymphocyte count was adjusted to 5 x 10 5 c e l l s per ml using 85% MEM and 15% autologous plasma for antigen stimulation (PPD and *** v a c c i n i a virus ). The cultures were incubated at 37 C i n Falcon 3033 tubes i n a t o t a l volume of 4 ml for 7 and 6 days respectively along with unstimulated controls. At the end of the incubation period, the mitogen stimulated cultures were incubated with 0.5 yCi of 3H-thymidine (New England Nuc-l e a r , s p e c i f i c a c t i v i t y 6.7 Ci/mmole) for 2 hours; antigen stimulated cultures were incubated with 2 pCi of 3H-thymidine for 4 hours. Further uptake of 3H-thymidine was blocked by the addition of 100 Vg of 'cold' thymidine to each culture. The culture tubes were centrifuged at 1,500 rpm for 10 minutes and the supernatant discarded. Five per cent t r i c h l o r o a c e t i c a c i d (TCA) was added to each tube to pre-c i p i t a t e the c e l l nucleoprotein. The p r e c i p i t a t e was centrifuged and washed once more with TCA followed by a f i n a l methanol wash. Hydroxide **** of hyamine was added to each tube. When the p r e c i p i t a t e had com-p l e t e l y dissolved, the contents were transferred to l i q u i d s c i n t i l l a t i o n Becton Dickinson ** Connaught Laboratories Ltd. (0.025 mg/4 ml) Connaught Laboratories Ltd. (0.032 doses) **** Packard 41 counting v i a l s along with two 0.3 ml washings of absolute ethanol. * F i f t e e n m i l l i l i t r e s of s c i n t i l l a t i o n s o l u t i o n was added to each v i a l . Counting was done i n a Picker Liquimat 220 s c i n t i l l a t i o n counter. A l l counts were corrected for quench using the external standard method. 3. SERUM IMMUNOGLOBULIN LEVELS These were determined using Hyland immunodiffusion plates. 4. CULTURE OF COLONY-FORMING UNITS Peripheral blood colony-forming units (CFU-C) were studied by the method of Kurnick and Robinson (123). S t e r i l e technique was used throughout. Five m i l l i l i t r e s of venous blood was placed i n t o 2001 Falcon tubes containing 150 units of heparin and sedimented by gravity at room temperature f o r 1 hour. A t o t a l white c e l l count was done on the w h i t e - c e l l r i c h supernatant. One m i l l i l i t r e aliquots of a 5 ml cell/media/agar suspension of 5 x 10 6 nucleated white c e l l s , McCoy's 5-A (modified) medium con-ta i n i n g enrichments, 15% f e t a l c a l f serum, and 0.5 ml of 3% agar were dispensed i n t o 4 Falcon 1008 p e t r i dishes. * 3,800 ml Toluene (Fisher D-289); 11.4 g PPO (Packard); 0.38 g POPOP (Packard). •kit 500 ml modified McCoy's 5A; 6.25 ml sodium pyruvate (100 mM); 3.7 ml sodium bicarbonate (7.5%); 2.5 ml Eagle's MEM vitamins (lOOx); 5 ml Eagle's MEM amino acids (50x); 2.5 ml Eagle's non-essential amino acids (lOOx); 2.5 ml glutamine (200 mM); 0.25 ml L-serine (21 mg/ml); 1.0 ml L-asparagine (10 mg/ml). 42 The dishes were incubated f o r 2 weeks i n a humidified 7.5% CO2 incubator before counting the t o t a l number of colonies per dish (the minimum c e l l requirement being 20 c e l l s per colony). 5. PHOTOGRAPHY OF CFU Colonies observed under the inverted microscope a f t e r 14 days of culture were 'picked' f o r phase contrast microscopy photographs by d i r e c t i n g a non-heparinized c a p i l l a r y pipette s l i g h t l y to the side of the desired colony on an angle, and, with the aid of bulb suction, quickly sucked up in t o the pipette. The colony-agar mixture was placed on a s l i d e and a cover s l i p placed over the mixture. By c a r e f u l l y watching the colony i t s p o s i t i o n could be marked, and, a f t e r sealing the cover s l i p with melted p a r a f f i n , photographs taken. 6. SPONTANEOUS DNA SYNTHESIS (BASELINE TRANSFORMATION) The uptake of 3H-thymidine by pe r i p h e r a l blood c e l l s was studied s e r i a l l y i n patients by mixing 0.05 ml of whole blood with 1 ml of MEM and adding 1 yCi of 3H-thymidine. Following a 3 hour incubation at 37°C, 2 drops of cold thymidine and 1 drop of zappoglobin were added to each tube. Within one minute of adding the zappoglobin, the c e l l suspension was transferred to 10 ml of normal s a l i n e and suction applied through a m i l l i p o r e GF f i l t e r u n i t . Following one further wash with s a l i n e , one d i s t i l l e d water wash, and one methyl alcohol wash, the f i l t e r pads were counted i n a l i q u i d s c i n t i l l a t i o n counter using the toluene-based s c i n t i l l a t o r . 43 7. AUTORADIOGRAPHS The procedure i s a modification of that described by Whitelaw (7-a) Preparation of Blood Films Two microcuries of 3H-thymidine was added to an EDTA vacutainer tube containing 6 ml of fresh venous blood. This mixture was then incubated at 37°C for 30 minutes a f t e r which the blood was spun for 15 minutes at 600 rpm. The buffy coat c e l l s were recovered and trans-f e r r e d t t o a second te s t tube where, i f necessary, plasma could be added to those suspensions too thick for spreading. A drop of the radioactive blood was placed on a s l i d e and spread with the edge of a second s l i d e to obtain an even d i s t r i b u t i o n of white c e l l s . The s l i d e s were a i r dried and then f i x e d i n 96% ethanol and 10% n e u t r a l phosphate buffered formalin (3:1) f o r 2 minutes, washed ** i n 0.01 M phosphate b u f f e r , and stored u n t i l autoradiographs could be; prepared. (124) . 10% Neutral Formalin: Commercial formalin (40%) D i s t i l l e d water Mix above and b u f f e r with: 100 ml 900 ml N a H 2 P 0 i + anhydrous N a 2 H P 0 i t anhydrous 3.5 g 6.5 g 0.01 M Phosphate Buffer: Stock: 0.1 M N a 2 H B 0 4 (14.2 g/1) 0.1 M K H 2 P O 4 (13.6 g/1) To make 3.5 1 buf f e r use: Stock Na 2 H P 0 i t Stock K H 2 P O 4 D i s t i l l e d water 250 ml 100 ml 3,150 ml 44 (7-b) Autoradiography In a dark room t h e i f i x e d s l i d e s were coated with nuclear emul-sion by gently dipping the s l i d e i n the l i q u i d emulsion and allowed to dry i n a v e r t i c a l p o s i t i o n f o r at least two hours. A f t e r trans-f e r r i n g the s l i d e s to p l a s t i c s l i d e boxes containing humi-caps as a source of capsulated drying agent, the boxes were sealed with e l e c t r i c a l tape and allowed to develop f o r three days at 4°C i n a lead l i n e d con-tain e r . (7-c) Development of Prepared Autoradiographs In the dark, the s l i d e s were developed f o r 355 minutes i n D-19 A A developer i n a free flowing 60 ± 1°F water bath, then rinsed gently A A * i n the water and fi x e d i n acid f i x a t i v e f o r 5 minutes. On s a t i s -factory c l e a r i n g of the nuclear emulsion, the s l i d e s were rinsed i n the water bath f o r a further 15 minutes before drying at 37°C f or 24 hours. (7-d) Staining The developed dry s l i d e s were then stained i n a 1:1 Wright's water s t a i n f o r 15 minutes, washed with tap water, rinsed b r i e f l y with A Kodak Nuclear Track Emulsion NTB2. A A Kodak. Prepare as directed and use as a 1:1 mixture of de-veloper and d i s t i l l e d water. A A A Kodak. Use as a 1:3 mixture of f i x a t i v e and d i s t i l l e d water. Wright's Stain Solution, Fisher S c i e n t i f i c Company SO-W-16. 45 concentrated Wright's s t a i n to remove p r e c i p i t a t e d s t a i n and counter-stained with buffered Giemsa for 30 minutes. 8. PREPARATION OF NEURAMINIDASE TREATED TUMOUR CELLS The method of preparation follows the technique used by S e i g l e r (125). Fresh tumour tissue rinsed free of blood was chopped, teased, and syringed into a s i n g l e c e l l suspension. The suspension was allowed to s e t t l e f o r f i v e minutes on i c e and the remaining c e l l s i n the super-natant were suspended i n a n t i b i o t i c - f r e e Eagle's MEM. The suspended c e l l s were centrifuged at 1,000 rpm and the p e l l e t resuspended i n 9 ml of medium. A c e l l count was performed and the suspended tumour c e l l s were d i l u t e d i f the c e l l concentration was greater than 2 x 10 7 per ml; the c e l l s were then i r r a d i a t e d with 10,000 rads from a Cobalt 60 source. To each 9 ml of i r r a d i a t e d c e l l suspension, 500 units of neura-minidase was added and the mixture incubated at 37°C for 30 minutes, a f t e r which the c e l l s were p e l l e t e d , resuspended i n 10 ml of medium and p e l l e t e d again. This f i n a l p e l l e t was resuspended i n 0.75 ml medium containing the desired dose of BCG and i n j e c t e d subcutaneously into the patient being treated. Giemsa's Blood Stain, Matheson Coleman & B e l l GX85 B527. One part Giemsa:Fifty parts b u f f e r (6 ml M/15 NaaHPO^, 44 ml M/15 KH 2P0 4). ** V i b r i o cholerae Neuraminidase. General Biochemicals. RESULTS 1. HEALTHY VOLUNTEERS As a q u a l i t y c o n t r o l blood studies were p e r i o d i c a l l y c a r r i e d out on healthy laboratory volunteers and did not at any time show any evidence of technical d i f f i c u l t i e s . 2. STUDENT NURSES Pre-BCG vaccination tuberculin t e s t i n g was simultaneously done with 5 TU PPD-Standard (USPHS), Connaught Bio-equivalent 12127-1, and CTg82099-1. These were repeated post-BCG and i n addition Connaught 250 TU 5018-1 was administered. No pre-testing with 250 TU was done as t h i s p r a c t i c e was discontinued by the D i v i s i o n of Tuberculosis Control i n B r i t i s h Columbia i n 1972. Reactions to PPD-Standard, Connaught Bio-equivalent and CTgg were considered p o s i t i v e at the 6 mm l e v e l while those at the 10 mm l e v e l to 250 TU were accepted as p o s i t i v e . Tables I and II compare sk i n t e s t r e s u l t s according to reaction s i z e i n millimetres between group A (intracutaneous BCG) and B ( o r a l BCG) nurses. The e f f i c a c y of intradermal BCG, as compared to o r a l BCG, to e l i c i t strong skin test responses to 5 TU PPD i s s t r i k i n g . In the intradermal group 91.2% of the reactions to 5 TU PPD were greater than 10 mm whereas only 20% of nurses receiving o r a l BCG showed reactions of t h i s magnitude. Reaction s i z e to 250 TU PPD was s i m i l a r l y stronger 46 TABLE I. Results of tuberculin t e s t i n g two groups of student nurses 10-13 weeks post-BCG vaccination showing per cent of group tested according to reaction s i z e . PPD-Staridard GROUP "A" Intradermal BCG (34) 0 mm 1-5 mm 6-9 mm 10 mm 47.1 17.6 8.8 26.5 91.4 GROUP "B" Oral BCG (35) 0 mm 1-5 mm 6-9 mm 10 mm 5.7 2.9 Bio-equivalent 12127-1 17.6 5.9 76.5 80.0 8.6 11.4 C T 6 8 2101-1 2.9 5.9 91.2 71.4 2.9 5.7 20.0 250TU 5018-1 100.0 5.7 5.7 88.6 Tested at 13 weeks. Tested at 10 weeks. TABLE I I . Results of tuberculin t e s t i n g two groups of student nurses 10-13 weeks post-BCG vaccination giving average s i z e of reaction i n mm. ft GROUP "A" Intradermal BCG (34) GROUP "B" Oral .BCG (35) Average Age (yrs) 19.0 18.9 PPD-Standard 4.9 0.7 Bio-equivalent 12127-1 12.3 2.8 C T 6 8 2101-1 17.0 3.5 250TU 5018-1 32.5 22.2 BCG Lesion (mm) 6.1 ft Tested at 13 weeks. *ft Tested at 10 weeks. 49 with 100% of nurses i n the intradermal group showing tuberculin s e n s i -t i v i t y . Eleven per cent of nurses receiving o r a l BCG f a i l e d to become tuberculin p o s i t i v e . Results of follow-up skin testing performed 6 months a f t e r i n i -t i a l s e n s i t i z a t i o n did not reconfirm the e f f i c a c y of intradermal BCG; responses of the intradermal.group had decreased while those of the o r a l group had increased (Table I I I ) . The one nurse retested who did not respond at a l l to e i t h e r 5 or 250 TU PPD was from the intradermal group. TABLE I I I . Results of tuberculin t e s t i n g two groups of student nurses 6 months post-BCG giving average s i z e of reaction i n mm. Group A Group B Intradermal BCG Oral BCG (31) (29) I n i t i a l 6 Month I n i t i a l 6 Month Response Response Response Response 5 TU 17.0 14.5 3.5 6.7 250 TU 32.5 18.5 22.5 16.0 Table IV shows the i n v i t r o PHA responses;-pre-vandipost-vaccination i n group A and B nurses. Within e i t h e r group, there was no s i g n i f i c a n t difference by the Student's t test (126,127) between the pre- and post-vaccination PHA response. Because there was no s i g n i f i c a n t difference before and a f t e r BCG vaccination, PHA r e s u l t s were averaged for the TABLE IV. PHA response i n group "A" and "B" student nurses pre- and post-BCG vaccination. Pre-BCG PHA Post-BCG PHA DPM x 1 0 _ 3 DPM x 10" 3 Group "A" 56 51 103 115 46 6 64 65 135 207 88 100 97 141 189 65 12 24 40 21 28 31 23 29 72 82 127 _ 109 _ 138 x = 78 _ 94 x = 79 x = 79 ± 9* Group "B" 34 27 73 127 48 55 140 108 59 95 57 69 34 54 60 38 77 60 66 57 38 37 35 84 83 114 42 30 38 _ 54 13 x = 57 _ 19 x = 63 x = 60 ± 5 Mean ± 1 standard deviation of the mean. 51 r e s u l t on each In d i v i d u a l . Although the mean PHA response i n group B was lower than group A t h i s difference was not s i g n i f i c a n t (p > 0.1). PPD cultures set up on these same i n d i v i d u a l s show responses seen i n Table V. Group A and group B show a s i g n i f i c a n t difference i n pre- and post-vaccination PPD i n v i t r o values (p < 0.001 and p < 0.005 re s p e c t i v e l y ) . Pre-vaccination PPD values between groups A and B do not d i f f e r s i g n i f i c a n t l y (p > 0.7) nor does post-vaccination PPD com-parisons 0> > 0.4). Contrary to previous studies done i n the laboratory (121), there was no s i g n i f i c a n t c o r r e l a t i o n between the average PHA and post-vacci-nation PPD i n v i t r o responses i n th i s group of nurses (r = 0.09, p > 0.8). 3. CLINICAL OBSERVATIONS IN LYMPHOMA PATIENTS Tables VI and VII b r i e f l y present the c l i n i c a l h i s t o r y of these patients. Patient 1. Six extradural recurrences, i n i t i a l l y i n the upper thoracic and c e r v i c a l area followed by progression to the middle thor-a c i c and lumbar regions, have occurred i n th i s patient while on BCG immunotherapy. The i n i t i a l three recurrences were treated with i r r a -d i a t i o n , cyclophosphamide, and v i n c r i s t i n e . The fourth recurrence was associated with "the appearance of a supraclavicular lymph node. Since the patient had had the maximum t o l e r a b l e dose of i r r a d i a t i o n and had f a i l e d to show a response to cyclophosphamide and v i n c r i s t i n e at the l a s t recurrence the lymph node was removed, treated with neuraminidase, and the treated c e l l s i n j e c t e d i n t o the l a t e r a l aspect of the thigh along with 0.3 mg of BCG. 52 TABLE V. PPD responses i n group "A" and "B" student nurses pre- and post-BCG vaccination. Pre-BCG PPD Post-BCG PPD DPM x 10" 3 DPM x 10" 3 Group "A" 49 195 77-8 114 16* 119 1* 210 71 262 85 130 10* 180 47 304 41 234 132 62 88 86 126 119 65 246 3* 115 1* 76 136 222 4 6 - ** 1 4 5 63 x = 59 ± 10 92 x = 158 ± Group "B" 72 19 91 108 5* 101 147 226 77 212 246 164 26* 154 13* 61 48 206 55 161 73 74 119 113 4* 77 47 112 24* 211 42 292 8* x = 65 ± 15 126 x = 142 ± Using values determined by previous studies (121) on the e f f e c t of intracutaneous BCG on i n v i t r o PPD responses only these i n d i v i d u a l s would be considered t r u l y tuberculin negative. Mean ± 1 standard deviation of the mean. TABLE VI. Lymphoma - non-randomized t r i a l . Recurrence Patient * Stage Diagnosis Primary Treatment+ a f t e r Immunotherapy Months Survival Comment 1. II I D MLLPD I + C 11-38 months 63 Now on chemotherapy 2. . III D MLLPD C - 0 51 D Disease free 3. II I D MLU I + C 0 39 Disease free 4. I D MLU I + C 0 37 Disease free 5. I D MLH I + C 0 32 Disease free 6. I D MLH I + C 0 32 Disease free 7. II D MLLPD I + C 6 weeks 10 died Recurred before immunized D = Dif f u s e + MLLPD = malignant lymphoma, lymphocytic, poorly d i f f e r e n t i a t e d MLU = malignant lymphoma, undifferentiated MLH = malignant lymphoma h i s t i o c y t i c £ I = I r r a d i a t i o n -r C = Chemotherapy TABLE VII. Lymphoma - randomized t r i a l . ^ Primary Months Patient Stage Diagnosis Treatment? Recurrence Survival Comment Treatment group 101 III D MLH 01 I N MLLPD 03 II D MLH 102 IV D MLLPD 103 IV D MLLPD 105 III N MLLPD 106 III D MLLPD 06 II D MLH Control group 02 II D MLH 04 II D MLH 05 II D MLLPD 104 III D MLH C + I 0 I + C 0 I + C 0 C 1 week? 4 months (I) C 3 months (I) C 8 months C + I 0 I + C 0 I + C 3 and 7 months I + C 2 months I + C 0 C 0 28 Disease free 26 Disease free 24 Disease free 23 Recurred before immu-nized. Now immunized and disease free 20 Again disease free 15 Again disease free 17 Disease free 12 Disease free 15 Deceased 7 Deceased 21 Disease free 15 Disease free D = Diffuse N = Nodular + MLH = malignant lymphoma, h i s t i o c y t i c MLLPD = malignant lymphoma, lymphocytic, poorly d i f f e r e n t i a t e d MLU = malignant lymphoma, undifferentiated -t- I = i r r a d i a t e d C = chemotherapy 55 A l o c a l reaction occurred w i t h i n f o r t y - e i g h t hours with r e l i e f of pain and within two weeks a draining sinus had become established at the s i t e of i n j e c t i o n which healed over within s i x weeks. Complete c l i n i c a l remission was associated with t h i s procedure. The patient gained eight pounds i n weight, the hemoglobin rose 1.5 g and he was able to return to work. A f t e r two months further relapses occurred which have, for the past sixteen months, been treated s u c c e s s f u l l y with i r r a d i a t i o n followed by prednisone and amethopterin. During this time, the BCG vaccinations have been discontinued due to the continued use of prednisone. Patients 2, 4, and 5. These patients are maintained on s c a r i -f i c a t i o n vaccination every s i x weeks with no recurrence to date. Patient 3. Having received repeated vaccinations over a period of a year, this patient developed a generalized macular rash associated with mild p r u r i t i s f i v e days a f t e r a vaccination. Five days a f t e r i t s appearance a biopsy was c a r r i e d out, both of the vaccination s i t e and of the area of skin involved with the generalized rash. No evidence of v a s c u l i t i s or c e l l u l a r i n f i l t r a t i o n was seen i n the biopsy from the macular rash and microscopically the s c a r i f i c a t i o n s i t e revealed a t y p i c a l granulomatous reaction to BCG with giant c e l l s . When the rash had s e t t l e d down, a further vaccination was c a r r i e d out using only 5 mg BCG, followed by an i d e n t i c a l rash. This cleared up i n f i v e weeks and the patient, since thatttmne, has been maintained on o r a l BCG at a dose l e v e l of 120 mg BCG every three weeks. There has been no further recurrence of the 56 generalized rash following the change to the o r a l route f o r BCG adminis-t r a t i o n , and i n vivo and i n v i t r o s e n s i t i z a t i o n remains high. Patient 6. BCG immunization occurred slowly i n t h i s patient. During the i n i t i a l four months of immunotherapy, a moderate e o s i n o p h i l i a (1944/cu mm) developed and as the e o s i n o p h i l i a gradually waned a more marked immunological reaction was evident at the s i t e of vaccination and the vaccinations were lengthened to every two weeks. At present, t h i s patient receives vaccinations every four weeks and remains c l i n i -c a l l y free of disease. Patient 7. This twenty year o l d woman presented with a superior mediastinal obstruction which responded dramatically to i r r a d i a t i o n . Following t h i s , she received a s i n g l e cycle of chemotherapy consisting of cyclophosphamide, v i n c r i s t i n e and prednisone. Two weeks following the chemotherapy BCG multiple s c a r i f i c a t i o n was commenced. However, she remained tuberculin negative and f a i l e d to show any immunological reaction to the vaccinations. At the s i x t h week there was c l i n i c a l evidence of upper abdominal recurrence and vaccination was discontinued. She f a i l e d , t h i s time, to a t t a i n a complete c l i n i c a l remission on chemo-therapy and i r r a d i a t i o n , did not receive further BCG vaccinations, and died ten months a f t e r diagnosis. Table VII shows the preliminary r e s u l t s i n the randomized t r i a l . Two stage IV patients (102 and 103) who received only chemotherapy to achieve a complete remission have recurred i n the abdomen where they had extensive presenting disease. Similar to patient 7, patient 102 57 had f a i l e d to immunize a f t e r four months. They have both responded w e l l to abdominal i r r a d i a t i o n . Stage III patient 105 who had only re-ceived chemotherapy to induce remission s i m i l a r l y recurred while on BCG immunotherapy. There has only been minimal t o x i c i t y to the BCG. Experience has shown that vaccination may be commenced two weeks a f t e r chemotherapy or i r r a d i a t i o n . The reactions observed havebbeen swelling and induration at the s i t e of vaccination followed by exudation and scabbing. As sen-s i t i z a t i o n progresses, the scabs l a s t f or four to f i v e weeks. Where possibl e , revaccination i s done only when the previous scab has come o f f . Frequently, there develops increased pigmentation at the s i t e of old vaccinations. Twenty-four hours following vaccination the s i t e s of previous vaccinations often become it c h y and s l i g h t l y erythematous but no u l c e r a t i o n occurs. The reactions always seem to be more severe on the thighs than on the arms. Small i n g u i n a l lymph nodes become tender and palpable (0.5 - 1.5 cm) f i v e to ten days following vaccination on the l a t e r a l aspect of the thighs. This reaction subsides i n two to three weeks but the nodes remain palpable f o r several months. A few patients f e e l f e b r i l e the night of the vaccination but are usually recovered by morning. A l l have been able to carry on t h e i r normal work or household a c t i v i t i e s . 4. CLINICAL OBSERVATIONS IN MELANOMA PATIENTS Table VIII shows the r e s u l t s of twenty-one patients treated i n the Immunotherapy C l i n i c . Of the twenty-one pat i e n t s , nineteen were TABLE VIII. Melanoma patients - treatment and c l i n i c a l responses. Responses # BCG Treatment Vaccinia-? Skin In Imniuno- Survival Patient Stage Test V i t r o i . c. m.s. or a l i n j . i . t . I r r . ther. (months) Comments 1. JM I + + _ _ + + _ Good 26 Died of ca. prostate and cardiac (age 85). 2. AL I - - + - + - + - Good 34+ Now tumour-free. 3. PB I - - + - + - + - Good 27+ Now tumour-free. 4. GH I + + + - + + - - N i l 6 Rapid progressive disease. 5. HH I + + + + - + - - Good 15+ Minimal residual.tumour. 6. BT I - + + + + - - - Poor 6 Progressive disease. 7. DD I + + - - + + - - Slight 9 Slig h t l o c a l regression. 8. H I - - + + + - - •- Poor 9 9. SF II - - + + + - - - Good 25+ Oral BCG - g . i . lesions only. Disease-free. 10. VM II - - + - + - - + ? 17+ Irr a d i a t i o n response. Disease-free. 11. EG II - - + - + - - + ? 15+ Ir r a d i a t i o n response. Disease-free. 12. LW II + + + Good 14 g . i . lesion only p a r t i a l l y resected. Nega-ti v e g . i . X-ray and l i v e r scan. Recent cerebral secondary i r r a d i a t e d . 13. JH III + + - - + + - + F a i r 21 Early good response. 14. MD III + + - - + - - + Poor 5 Progressive disease. 15. MF III - + + - + + - - N i l 4.5 Unable to se n s i t i z e . 16. GM III + + - - + - - - Good 14+ Good l o c a l response. Recent v i s c e r a l spread 17. MG IV + - - - + + - - Poor 6 18. GG IV - - + + + + - + Poor 4 Progressive disease. Unable to s e n s i t i z e . 19. IB IV + + - - + + - - S l i g h t 2.5 Slight l o c a l regression. Died cerebral haemorrhage into tumour. 20. PW IV - - + - + + - Poor 16+ 21. PB IV — + — — + + - N i l 4 Slight radiation response of subcutaneous le s i o n . * Stage I - disease c l i n i c a l l y l o c a l i z e d to no further than the regional lymph nodes when f i r s t seen. Stage II = disease c l i n i c a l l y l o c a l i z e d to a sin g l e region beyond the regional lymph nodes. Stage III = disease c l i n i c a l l y l o c a l i z e d to the subcutaneous tissue or lymph nodes beyond regional drainage. Stage IV = disease with v i s c e r a l involvement when f i r s t seen. + i . e . = intracutaneous s e n s i t i z a t i o n . m.s. = multiple s c a r i f i c a t i o n s to limbs, o r a l = o r a l BCG (see Table IX). i n j . = injected into tumour (see Table X) . i . t . = intratumour i n j e c t i o n of vacc i n i a . # I r r . = i r r a d i a t i o n to some le s i o n s . immunother. = immunotherapy. 00 **** 59 s u i t a b l e for assessment to immunotherapy and of the nineteen, eleven showed l i t t l e or no response (Patients 4, 6, 7, 8, 14, 15, 17, 18, 19, 20, 21) while eight (Patients 1, 2, 3, 5, 9, 12, 13, 16) have shown some response, defined i n seven by regression of the tumour by f i f t y per cent or more and i n one by extended c l i n i c a l w e l l being (Patient 9). Of the eight responders, four were i n Stage I having had the primary melanoma on the foot with metastatic disease no further than the i n -guinal lymph nodes. Three patients were treated i n i t i a l l y with d i r e c t i n j e c t i o n of v a c c i n i a virus i n t o tumour nodules. A l l responded w e l l i n i t i a l l y . A l l patients with involvement of l i v e r , spleen, bone and b r a i n f a i l e d to respond to immunotherapy. Table IX shows the t o t a l o r a l BCG dose over a period of months. Table X shows t o t a l milligrams of BCG i n j e c t e d i n t o tumour nodules over a period of months. Patient 1. This e i g h t y - f i v e year old native Indian had exten-sive subcutaneous melanoma. For the f i r s t twelve months of therapy there was excellent (80%) regression following multiple intratumour i n j e c t i o n s of v a c c i n i a v i r u s . The tumour then became c l i n i c a l l y r e s i s -tant to v a c c i n i a virus i n j e c t i o n s despite the fact that there was no evidence of blocking antibody i n the patient's serum which would i n h i b i t the i n v i t r o stimulation of normal lymphocytes s e n s i t i v e to v a c c i n i a v i r u s . Marked v i t i l i g o developed i n the areas of previous tumour growth. A continuation of immunotherapy with BCG and PPD i n j e c t i o n s gave good cont r o l although the tumour was not eradicated nor did there appear to be as much v i t i l i g o i n the areas regressing under t h i s therapy as with v a c c i n i a virus therapy. 60 TABLE IX. Milligrams of BCG taken o r a l l y by melanoma patients during immunotherapy treatment. Patient T o t a l Oral BCG Months 1 0 .' 4 2 680 14 3 560 13 4 100 2 5 40 1 6 2,980 7 7 2,405 6 8 2,080 6 9 4,525 22 10 435 5 11 2,160 10 12 4,020 10 13 2,250 9 14 600 3 15 280 3 16 3,800 9 17 440 4 18 680 3 19 120 2 20 130 2 21 1,000 4 TABLE X. Milligrams of BCG d i r e c t l y i n j e c t e d into melanomatous nodules during immunotherapy treatment. BCG Tumour Number of Months Patient I n j e c t i o n Administered 1 6.25 13 4 0.40 1 5 1.90 2 7 0.60 4 13 27755 14 15 4.10 4 17 0.90 4 18 0.40 1 19 0.60 2 20 0.50 2 62 Patient 2. This f i f t y - t w o year o l d white woman had multiple metastatic nodules measuring from 0.25 - 1.0 cm i n diameter on the dorsum of the foot as w e l l as on the lower part of the leg which responded to multiple v a c c i n i a virus i n j e c t i o n s but tended to recur over the f i r s t sixteen months. She was then tumour-free f o r s i x months but a further nodule appeared on the dorsum of the foot which continued to grow despite repeated i n j e c t i o n s of v a c c i n i a virus up to eight vac-c i n a t i n g doses. Thirteen months ago th i s 1.5 cm diameter nodule was s u r g i c a l l y removed and was shown microscopically to be i n f i l t r a t e d with inflammatory c e l l s , predominantly plasma c e l l s . A s i n g l e c e l l suspension prepared from the tumour f a i l e d to reveal v i a b l e tumour c e l l s . Five previous s i n g l e c e l l preparations from subcutaneous meta-s t a t i c melanoma biopsy material contained from 30 - 50% vi a b l e c e l l s . This patient i s now considered to be r e s i s t a n t to v a c c i n i a virus therapy. She i s being maintained on a prophylactic regime of o r a l BCG, having been s e n s i t i z e d intracutaneously when she was f i r s t seen i n the Immuno-therapy C l i n i c . Patient 3. This f i f t y - s e v e n year old white woman has shown a good response i n the s i x subcutaneous tumours on the dorsum of the foot. A l l tumours were less than 1 cm i n diameter. Two tumours have tended to recur repeatedly; one of these being i n the skin g r a f t placed at the s i t e of removal of the primary tumour. Tumour regression i n this patient has been associated with halo formation about the tumour. The l a s t tumour recurrence required f i v e times as large a dose of virus to produce regression and the patient i s considered to be developing 63 resistance to v a c c i n i a virus therapy. This patient, also, has been p r o p h y l a c t i c a l l y immunized to BCG and immunity i s being maintained by the use of o r a l BCG. Patient 5. This seventy-seven year old white male was referred to the Immunotherapy C l i n i c following p a r t i a l s u r g i c a l removal of re-current metastatic melanoma i n the i n g u i n a l nodes. The patient was immunized with intracutaneous BCG and t h i s was followed with repeated i n j e c t i o n s of 0.1 - 0.3 mg of BCG i n t o the i n g u i n a l tumour. For the f i r s t three months the tumour grew, reaching a s i z e of 4.0 x 6.0 cm. Because of the s e v e r i t y of systemic reactions, the regimen was changed to the i n j e c t i o n of PPD, using a dose of 5,000 TU. These i n j e c t i o n s were given every two weeks and i n s i x weeks the tumour had regressed to 2.5 x 3.5 cm. Twenty-one months a f t e r r e f e r r a l to the C l i n i c , no d e f i n i t e tumour could be palpated although the area was indurated from the s u r g i c a l procedure. Patient 9. This thirty-two year old white woman had a malig-nant melanoma removed from the l e f t upper arm i n June, 1970. She was w e l l u n t i l November, 19 71 when she developed p a r t i a l small bowel ob-s t r u c t i o n and a laparotomy was found to have multiple metastases to the small bowel. The l i v e r was not involved. Two enteroanastomoses were performed but the tumour was not resected. Post-operative recovery was uneventful and f o r some weeks the patient improved but symptoms of.abdominal pain, anorexia and weight loss recurred and she was re-ferred to the Immunotherapy C l i n i c i n January, 1972. Her further 64 c l i n i c a l course i s i l l u s t r a t e d i n Figure 1. Three procedures were ca r r i e d out. She received a 300 ml blood transfusion from patient 2 (at t h i s time patient 2 s t i l l had several tumours and was continuing to receive treatment for continued recurrences). The purpose of the blood transfusion was to give rapid immunization to v a c c i n i a virus and BCG. Along with t h i s transfusion the patient was immunized with intracutaneous BCG and also revaccinated. For the next f i v e weeks she received increasing subcutaneous doses of l i v e v a c c i n i a virus to a f i n a l s i n g l e dose of 10 vaccinating doses. She was also s t a r t e d on o r a l BCG, s t a r t i n g with a dose of 5 mg and increasing i t to 40, 80, and 120 mg; the doses being given twice weekly. Her c l a s s i c response to the o r a l BCG was that twentySfour to f o r t y - e i g h t hours a f t e r re-ceiving o r a l BCG, pain developed over the larges t tumour mass i n i t i a l l y and with continuing therapy i n a l l of the palpable tumours. Four weeks af t e r commencing the BCG she again developed p a r t i a l obstruction and the dose of o r a l BCG was reduced. The dose was then monitored according to how much the pain induced by the previous dose had improved. As treatment continued, i n addition to the pain i n t h i s large f i x e d tumour i n the l e f t lower quadrant, tender masses i n other areas of the abdomen became palpable over the course of the next several months. She con-tinued to recover c l i n i c a l l y i n the summer of 1972 and the BCG was discontinued for s i x weeks. Further o r a l BCG given i n October, 1972 resulted i n some abdominal discomfort and a questionable mass was palp-able i n the r i g h t lower quadrant. The p o s s i b i l i t y of tuberculous adeni-t i s was entertained and BCG was discontinued. The firmness and increasing Figure 1. C l i n i c a l improvement while on immunotherapy i n patient 9. ON 66 s i z e of the mass resulted i n r e - i n s t i t u t i o n of o r a l BCG but the patient again developed p a r t i a l i n t e s t i n a l obstruction requiring laparotomy i n January, 19 73. Once again, j u s t p r i o r to surgery, the administration of o r a l BCG resulted i n an increase of pain and tenderness over the abdominal tumour. At laparotomy two new metastatic melanomas were found, however, a l l the previous melanoma tumours had disappeared leaving only s l i g h t r e s i d u a l pigment. The two new tumours were resected and the post-operative recovery was uneventful. The patient was maintained on o r a l BCG; 160 mg every t h i r d week, and was without evidence of further recurrence nineteen months l a t e r . Patient 12. In 1965 t h i s f i f t y - f o u r year old white male had a malignant melanoma removed from h i s back. He had a recurrence i n 1968 i n the righ t a x i l l a and i n September, 1972 developed abdominal pain and a laparotomy i n November, 1972 revealed a large metastatic melanoma tumour i n the upper jejunum. The tumour i n the jejunum was resected but involved nodes surrounding the superior mesenteric artery could not be removed. Post-operatively the patient was r e f e r r e d to the Immunotherapy C l i n i c and for eleven months received 120 mg of BCG o r a l l y once a week. Although he continued without c l i n i c a l evidence of bowel recurrence and had a negative small bowel followTthrough study and l i v e r scan, he developed evidence of a cerebral secondary and died. Patient 13. This twenty-seven year old white woman presented i n 19 72 with numerous widely spread subcutaneous metastatic nodules that had recurred following removal of a primary melanoma from the 67 l e f t l e g . During the f i r s t f i v e months the patient made a good c l i n i -c a l response to repeated i n j e c t i o n of tumours with BCG. However, mul-t i p l e draining abscesses developed and anti-tuberculous therapy was i n s t i t u t e d . Because tumours continued to appear, one was removed, treated with neuraminidase, i r r a d i a t e d , and rei n j e c t e d along with BCG. This therapy, combined with intratumour i n j e c t i o n s of BCG as w e l l as o r a l BCG, l e d to tumour regression with no new nodules f o r four months. When new nodules appeared, a nodule was again removed and the above procedure repeated. A shorter remission of three months was accomp-l i s h e d . New nodules again became troublesome and one further neura-minidase treatment was c a r r i e d out. However, a d d i t i o n a l tumours con-tinued to appear, some of which were removed s u r g i c a l l y . The patient was maintained on o r a l BCG throughout. Nineteen months a f t e r r e f e r r a l to the c l i n i c the patient was treated with dimethyl t r i a z i n o imidazole carboxamide (DIC) but succumbed to her disease two months l a t e r . Patient 16. This s i x t y - f o u r year o l d male had a malignant melanoma removed from the l e f t ear twenty years p r i o r to r e f e r r a l to the Immunotherapy C l i n i c where he presented with multiple small sub-cutaneous nodules measuring from 0.2 - 0.4 cm i n diameter. He was tuberculin p o s i t i v e on s k i n t e s t i n g and an e f f o r t was made to maintain his disease with o r a l BCG alone. Over a period of three months he received 1.8 g BCG o r a l l y during which time the lesions did not change i n s i z e but then showed evidence of progression. For the next seven months the patient received 120 mg BCG o r a l l y every other week and simultaneously the lesions were i n j e c t e d with between 100 - 300 TU 68 PPD per dose. The i n j e c t e d lesions t o t a l l y regressed, however, further new lesions continued to appear. During the eighth month, the response diminished and the patient showed evidence of more extensive recurrence of disease which was unresponsive to DIC. 5. LABORATORY OBSERVATIONS (5-a) Lymphocyte Stimulation i n Lymphoma Patients Pe r i p h e r a l blood responses to i i i v i t r o lymphocyte stimulation have been followed pre-treatment (where possible) and every four to s i x weeks thereafter i n most lymphoma patients. Table XI presents the i n v i t r o responses to the general mito-gens: PHA, PW, and Con-A, and to the antigen, PPD. These responses represent the i n d i v i d u a l s ' mean response ± one standard deviation of the mean, before and during t h e i r immunotherapy treatment, regardless of any chemo- or radiotherapy the patient may have received. The range of responses observed within the group i s large. Consequently, the i n d i v i d u a l s ' average response to stimulation does not allow the c l i n i -cian to s e l e c t those patients responding w e l l to immunotherapy nor to s e l e c t those patients vulnerable to relapse. For example, patient 01 has not yet had a relapse i n s p i t e of very low antigen and mitogen responses. On the other hand, patient 1 had s i x recurrences while demonstrating good i n v i t r o responses. As both patients 01 and 1 had received i r r a d i a t i o n , the low response of patient 01 could not be a t t r i b u t e d to t h i s case. Figure 2, showing the s e r i a l PHA, PW and 69 TABLE XI. In v i t r o responses i n lymphoma pa t i e n t s . Patient Sex PHA PW Con-A PPD Non-Randomized Series 1 M 52 + A 6 21 + 2 70 + 8 97 + 16 2 M 101 + 7 39 + 4 104 + 10 233 + 31 3 F 96 + 10 63 + 8 117 + 14 177 + 25 4 M 41 + 5 11 + 2 32 + 6 264 + 58 5 F 84 + 10 44 + 5 93 + 6 242 + 50 6 F 95 + 22 12 + 2 66 + 7 75 + 16 Randomized Series 101 M 36 + 5 28 + 4 75 + 5 178 + 36 01 F 4 + 1 16 + 2 44 + 3 26 + 7 03 M 22 + 13 11 + 3 46 + 10 75 + 21 102 F 77 + 13 30 + 5 63 + 9 • 90 + 30 103 F 34 + 6 25 + 5 58 + 8 166 + 42 105 M 20 + 3 13 + 3 35 + 10 150 + 48 106 M 15 + 4 26 + 5 29 + 8 116 + 13 06 M 64 + 17 28 + 5 70 + 10 95 + 23 DPM x 10 3 ± 1 standard deviation of the mean. Normal values are: PHA 105 ± 5; PW 63 ± 7; Con-A 98 ± 7; PPD P o s i t i v e > 30. Figure 2. Monthly f l u c t u a t i o n s i n PHA and PPD responses i n patient 3. o 71 PPD observations i n patient 3, i l l u s t r a t e s the type of monthly f l u c -tuations observed i n a l l patients studied. The large response range, p a r t i c u l a r l y to the general mitogens, i s not representative of lymphoma patients as i t i s also evident within a group of healthy i n d i v i d u a l s (Table IV). No pattern emerges from grouping patients' i n v i t r o responses according to disease stage or h i s t o l o g i c a l diagnosis (Table XII). Because of small sample s i z e s , the responses of the less innocuous stages (I and II) were compared to those patients c l a s s i f i e d as stage III or IV. None of the responses showed a s i g n i f i c a n t difference by the Student's t test (p > 0.1 i n a l l cases). Again because of the small sample s i z e , only the h i s t o l o g i c a l c l a s s i f i c a t i o n of MLLPD and MLH were compared. No s t a t i s t i c a l difference was observed (p > 0.1 i n a l l cases); nor did mean mitogen and antigen responses d i f f e r s i g n i -f i c a n t l y i n those patients r e c e i v i n g i r r a d i a t i o n i n addition to chemo-therapy to induce a complete primary c l i n i c a l remission (Table XII). Although the mean mitogen responses i n female lymphoma patients was birgbsr than t h e i r male counterparts, the difference was not s i g n i f i c a n t ; nor was there a s i g n i f i c a n t difference i n PPD values between the two sexes (Table XII). Although the average responses between the two i n i t i a l t r e a t -ment groups did not d i f f e r s i g n i f i c a n t l y on an i n d i v i d u a l b a s i s , abdo-minal i r r a d i a t i o n appeared to suppress both mitogen and PPD responses more severely than chemotherapy alone. The suppression of response to Con-A appears to be less marked than that to PHA and PW. Recovery 72 TABLE XII. In v i t r o responses i n lymphoma groups. PHA PW Con-A PPD Stage ft ftft I + II (6) 52 ± 13 20 ± 5 59 ± 8 130 ± 37 III + IV (8) 54 ± 11 31 ± 4 69 ± 10 151 ± 16 H i s t o l o g i c a l C l a s s i f i c a t i o n MLLPD (7) 43 ± 13 24 ± 3 58 ± 8 125 ± 23 MLH (5) 60 ± 13 25 ± 5 70 ± 7 133 ± 30 Non-Immunotherapy Treatment Chemotherapy + I r r a -d i a t i o n (10) 51 ± 10 26 ± 5 64 ± 8 135 ± 23 Chemotherapy (4) 58 ± 18 27 ± 5 65 ± 13 160 ± 25 Sex Male (8) 44 ± 10 22 ± 3 58 ± 9 151 ± 23 Female (6) 65 ± 14 32 ± 7 74 ± 10 119 ± 28 Number of patients. ftft o DPM x 10" 3 ±:1 standard deviation of the mean. 73 of responses was sometimes evident four to s i x months a f t e r i r r a d i a t i o n and/or chemotherapy as i n patients 2 and 3 (Figures 3 and 4)• A f a i l u r e , however, of the PHA response to recover despite adequate responses to PPD was shown by patient 03 (Figure 5), while patient 01 f a i l e d to show a recovery to e i t h e r stimulation(Figure 6). S e r i a l r e s u l t s of Con-A and PW stimulation showed equally divergent patterns. Continued observations of the mitogen and PPD responses did not show a decline that might have allowed p r e d i c t i o n of recurrence i n patients 1, 102, 103, and 105. The s e r i a l i n v i t r o responses to PPD, PHA, Con-A and PW were reviewed f o r c o r r e l a t i o n of responses with the following r e s u l t s : PPD and PHA r = 0. 30, P < 0.001 PPD and PW r = 0. 20, P < 0.020 PPD and Con- •A r = 0. 20, P < 0.020 PHA and PW r = 0. 68, P < 0.001 PHA and Con- •A r = 0. 59, P < 0.001 PW and Con- •A r = 0. 76, P < 0.001 Table XIII presents the routine hematological p i c t u r e of the i n d i v i d u a l lymphoma patients averaged during t h e i r treatment at the Immunotherapy C l i n i c while receiving immunotherapy. (5-b) Lymphocyte Stimulation i n Melanoma Patients P e r i o d i c lymphocyte stimulation studies were done while melanoma patients were attendingethe C l i n i c . Table XIV presents the patients' mean i n v i t r o responses to the three mitogens and to the antigen, PPD. Six patients ('other') were not s u i t a b l e for assessment to immunotherapy treatment. This group had received s u r g i c a l or r a d i a t i o n treatment for t h e i r primary l e s i o n and during the time of observation #2 MD 300 —i 250 - \ 2 3 4 5 6 7 8 Months Figure 3. Recovery of i n v i t r o responses following chemotherapy (normal mean PHA = 105 x 10" 3 DPM: p o s i t i v e PPD = >30 x 10" 3 DPM). Figure 4. Recovery of i n v i t r o responses following i r r a d i a t i o n and chemotherapy. (normal mean PHA = 105 x 10" 3 DPM: p o s i t i v e PPD = > 30 x 10" 3 DPM). Figure 5. Divergence of i n v i t r o responses following i r r a d i a t i o n and chemotherapy. (normal mean PHA = 105 x 1 0 - 3 DPM: p o s i t i v e PPD = > 30 x 1 0 - 3 DPM). 300 ro b 250 H 200 H * 150-1 o 100 Irradiation ( and — • Chemotherapy 50 H Immunotherapy PPD r n — i i i I^ I ! 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Months Figure 6. F a i l u r e of i n v i t r o responses to recover following i r r a d i a t i o n and chemotherapy i n patient 01 (normal mean PHA = 105 x 10 DPM p o s i t i v e PPD > 30 x 1 0 - 3 DPM). TABLE XIII. Peripheral white blood c e l l counts on lymphoma patients. Absolute Lympho-Patient WBC/cu mm cytes/cu mm Non-Randomized 1 4,100 + 210 1,184 + 64 2 4,200 + 307 1,476 + 98 3 8,600 + 793 2,462 + 153 4 6,900 + 685 2,743 + 247 5 3,240 + 168 1,026 + 64 6 3,900 + 168 849 + 62 x = 5,156 + 788 1,623 + 296 Randomized 101 5,800 ± 154 1,456 ± 121 01 4,200 ± 145 653 ± 58 03 6,100 ± 272 1,479 ± 192 102 4,625 ± 281 1,534 ± 255 103 3,900 ± 185 851 ± 131 105 7,100±1,066 2,645±1,060 106 4,850 ± 223 1,388 ± 101 06 5,100 ± 461 1,257 ± 98 x = 5,209 ± 353 1,407 ± 196 Mean response ± 1 standard deviation of the mean. Normal WBC/cu mm = 4,000 - 10,000. Normal absolute lymphocytes = 1,500 -3,000. 79 TABLE XIV. In v i t r o responses i n melanoma patients. Patient PHA PW Con-A PPD Responders 13 133 ±= * - 8 72 + 3 108 + 10 338 + 46 16 27 ± 7 19 + 4 49 + 10 163 + 67 2 65 ± 5 29 + 2 54 + 4 258 + 45 5 17 ± 3 11 + 1 34 + 8 258 + 42 9 141 ± 12 68 + 5 108 + 7 186 + 33 3 32 ± 11 24 + 3 76 + 9 51 + 14 1 27 ± 3 7 + 2 25 + 9 277 + 55 12 42 ± 10 33 + 4 69 + 2 165 + 51 >n-Responders 8 51 ± 9 29 + 4 97 + 13 81 + 27 4 39 ± 14 23 + 4 77 + 15 479 + 94 18 10 ± 2 48 + 9 90 + 11 18 + 6 17 1 ± 1 10 + 3 25 + 6 31 + 23 15 11 ± 3 30 + 2 61 + 5 71 + 14 7 96 ± 20 38 + 6 121 + 4 150 + 33 14 36 ± 20 50 + 17 111 + 16 112 + 54 19 6 ± 3 36 + 4 58 + 10 127 + 20 21 66± 3 16 + 4 52 + 11 21 + 18 20 3 ± 1 23 + 5 64 + 10 8 + 4 6 55 ± 15 44 + 4 107 + 7 76 + 9 )ther' 25 83 ± 20 74 + 7 125 + 9 293 + 49 10 50 ± 15 34 + 5 98 + 14 142 + 16 22 52 ± 15 75 + 10 169 + 7 134 + 36 23 90 ± 16 33 + 11 83 + 17 370 + 97 11 63 ± 21 52 + 13 88 + 18 192 + 50 24 39 ± 12 59 + 3 125 + 9 131 + 43 DPM x 10" 3 ± 1 standard deviation of the mean. 80 were disease free. Tuberculin negative patients were vaccinated with BCG and when recurrence was evident, BCG immunotherapy was i n s t i t u t e d . Table XV presents the mean mitogen and antigen i n v i t r o responses of the melanoma patients c l a s s i f i e d according to group or disease stage. Although the mean PHA response appears much lower i n non-responders the difference i s not s i g n i f i c a n t ; nor are the PW or Con-A responses s i g n i f i c a n t l y d i f f e r e n t . The PPD response between responders and non-responders does d i f f e r (p < 0.05). Although the mean response of non-responders i s low, compared to normal tuberculin p o s i t i v e i n d i v i d u a l s i t i s not an unreasonable response. Within the responder group, mitogen responses, but not antigen responses, were s i g n i f i c a n t l y lower i n male patients (p < 0.05 for a l l mitogens. In the group 'other', sex did not influence the l e v e l of response to e i t h e r mitogens or antigen (Table XVI). Only two non-responders were female. An analysisoof variance on the mitogen and antigen responses i n the grouped and staged melanoma patients showed the r e s u l t s i n Table XVII. The s e r i a l i n v i t r o responsestto PPD, PHA, PW, and Con-A were reviewed for c o r r e l a t i o n of responses with the following r e s u l t s : PPD and PHA r = 0.24, p < 0.001 PPD and PW r = 0.08, p < 0.250 PPD and Con-A r = 0.05, p < 0.500 PHA and PW r = 0.57, p < 0.001 PHA and Con-A r = 0.36, p < 0.001 PW and Con-A r = 0.56, p < 0.001 TABLE XV. In v i t r o responses of melanoma patients according to group and stage of disease. Group PHA PW Con--A PPD Responders 61 + * 16 33 + 8 65 + 10 212 + 30 Non-responders 29 + 9 32 + 4 78 + 8 107 + 38 'Other' 63 + 8 55 + 7 115 + 4 210 + 38 Stage PHA PW Con--A PPD I 48 + 8 26 + 4 74 + 11 204 + 48 II 76 + 16 52 + 8 98 + 9 196 + 24 III 52 + 24 43 + 10 82 + 14 171 + 51 IV 5 + 2 27 + 6 58 + 6 41 + 20 * _3 DPM x 10 ± 1 standard deviation of the mean. TABLE XVI. In v i t r o responses i n two groups of melanoma patients divided according to sex. PHA PW Con-A PPD Responders Male 2 8 + 5 * 1 8 + 5 4 4 + 9 2 1 6 + 2 5 Female 9 3 + 2 3 4 8 + 1 1 8 6 + 1 1 2 0 8 + 5 3 Other Male 6 0 + 1 3 5 6 + 9 1 2 5 + 2 0 2 1 2 + 6 5 FeEemale 6 5 + 9 5 3 + 1 0 1 0 4 + 8 2 0 9 + 3 7 DPM x 1 0 j + 1 standard deviation of the mean. TABLE XVII. Analysis of variance of i n v i t r o responses of grouped and staged melanoma patients. Source of Va r i a t i o n df Sum of Squares Mean Square F Groups A. PHA among 2 6,790 3,395 2.66 within 22 28,032 1,274 Tot a l 24 34,822 B. PW among 2 2,297 1,148 3.32 within 22 7,593 345 Tot a l 24 9,890 C. Con-A among 2 9,652 4,826 * 4.15 within 22 25,560 1,161 Total 24 35,212 D. PPD among 2 67,318 33,659 2.65 within 22 279,380 12,699 Tot a l 24 346,698 Stages A. PHA among 3 12,904 4,301 3.90 within 18 19,832 1,101 Total 21 32,736 B. PW among 3 2,779 926 * 3.28 within 18 5,083 282 Tot a l 21 7,862 C. Con-A among 3 4,155 1,385 1.63 within 18 15,216 845 Tot a l 21 19,371 D. PPD among 3 92,779 30,926 2.66 within 18 208,956 11,608 Total 21 301,735 * 0.025 < p < 0.05 84 Figures 7, 8, 9, and 10 show the mitogen and PPD responses of responding and non-responding melanoma patients during the f i r s t eleven months of immunotherapy treatment. Figures 11, 12, and 13 show the white blood count (WBC), lymphocyte count and monocyte count i n the same group of patients. (5-c) Immunoglobulin Levels on Lymphoma and Melanoma Patients Serum IgG, IgA, and IgM l e v e l s were determined i n three lymphoma and four melanoma patients receiving continuous BCG immunotherapy for between f i v e and twenty-nine months. No s i g n i f i c a n t v a r i a t i o n was found during t h i s time period i n any i n d i v i d u a l although the melanoma patie n t s , as a group, had a s i g n i f i c a n t l y higher IgG l e v e l compared to lymphoma patients (p < 0.02). (5-d) Spontaneous DNA Synthesis (Baseline Transformation) An increase above the laboratory value f o r a group of normal i n d i v i d u a l s has been observed i n two circumstances. (1) Following BCG treatment, da i l y studies have shown increased 3H-thymidine uptake by unstimulated p e r i p h e r a l blood white c e l l s i n v i t r o which follows the s e v e r i t y of the c l i n i c a l response to vaccination peaking two to s i x days a f t e r the administration of BCG. A higher isotope uptake occurred following d i r e c t i n j e c t i o n of BCG i n t o tumours as compared to scratch or o r a l vaccination; the l a t t e r g i v ing the l e a s t release of DNA synthesizing c e l l s (Table XVIII). (2) The second type of response occasionally observed was that of a gradual continuous r i s e i n spon-taneous transformation which, upon reaching a plateau, then declined Figure 8. In v i t r o responses to Pokeweed expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment. Abscissa = months Ordinate = DPM x 10~ 3 0 0 ON Figure 9. In v i t r o responses to Concanavalin-A expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment. Abscissa = months Ordinate = DPM x 10~ 3 00 Figure 10. In v i t r o responses to PPD expressed as a percent of the mean normal value during the f i r s t 11 months of immunotherapy treatment. Abscissa = months Ordinate = DPM x 10"3 oo oo RESPONDERS (8) NON-RESPONDERS (11) Figure 11. Total white c e l l counts during the f i r s t 11 months of immunotherapy treatment. Abscissa = months Ordinate = WBC/mm3 oo >X5 Figure 12. Absolute lymphocyte counts per cu mm during the f i r s t 11 months of immunotherapy treatment. VO o — — RESPONDERS (8) 8OO-1 NON- RESPONDERS (11) Figure 13. Absolute monocyte counts per cu mm during the f i r s t 11 months o immunotherapy treatment. 92 TABLE XVIII. E f f e c t of BCG treatment modality on baseline transformation and CFU-C l e v e l s . Method of Assessment Normal Value Inj ection Treatment Scratch Oral Pre Post Pre Post Pre Post 3H-thymidine incorporation 275 ± 15 325 900 275 650 240 470 Colonies/10 6 c e l l s plated 10.5 ± 1 . 3 12 65 22 3.5 11 Mean ± 1 standard deviation of the mean. 93 to within normal values. This pattern of response preceded the death of the patients (Table XIX). The marked difference between baseline transformation l e v e l s i n responding and non-responding melanoma patients i s due p r i m a r i l y to this continual r i s e i n spontaneous transformation preceding the death of the patients e x h i b i t i n g t h i s response (Figure 14). (5-e) Autoradiographs Autoradiographs of blood smears taken at regular i n t e r v a l s showed a s i g n i f i c a n t increase above background l e v e l only i n those patients whose spontaneous transformation rate rose three to four times above the normal mean response. Table XIX shows the number of l a b e l l e d c e l l s per 500 mononuclear c e l l s counted i n the two melanoma patients whose spontaneous transformation rate rose alarmingly. One thousand white blood c e l l s were counted where possible; however, the counts are reported as per 500 c e l l s to overcome discrepancies i n the cases where there were a paucity of mononuclear c e l l s . A l a b e l l e d c e l l was defined as a c e l l showing a minimum of 5 grains over the nucleus. Normal in d i v i d u a l s not infrequently had l a b e l l e d c e l l s present i n smears. This l a b e l l i n g index, however, was not above 2/500 mononuclear c e l l s . Figures 15 and 16 show the type of c e l l l a b e l l e d . The c e l l seen i n Figure 15 i s considered to be a large transformed lymphocyte while that i n Figure 16 i s regarded as a 'Turk' c e l l . (5-f) Colony Forming Units A f t e r two weeks of culture, t y p i c a l colonies were composed of e i t h e r macrophages, eosinophils or granulocytes, although i n a few 94 TABLE XIX. S e r i a l spontaneous baseline transformation and autoradiograph l a b e l l e d c e l l s i n two melanoma patients. Autoradiographs* Spontaneous Patient Date Transform- Grain D i s t r i b u t i o n / C e l l mation DPM Tot a l < 2 5 2 5 _ 5 Q > 5 Q 9 8 274 0.5 9 .12 1,716 3.2 1.6 1.6 9 .19 293 0 9 22 363 4 2 2 9 26 224 .5 10:20 1,040 8.5 1.5 3 4 10:27 3,508 12 1 5.5 5.5 11:3 8,083 11 .5 4.5 6 11:4 8,877 13.5 .5 3.5 9.5 11:5 4,503 14 .5 2.5 11 11:10 4,535 6 - 2 4 1 1 : 1 7 ** 769 3 - - 3 12:4 - 1:10 488 3 - 1.5 1.5 6 28 - 7:21 442 ND 8 1 756 2 1 1 8 4 2,171 5 3 2 8 8 3,807 20 3 11 6 8 11 3,149 12.5 2.5 7.5 2.5 8 18 4,128 13 3.5 6 3.5 8 22 4,500 12 2 8 2 8 29 2,784 8.5 2.5 3.5 2.5 9 1,578 4 2 1 1 9 11 1,120 5.5 1 2 2.5 # of l a b e l l e d cells/500 mononuclear c e l l s - "Normal" l a b e l l i n g index = 2/500. ** Died same month. RESPONDERS (8) 5 6 7 8 9 10 Figure 14. Spontaneous uptake of 3H-thymidine i n the p e r i p h e r a l blood of melanoma patients expressed as a percent of normal. Abscissa = months Ordinate = % of normal 5 Figure 15. Autoradiograph on p e r i p h e r a l blood buffy coat showing a l a b e l l e d large transformed lymphocyte. ON Figure 16. Autoradiograph on peripheral blood buffy coat showing a l a b e l l e d 'Turk' c e l l . VO 98 instances mixed populations were observed. While 20 c e l l s were required to be considered a colony, one could often observe colonies of more than 500 c e l l s . Figures 17 - 24 are representative of the type of c e l l s observed i n the i n d i v i d u a l colonies. Figures 17 and 18 show c e l l s picked from a very t i g h t colony as seen by the inverted microscope. By phase microscopy these c e l l s show a c l e a r l y o utlined nuclear membrane, d u l l granules, and dense areas of chromatin which are only present i n macrophages. Figures 19 and 20 show c e l l s picked from a f a i r l y loose colony l y i n g s u p e r f i c i a l l y i n the agar as seen by the inverted microscope. Phase microscopy revealed a colony of developing eosinophils with c e l l s varying from very early agranular c e l l s to densely granulated c e l l s . In 425 c e l l s counted at l e a s t 6 mitosis were seen. Figure 21 shows two c e l l types a r i s i n g i n a s i n g l e colony. The o v e r a l l appearance under the inverted microscope was of a l i g h t e r and less dense colony than the t y p i c a l eosinophil colony. Phase micro-scopy revealed a mixed eosinophil and neutrophil colony. Figures 22 and 23 also show eosinophils but picked from other colonies. Figure 24 shows c e l l s from a colony s i m i l a r i n appearance to that of Figures 17 and 18 when viewed under the inverted microscope. The periphery of the colony was, however, looser. By phase microscopy the colony consisted of more than 600 macrophages of which 3% were i n mitosis. Figure 20. Phase microscopy of eosinophils showing development of granules. o t o Figure 22. Phase microscopy of an e o s i n o p h i l colony showing an agranular c e l l i n the centre. o 107 The mean number of CFU-C i n healthy laboratory volunteers was 10.5. Seven male i n d i v i d u a l s included i n t h i s normal population had a mean of 17.2 CFU-C as compared to a mean of 9.2 CFU-C for t h i r t y -eight females. This marked difference was not s t a t i s t i c a l l y s i g n i f i -cant. Female patients of both melanoma and lymphoma groups showed a s i g n i f i c a n t l y lower number of CFU-C than t h e i r male counterparts (Table XX) . TABLE XX. CFU-C i n male and female patients. Patients I'aMale Female Lymphoma 15.2 ± 2.3 6.5 ± 1.0 p < 0 . 0 1 Melanoma 9.0 ± 1.4 6.1 ± 1.0 p < 0.05 Me an CFU—C i 1 standard deviation of the mean. Neither patient population d i f f e r e d s i g n i f i c a n t l y from the normal popu-l a t i o n considered as a whole nor wheri compared by sex. Colony forming units i n female' patients were s i g n i f i c a n t l y lower than both normal females (p < 0.02) and the whole normal population (p < 0.01). Lymphoma females d i f f e r e d only to the t o t a l normal population (p < 0.05). No difference was observed between the female population of e i t h e r patient group. Male melanoma patients were s i g n i f i c a n t l y lower than male lym-phoma patients (p < 0.05). A comparison of responders and non-responders 108 did not show a s i g n i f i c a n t difference. This comparison was made on responders excluding melanoma patient 13 whose responses were high when studied on a d a i l y basis and would have, i f included with respon-ders, made the two groups s i g n i f i c a n t l y d i f f e r e n t . The magnitude of response sometimes followed the spontaneous baseline transformation rate (Figure 25) and the c l i n i c a l response of those patients responding w e l l to immunotherapy. Figure 26 shows a t y p i c a l p o s i t i v e response of peripheral blood CFU-C following the intratumour i n j e c t i o n of BCG i n melanoma patient 13. In conjunction with the elevated CFU-C tumour regression was observed. 6. CLINICAL STATUS OF THE PATIENTS STUDIED NINE MONTHS FOLLOWING  COMPLETION OF IN VITRO STUDIES The c l i n i c a l status of the patients studied i s , nine months following completion of these i n v i t r o studies, as follows. A l l non-randomized lymphoma patients are considered to be disease-free. Patient 1 i s being maintained on chemotherapy alone. Patients 2 - 6 ureceive no maintenance therapy. A l l randomized lymphoma patients i n the treatment group are considered to be disease-free. Patients 101, 01, and 03 receive no maintenance therapy. Patients 102, 105, and 106 are s t i l l r e c e iving maintenance BCG immunotherapy. Patient 103 recurred again at 22 months s u r v i v a l . Control i n t h i s case i s being maintained by chemotherapy. Patient 06 i s completing chemotherapy f o r h i s f i r s t recurrence. I t i s intended to r e i n s t i t u t e BCG immunotherapy i n patient 06 following his chemotherapy. Figure 25. Ef f e c t of BCG vaccination on two i n v i t r o parameters measured da i l y f o r one week following immunotherapy. Figure 26. E f f e c t of intratumour i n j e c t i o n of BCG on per i p h e r a l blood CFU-C. I l l In the randomized control lymphoma pat i e n t s , only patient 104 remains disease-free. Patient 05 recurred at 30 months s u r v i v a l . His disease i s being c o n t r o l l e d by chemotherapy. No melanoma patient c l a s s i f i e d as a non-responder i s a l i v e at the present time (June, 19 75). F i f t y percent of responders (patients 2, 3, 5, and 9) are a l i v e and tumour-free at present. Patient 9, however, had presented i n March 1975 with a further recurrence. Following her h o s p i t a l i z a t i o n f or recurrence i n January, 19 73, she had been maintained on o r a l BCG; 160 mg every s i x weeks. Contrary to her previous recurrences which were confined to the gut, the l a t e s t metastatic focus was located on the anterior aspect of the neck. The nodule was s u r g i c a l l y removed and confirmed to be melanoma by h i s t o l o g i c a l examination. There was, however, a marked granulomatous reactive process associated with the tumour suggesting the patient was making an immunological response. Seven weeks following i t s removal a 4 mm nodule was discovered i n the l a t e r a l quadrant of the l e f t breast. Following two intratumour i n j e c t i o n s of v a c c i n i a virus to a t o t a l of 4 vaccinating doses the nodule could no longer be palpated. No further melanomatous nodules have appeared i n the succeeding 8 weeks. At the present time, t h i s patient i s being maintained weekly on a l t e r n a t i n g o r a l BCG, subcutaneous v a c c i n i a v i r u s , and subcutaneous Corynebacterium  parvum. Of the melanoma patients 'other', patients 10 and 22 have died. Patient 11 i s i n the terminal stage of her disease. Patients 23, 24, and 25 have not been seen i n the Immunotherapy C l i n i c recently and are presumed to be a l i v e and w e l l . DISCUSSION The student nurses study was undertaken f or three reasons. (1) No current research i s a v a i l a b l e on the use of o r a l BCG i n adults, p a r t i c u l a r l y i n i n v i t r o assays of e f f e c t i v e s e n s i t i z a t i o n . The use of o r a l BCG as a means of inducing s e n s i t i z a t i o n i s a return to the o r i g i n a l method of vaccination as described by Calmette and Guerin. This mode of BCG administration, although popular i n South America (128) has not found favour i n the North American continent i n s p i t e of i t s confirmed e f f i c a c y i n protection against tuberculosis. (2) With mounting evidence f o r the importance of d i r e c t BCG-tumour contact f o r e f f e c t i v e immunotherapy (74), o r a l BCG seemed a l o g i c a l way of attacking a tumour in v o l v i n g the alimentary t r a c t . (3) In order to stimulate the maximum amount of lymphoid t i s s u e , o r a l BCG was entered i n t o the therapeutic regimen of melanoma and lymphoma patients whose disease did not n e c e s s a r i l y involve the gut. As a l l tuberculin-negative student nurses entering t r a i n i n g at the Vancouver General H o s p i t a l receive BCG vaccination, a ready population was on hand i n which to compare the e f f i c a c y of o r a l BCG to the regular intracutaneous method of inducing s e n s i t i v i t y . By show i n vivo and i n v i t r o evidence of conversion i n these nurses, we could assume that i n a population of immunocompetent cancer patients s i m i l a r s e n s i t i z a t i o n could be attained. 112 113 There were two unfortunate aspects i n t h i s study. F i r s t l y , the B.C. D i v i s i o n of Tuberculosis Control had discontinued 250 TU skin t e s t i n g . Secondly, the pre-vaccination blood specimen was drawn f o l -lowing i n i t i a l skin t e s t i n g . The f a i l u r e to skin test i n i t i a l l y with 250 TU PPD allowed the i n c l u s i o n of g i r l s who would be considered tuber-c u l i n p o s i t i v e by our normal laboratory standards. Reports i n the l i t e r a t u r e have suggested that skin t e s t i n g may a f f e c t the i n v i t r o response to the p a r t i c u l a r antigen used. K e l l y et_ a l . (129) found skin test negative lymphocytes become p o s i t i v e a f t e r intradermal t e s t i n g ; they also found the response of skin test p o s i t i v e i n d i v i d u a l s enhanced. Matsaniotis et a l . (130) also f e e l that, i n tuberculin p o s i t i v e i n d i -viduals as w e l l as tuberculin negative i n d i v i d u a l s , increased a c t i v i t y may be due to skin t e s t i n g . Kerby (131) found a transient but s i g n i -f i c a n t r i s e i n the i n v i t r o transformation response to tuberculin a f t e r a p o s i t i v e skin test reaction which l a s t e d only two weeks. In t h i s laboratory, i n v i t r o responses of less than 30,000 DPM are considered negative; although a group of student nurses studied previously (121) had shown a pre-vaccination l e v e l twice t h i s value. Using the lower l e v e l to i n d i c a t e tuberculin p o s i t i v i t y , 70% of g i r l s studied were i n fac t p o s i t i v e i n i t i a l l y . These pre-specimen values do not allow one to say conclusively that e i t h e r o r a l or parental BCG d e f i n i t e l y converted i n vivo or i n v i t r o responses although 27/35 i n v i t r o responses were enhanced following e i t h e r intradermal or o r a l BCG. The dichotomy between responses jLn v i t r o and i n vivo agrees with previous work done i n this laboratory (121). The mean skin t e s t 114 responses i n the two groups show a c l e a r cut difference between the vaccination method used. In vivo s e n s i t i z a t i o n was much weaker 10 weeks following o r a l BCG being detectable only at the 250 TU l e v e l . This l e v e l of response was s i m i l a r to i n vivo responses observed pre-viously following intradermal vaccination with only o n e - f i f t h the regu-l a r dose of BCG (121). In v i t r o t e s t i n g at the same time, however, showed no s i g n i f i c a n t difference between vaccination method, responses being enhanced to the same degree i n both groups. Follow-up skin t e s t i n g at s i x months, however, showed elevated i n vivo responses to 5 TU when compared to i n i t i a l responses i n the nurses who had received o r a l BCG while responses to 5 TU i n the o r i g i n a l intradermal group had decreased s l i g h t l y . At the s i x t h month follow-up, two of the nurses who had o r i g i n a l l y received o r a l BCG had i n the meantime received intradermal BCG because of t h e i r i n i t i a l low response. Their responses do not s i g n i f i c a n t l y a f f e c t the elevated responses to 5 TU PPD seen at s i x months. Responses to 250 TU at s i x months had waned much more i n the o r i g i n a l intradermal group. On the basis of these r e s u l t s , we f e e l confident that o r a l BCG i s an e f f e c t i v e way of s e n s i t i z i n g immunocompetent adults. Previous work i n the laboratory on student nurses had shown some degree of c o r r e l a t i o n following BCG vaccination between the PHA responses and the response to PPD (121) that suggested those i n d i v i -duals with high l e v e l s of general stimulation had a greater number of c e l l s responsive to antigen, or, conversely a population of c e l l s which appeared to have an enhanced responsiveness to s p e c i f i c antigen. 115 Why t h i s group of student nurses did not show th i s c o r r e l a t i o n i s not apparent, although the o r i g i n a l observation was observed only when using the regular dose of BCG for intradermal vaccination. Of p a r t i -cular i n t e r e s t i s the fact that both lymphoma and melanoma patients did show a c o r r e l a t i o n between PHA and PPD i n v i t r o responses. Lymphoma patients s i m i l a r l y showed c o r r e l a t i v e responses between the other two mitogens and PPD as w e l l as among the mitogens themselves. Melanoma patients, on the other hand, f a i l e d to show a c o r r e l a t i o n between the other two mitogens and PPD. Perhaps t h i s could be due e i t h e r to more extensive disease or a more var i a b l e treatment regimen. The i n v i t r o lymphocyte responses to mitogens and PPD following i n i t i a l remission induction i n our lymphoma patients show a widely divergent pattern of recovery. The continuing remissions i n those patients whose responses did not improve suggest that neither of these parameters w i l l be of help i n p r e d i c t i n g early recurrence. PHA responses of randomized relapse patients as a group compared to remission patients are i n f a c t twice as high. Furthermore, i n patients 1, 02, 102, and 103 who had recurrences the PHA and PPD i n v i t r o lymphocyte cultures were performed one month or less p r i o r to the recurrence. The PHA responses i n these patients showed no s i g n i f i c a n t decrease from e a r l i e r observations; nor did i n v i t r o PPD responses. Neither disease stage, h i s t o l o g i c a l c l a s s i f i c a t i o n , non-immunotherapy treatment nor sex a f f e c t e d the mitogen and antigen responses. Responses to mitogens i n the ran-dominzed patients are generally lower than healthy i n d i v i d u a l s . The moderate degree of c o r r e l a t i o n observed between PHA, PW, and Con-A responses done simultaneously on patients over a period of 116 months suggest that the mitogens are a l l r e f l e c t i n g a general l e v e l of lymphocyte response or that they represent the stimulation of p a r t i a l l y overlapping populations. The presence or absence of a lymphocytopenia (defined i n most reports as less than 1,000 per cu mm) has been reported (98,99,132,133) to be of prognostic value i n a v a r i e t y of cancers. Only one of our lym-phoma patients (103) who presented with recurrence has had a consistent lymphopenia. Radiotherapy (a known potent causeoof lymphocytopenia (113,134)) had not been used for i n i t i a l treatment i n t h i s lady. Of i n t e r e s t i s patient 01 who shows not only the most marked lymphocyto-penia but also the most depressed mitogen and PPD i n v i t r o responses. In s p i t e of t h i s , the patient continues to do w e l l c l i n i c a l l y . Levy and Kaplan (135), however, found no c o r r e l a t i o n between absolute lympho-cyte counts and PHA stimulation i n untreated Hodgkin's patients. They f e e l the defect they detected was a r e s u l t of the depletion or malfunc-t i o n of a s p e c i f i c subpopulation of lymphocytes. I f , i n patient 01, there i s a defect i n a lymphocyte subpopulation i t has not affected her general well-being. The PHA response i n melanoma patients also showed a large v a r i -a b i l i t y . However, i n general, patients presenting with a PHA response of less than 40% of normal f a i l e d to respond to treatment. Age i s known to depress immune responses (136,137,138), however, i t i s doubtful whether t h i s could account for the low PHA response i n the one e l d e r l y s u r viving melanoma responder (patient 5) as h i s i n v i t r o PPD responses were very high. As mentioned, PHA and Con-A are s a i d to measure T - c e l l 117 responses. That they are two d i f f e r e n t populations i s suggested by the observation that non-responders showed a low PHA but normal Con-A even a few days before death. I t would be d i f f i c u l t to ascribe such a dichotomy of response to a serum factor. Melanoma patients f a i l i n g to show eit h e r skin s e n s i t i v i t y or i n v i t r o lymphocyte response to PPD i n v a r i a b l y f a i l e d to make a c l i n i c a l response. Of i n t e r e s t here are the observations of I s r a e l et_ a l . (100) who found a p o s i t i v e tuberculin skin test to be of s i g n i f i c a n t advantage i n s u r v i v a l of patients with early bronchogenic carcinoma. On the other hand, a high degree of s e n s i t i v i t y to PPD, both i n v i t r o and i n vivo does not assure a good c l i n i c a l response (patients 4 and 19). The severest reactions to BCG i n j e c t i o n occurred i n these highly s e n s i -t i z e d patients and i n patient 16 who was very poorly s e n s i t i z e d (both i n vivo and i n v i t r o ) . A l l f a i l e d to make a c l i n i c a l response. During BCG immunotherapy, Falk (139) has observed a trend towards elevatedllgA immunoglobulin l e v e l s . Although IgG le v e l s were higher i n our melanoma patients than lymphoma patients, no change was observed i n any of the three classes of immunoglobulin tested. Our r e s u l t s agree with those of Bluming e_t a l . (104) . Although S e i g l e r et_ a l . (106) did not observe changes i n immunoglobulin l e v e l s during immunotherapy treatment, they f e l t there was a trend towards elevated IgG and IgM le v e l s which they explained as being the r e s u l t of s u r v i v a l and s e l e c t i o n of patients with elevated serum immunoglobulin l e v e l s . Following antigenic challenge, DNA synthesizing c e l l s capable of attacking the challenge source appear inbboth draining nodes and 118 peripheral blood (140,141,142). The increased incidence of these meta-b o l i c a l l y active c e l l s (as measured by ^H-thymidine uptake) has been used by Harris (143) to f o r e t e l l an impending r e j e c t i o n c r i s i s . Dwyer and Mackay (144) found that the early c e l l u l a r response to immunization with f l a g e l l i n included the appearance i n human peripheral blood of b l a s t - l i k e c e l l s which could bind antigen. Their appearance preceded the peak t i t r e of c i r c u l a t i n g antibody by several days. Alexander and H a l l (140) have termed these c e l l s , a r i s i n g following antigenic stimu-l a t i o n , 'immunoblasts.' Their studies have shown that (1) they leave the blood stream r a p i d l y so that very few are present at any one time; (2) they do not p r e f e r e n t i a l l y enter lymphoid organs as such, and for t h i s reason very few r e c i r c u l a t e from blood to lymph and (3) they appear to enter the extravascular tissues generally, and, i n p a r t i c u l a r , the lamina prop r i a of the gut. Would an assay system for DNA synthesizing c e l l s be useful i n assessing immunocompetence i n cancer patients? Was i t possible that the l e v e l of spontaneous DNA synthesis would be elevated due to i n vivo stimulation by TSTA i n melanoma and lymphoma patients? Matchett et a l . (108) have found patients with Hodgkin's disease to have an increased number of c e l l s a c t i v e l y synthesizing DNA c i r c u l a t i n g i n the peripheral blood. Associated with this was a decreased response of p e r i p h e r a l blood lymphocytes to PHA stimulation. They hypothesized that the c i r c u -l a t i n g T-lymphocytes were stimulated by the presence of a c t i v e disease thus depleting the e f f e c t i v e c e l l pool by making such transformed lympho-cytes r e f r a c t o r y to further stimulation. Similar observations of 119 increased spontaneous DNA synthesis by the leukocytes i n Hodgkin's disease has been reported by Crowther e_t a l (145) . Our lymphoma patients never presented with elevated l e v e l s of spontaneous transformation during t h e i r routine v i s i t s to the Immuno-therapy C l i n i c . This was not e n t i r e l y unexpected because, for the most part, these patients lacked c l i n i c a l l y - a p p a r e n t disease. Elevated transformation l e v e l s were not observed e i t h e r when the patients de-veloped recurrence. This could be a matter of sample timing. Alexander and H a l l ' s studies (140,141,142) would suggest that by the time c l i n i -c a l evidence of recurrence was apparent the normal release of immuno-bl a s t s by the regional nodes had been i n t e r f e r r e d with by the continual bombardment of the node by TSTA. Such immobilized immunoblasts "undergo d i f f e r e n t i a t i o n to plasma c e l l s , the presence of which i s a c h a r a c t e r i s -t i c feature of these nodes. In other words, the e f f e c t of the tumour i s not to cause immune p a r a l y s i s , i n the accepted sense of the word, but to i n t e r f e r e with the normal migration of immunologically active c e l l s " (142). Such a s i t u a t i o n would be expected to lead to an impair-ment of the host reaction against the tumour, since the immunoblasts can only act i f they leave the node and reach the tumour. Our sporadic t e s t i n g f or increased transformation of peripheral blood white c e l l s was of l i t t l e value i n these lymphoma patients. Contrary to lymphoma pat i e n t s , melanoma patients d i d occasion-a l l y show an above normal r i s e i n transformation l e v e l s . These patients a l l had i n common large tumour mass. At no time were r i s e s i n spon-taneous baseline l e v e l s associated with e i t h e r extraneous i n f e c t i o n or 120 with the appearance of immature granulocytes (vide i n f r a ) both of which are known to increase 3H-thymidine uptake i n white blood c e l l s (146, 147). In such patients i t i s possible that the release of tumour a n t i -gen systemically resulted i n the stimulation of uninvolved nodes and subsequent release of immunoblasts accounting for the increased 3H-thymidine uptake. However, with the continued breakdown of tumour tissue and the release of tumour antigen, those areas s t i l l capable of releasing the immunoblasts would eventually become immunologically i n e r t by the prolonged exposure to excessive antigen. No obvious c l i n i c a l b enefit was observed i n those patients e x h i b i t i n g t h i s apparent immune response probably due to the enormous load of tumour present. In view of the observation (142) that these immunoblasts can d i f f e r e n t i a t e into plasma c e l l s i t i s not improbable that these c e l l s could be producing blocking or enhancing antibody thereby preventing any e f f e c t i v e anti-tumour response that the host could muster. The uptake of 3H-thymidine i n unstimulated p e r i p h e r a l blood of responders and non-responders showed a marked r i s e i n the second to f i f t h month i n the l a t t e r group. As t h i s was l a r g e l y due to high values i n three patients with obvious advanced disease t h i s parameter i s of no prognostic s i g n i f i c a n c e . In the days immediately following BCG stimulation, the occur-rence of elevated l e v e l s of spontaneous transformation would suggest adequate l e v e l s of immunocompetence i n a good percentage of cancer patients although animal work has shown normal responses to sheep erythrocytes and k i l l e d B r u c e l l a abortus organisms i n the face of 121 d e c l i n i n g immune- re a c t i v i t y of lymphoid tissue l a t e i n tumour growth. Those patients who did not show an elevated l e v e l of transformation also f a i l e d to show evidence of c l i n i c a l response. Having been released following BCG stimuation, these immunoblasts could represent e f f e c t o r c e l l s which could attack foreign tumour c e l l s n o n - s p e c i f i c a l l y . Both lymphoma and melanoma patients were found to release immunoblasts i n proportion to the amount of BCG stimulation. Tumour l o c a l i z a t i o n could also determine the effectiveness of the c e l l s . Melanoma patient 9 showed an excellent c l i n i c a l response following o r a l BCG i n s p i t e of a d a i l y post-therapy uptake of 3H-thymidine l i t t l e above normal. In t h i s s i t u a t i o n tumour regression may have been the r e s u l t of concen-t r a t i n g the immunoblasts i n the lamina prop r i a of the gut where her disease was l o c a l i z e d . In conclusion, baseline transformation has a l i m i t e d usefulness i n evaluating patients' immediate response to BCG stimulation. Regression of tumour lesions at s i t e s distant from the l e s i o n receiving the BCG i n j e c t i o n have been w e l l documented (10, 148). The reason f o r t h e i r regression could be the r e s u l t of the re-lease of these immunoblasts into the systemic c i r c u l a t i o n . Following BCG antigenic stimulation, d a i l y autoradiographic studies on peripheral blood smears did not c o r r e l a t e with elevated l e v e l s of spontaneous transformation. L a b e l l i n g time was chosen to be as short as possible to allow i n d i v i d u a l grain counts to be done. Had the isotope been incubated f o r a longer period of time, perhaps more c e l l s i n the 'S' phase would have been l a b e l l e d and some corre-l a t i o n observed. As i t was, only where spontaneous transformation 122 l e v e l s rose two to three times above the upper l i m i t s of normal were l a b e l l e d b l a s t s s i g n i f i c a n t l y present. These b l a s t s were t y p i c a l of transformed lymphocytes with b a s o p h i l i c cytoplasm and one or more n u c l e o l i . S i g n i f i c a n t numbers of l a b e l l e d c e l l s were only seen i n patients whose outcome was r a p i d l y f a t a l . The c e l l s were not believed to be myelocytic and not representing a leukoerythroblastic picture known for i t s poor prognosis. As mentioned, the use of BCG, e i t h e r as a means of primary s e n s i t i z a t i o n or as an adjunct i n tumour e r a d i c a t i o n , i s associated with the development of tuberculin-type delayed h y p e r s e n s i t i v i t y . Macroscopically, 24 hours a f t e r antigen i n j e c t i o n , erythema and indura-t i o n develop reaching a peak at 72 hours. H i s t o l o g i c a l l y (149) there i s d i l a t i o n of c a p i l l a r i e s accounting for the erythema which develops and a c e l l u l a r i n f i l t r a t e across the walls of venules into the s i t e of antigen deposition r e s u l t i n g i n induration. While the predominant c e l l s at the onset are polymorphonuclear c e l l s , they have been replaced at 24 hours by mononuclear c e l l s . Evidence has accumulated that these i n f i l t r a t i n g c e l l s are not tis s u e macrophages but c e l l s a r i s i n g from the' c i r c u l a t i n g pool which i n f i l t r a t e the test s i t e . From the work by Lubaroff and Waksman (150), c i r c u l a t i n g c e l l s which i n f i l t r a t e into the test s i t e are believed to originate i n the bone marrow. I t has also been shown that 80% of the i n f i l t r a t i n g c e l l s belong to the monocyte/macrophage class which has been s e l e c t i v e l y concentrated at the s i t e , perhaps by MIF. These c e l l s represent the non-specific element of the response a r i s i n g at 123 the t e s t s i t e following the i n i t i a l i n t e r a c t i o n between antigen and a few s e n s i t i z e d c e l l s . In order to maintain an adequate host-tumour balance or to eradicate the neoplasm completely, tumour immunity, l i k e BCG-induced delayed h y p e r s e n s i t i v i t y reactions, requires a continual supply of e f f e c t o r c e l l s . Such e f f e c t o r c e l l s would, i n a l l p r o b a b i l i t y , a r i s e from some pluripotent stem c e l l normally leading a quiescent existence i n the bone marrow. With the development of s o f t agar techniques, i t has been possible to grow these p r i m i t i v e c e l l s i n v i t r o (58). These techniques have shown that low l e v e l s of such progenitor c e l l s are also c i r c u l a t i n g i n the peripheral blood (151,152,153). Both animal and human experiments have c l e a r l y shown these p e r i p h e r a l blood c e l l s to have a p l u r i - or m u l t i - p o t e n t i a l c a p a b i l i t y . Transplantation of buffy coat c e l l s from rodent blood protects and repopulates the hema-t o p o i e t i c system of l e t h a l l y i r r a d i a t e d r e c i p i e n t s (151). S i m i l a r l y , i n human beings, allogeneic marrow engraftment has been observed a f t e r the transfusion of p e r i p h e r a l blood leukocytes from patients with chronic myelogenous leukemia to patients with myelosuppression (154). I t was postulated that immunocompetent cancer p a t i e n t s , being stimulated to mobilize t h e i r defensive army of e f f e c t o r c e l l s by repeated BCG vaccination, would have elevated numbers of these c i r c u l a t i n g pro-genitor c e l l s . This would be analogous to the f i n d i n g of nucleated red blood c e l l s under conditions of active erythropoiesis. Quanti-t a t i v e l y , f l u c t u a t i o n s i n progenitor c e l l populations would be greater i n the bone marrow. As one could not j u s t i f y subjecting patients to 124 bone marrow a s p i r a t i o n s o l e l y f o r research purposes and as i t i s the systemic response that determines the ultimate outcome i n the host-tumour i n t e r p l a y , p e r i p h e r a l blood observations were made with the fin d i n g of elevated numbers of colony-forming c e l l s within seven days following BCG treatment. The number of c e l l s c i r c u l a t i n g i n the p e r i p h e r a l blood found to be capable of forming colonies i n t h i s laboratory agrees with figures reported by Chervenick and Boggs (151) and i s s l i g h t l y higher than found by Kurnick and Robinson (152). The type of c e l l s observed i n the colonies i n both our laboratory normals and cancer patients also agrees with other i n v e s t i g a t o r s . Excluding cases of extensive meta-s t a t i c disease, the fact that these lymphoma and melanoma patients could, i n s p i t e of t h e i r disease, form normal colonies lends c r e d i t to t h e i r immune c a p a b i l i t y . In contrast, i t has been reported that patients with chronic lymphocytic leukemia f a i l to show a p r o l i f e r a t i o n of p e r i p h e r a l progenitor c e l l s i n v i t r o (153). Colonies developing from the blood of normal i n d i v i d u a l s and cancer patient show c e l l s i n d i f f e r e n t stages of maturation as w e l l as numerous m i t o t i c f i g u r e s . There did not appear to be a l a r g e r percentage of small colonies i n cancer patients except i n those with extensive disease suggesting per-haps a reduced p r o l i f e r a t i v e c a p a b i l i t y i n t h i s s i t u a t i o n . Marked er y t h r o p o i e t i c differences between normal adult male and female are w e l l documented. Similar differences appear to occur i n normal human granulopoiesis. Rosenblum et a l . (155)' have observed a greater granulocyte p r o l i f e r a t i v e capacity i n normal male marrow 125 compared to female marrow which was dependent on the hormone influence of androgen. S i m i l a r l y , Rubin (156) has observed such a difference using p e r i p h e r a l blood cultures of colony-forming c e l l s . Our laboratory normal population did not show th i s sex v a r i a t i o n . In a l l p r o b a b i l i t y t h i s was due to the very unequal sample sizes i n the two groups which made i t d i f f i c u l t to i n t e r p r e t s i g n i f i c a n t differences i n the patient population. Both melanoma and lymphoma patients showed a lower number of colony-forming c e l l s i n the female population as compared to t h e i r male counterparts. The s i g n i f i c a n t l y lower number of colony-forming c e l l s i n male melanoma patients compared to male lymphoma patients suggests a lowered immunocompetence l e v e l i n t h i s group due to t h e i r more extensive presenting disease; the female melanoma patients, on the whole, had less extensive disease. Although there was no s t a t i s -t i c a l l y s i g n i f i c a n t difference i n the number of colony-forming c e l l s between responders and non-responders, the patients who f a i l e d to respond c l i n i c a l l y did not show a r i s e i n colony-forming c e l l s following BCG i n j e c t i o n . Animal work has provided evidence f o r the migration of multi-p o t e n t i a l c e l l s going to colonize other areas of the body (157,158). Toujas jet a l . (157) reported that a f t e r i n j e c t i o n of Brucellae abortus  B19 there occurred a clearcut f a l l i n the number of c e l l s i n the marrow followed by increased spleen c e l l u l a r i t y . They reported (158), also, that "Bennet and Cudcowicz have studied a s i m i l a r marrow c e l l u l a r i t y following i n j e c t i o n of Corynebacterium parvum. I t has been shown that this adjuvant causes the t o t a l number of c e l l s as w e l l as the number 126 of colony-forming c e l l s i n the marrow to f a l l and to promote t h e i r increase i n the spleen. Barnes and Loutet have demonstrated that the number of colony-forming c e l l s i n the blood i s increased i n animals given Bordetella p e r t u s s i s " as has Monette et a l . (159). Work by Toujas et a l . (158) showed a coincidence between the period of increased immune response (as measured by antibody formation to sheep red blood c e l l s ) , increased m u l t i p o t e n t i a l stem c e l l s , and prolonged s u r v i v a l of immunized animals grafted with L 1210 leukemia. Using BCG to provoke a non-specific immunological stimulation i n mice, Toujas et_ a l . (158) found colony-forming c e l l augmentation to be much slower than that produced by B r u c e l l a abortus or Corynebacterium  granulosum. They f e e l the i n i t i a l f a l l i n colony-forming c e l l s , par-t i c u l a r l y i n the bone marrow, to be due to stem c e l l d i f f e r e n t i a t i o n and migration and that the acceleration i n the d i f f e r e n t i a t i o n of stem c e l l s and subsequent numerical increase contributes to the a v a i l a b i l i t y of immunocompetent c e l l s — b o t h monocytes and non-committed lymphoid precursor c e l l s . I t i s not known whether b a c t e r i a l immunostimulants d i r e c t l y induce p r o l i f e r a t i o n of progenitor c e l l s or act through intermediaries. Colony stimulating f a c t o r (CSF) could be such an intermediary. This i s an operational term given to a family of glycoprotein molecules having a molecular weight of about 45,000 which i s demonstrable i n the serum and urine of both normal human beings and animals. I t appears to be a humoral regulator of granulopoiesis and macrophage formation. Metcalf (160) has shown that b a c t e r i a l antigens i n j e c t e d into mice e l i c i t a dramatic r i s e i n the a b i l i t y of serum from such mice to 127 stimulate colony formation i n v i t r o by normal bone marrow c e l l s . This r i s e i s detectable i n the serum within one hour of i n j e c t i o n returning to near normal l e v e l s within twenty-four hours. "Since CSF appears to be a humoral regulator of granulopoiesis and monocyte formation i n vivo i t seems l i k e l y that the increased serum CSF l e v e l s developing i n response to antigens may mediate the increased production of granu-locytes and monocyte/macrophages known to occur a f t e r antigenic stimu-l a t i o n or b a c t e r i a l i n f e c t i o n " (160). In human beings the p e r i p h e r a l blood monocyte as w e l l as i t s more mature d e r i v a t i v e , the t i s s u e macrophage have been suggested as sources of CSF (161). Goldie et a l . (1962) suggest that the t i s s u e macrophage functions "both as a phagocyte and as a s u r v e i l l a n c e c e l l able to r e c r u i t granulocytes f o r host defence." Parker and Metcalf (1963), and more recently C l i n e and Goldie (164), have suggested that activated lymphocytes (both 'T' and 'Bj) may themselves be capable of synthesizing s i g n i f i c a n t amounts of CSF i n mice. The former group of investigators suggest that lymphoid c e l l s a c t i v e l y engaged i n immuno-l o g i c or inflammatory /responses "provide a mechanism whereby a sustained p r o l i f e r a t i o n of granulocytic and macrophage precursor populations can be stimulated." Whether the release of colony-forming c e l l s i s a d i r e c t r e s u l t of antigen stimulation or depends on an intermediary i s at t h i s point i r r e l e v a n t . The f a c t remains that our laboratory observations r a i s e the p o s s i b i l i t y that the increased release of colony forming c e l l s plays an important role i n BCG-induced immunopotentiation. In view 128 of the report by Evans and Alexander (44) who showed that macrophages from animals immunized to BCG could be activated by incubation with PPD and that these 'armed' macrophages were then capable of nonspeci-f i c a l l y k i l l i n g tumour, i t i s not improbable that colony-forming c e l l s play an important r o l e i n tumour r e j e c t i o n as one of the main colony types formed are macrophages. The release of macrophage colony-forming c e l l s would also f i t w e l l with the report of Hanna e_t a l . (19) who showed that BCG-induced tumour r e j e c t i o n i n animals i s associated with an intense p r o l i f e r a t i o n of r e t i c u l o e n d o t h e l i a l components and h i s t i o c y t e s . This release of colony-forming c e l l s from the bone marrow could thus represent an eco-nomy of numbers while providing f o r high l o c a l i z e d p r o l i f e r a t i v e e f f e c -tiveness. Local p r o l i f e r a t i o n might also o f f e r an explanation f o r the continuation of acute r e j e c t i o n i n kidney transplant patients even when t h e i r p e r i p h e r a l white blood c e l l s appear to be acutely suppressed by drug therapy. C l i n i c a l l y , i n the patients studied here, immunotherapy seems to o f f e r b e n e f i c i a l r e s u l t s . Recently, Beard and F a i r l e y (165) have suggested that i t i s easier to obtain a second remission i n acute leukemia patients who have received immunotherapy. Our three lymphoma patients who recurred while receiving immunotherapy again attained a complete remission while only one out of the three non-immunotherapy (control) patients who recurred achieved a second complete remission. Some patients who can be immunized to v a c c i n i a v i r u s appear able to a t t a i n s a t i s f a c t o r y c l i n i c a l responses while t h e i r disease i s 129 w e l l l o c a l i z e d . The reason for the development of c l i n i c a l resistance to t h i s type of therapy remains obscure. Two patients (melanoma patients 1 and 2) have developed such resistance a f t e r 12 and 22 months respec-t i v e l y . The c l i n i c a l symptomatology to v a c c i n i a virus i n j e c t i o n , both l o c a l l y and systemically, was the same i n these patients, not only while they were responding s a t i s f a c t o r i l y but also when they ceased to respond. Resistance to v a c c i n i a virus therapy did not i n t e r f e r e with the response to BCG and/or PPD. In view of the use of a s e n s i t i z i n g transfusion from patient 2 and the i n i t i a l use of subcutaneous l i v e v a c c i n i a virus therapy, i t i s d i f f i c u l t to a t t r i b u t e with certainty the i n i t i a l response i n this patient to the o r a l BCG. However, pain i n the abdominal masses 24 -48 hours following each administration of o r a l BCG suggested that a tumour-associated inflammatory reaction was occurring. With the d i s -appearance of tumour, no further abdominal pain was noted. When an abdominal tumour did recur, pain again developed following o r a l BCG. The second patient with r e s i d u a l melanoma following small bowel resection (patient 12) showed no c l i n i c a l or radiographic evidence of l o c a l spread for one year following the incomplete resection, giving support to t h i s mode of therapy for metastases l o c a l i z e d to the bowel. This patient l a t e r developed cerebral metastases. However, i n patients with advanced progressive disease o r a l BCG did not prevent the develop-ment of bowel metastases. We do not have s u f f i c i e n t data to assess whether o r a l BCG would be h e l p f u l i n preventing the recurrence of tumour where i t has c l i n i c a l l y 130 been completely excised or treated by i r r a d i a t i o n as i n patients numbers 10 and 11, for although they remain w e l l at 17 and 15 months, th i s may represent the natural course of t h i s unpredictable tumour (see r e s u l t s , p.111). In other patients the use of o r a l BCG alone f a i l e d to h a l t the progression of tumours that were extending at the time of i t s ad-m i n i s t r a t i o n . I t i s impossible to i n t e r p r e t the apparent s t a b i l i z a t i o n f o r 2 -3 months, followed by progression i n t h i s disease. Because of the draining sinuses that occurred following the i n j e c t i o n of l i v e BCG, we are exploring the use of simultaneous o r a l BCG and PPD i n j e c t i o n into the tumour. The o r a l BCG appears to ' f l a r e ' the reaction to PPD. We have found o r a l BCG i n patients with e i t h e r melanoma or lymphoma to be almost free of adverse side e f f e c t s but such immunization i s r e l a t i v e l y slow compared to intracutaneous v a c c i -nation and the degree of s e n s i t i v i t y may be l e s s . We have no evidence to suggest, however, that a high degree of s e n s i t i z a t i o n to PPD i s ne c e s s a r i l y a good thing i n BCG immunotherapy. Autopsies on seven patients with advanced melanoma a f t e r exten-sive intratumour and o r a l BCG have shown no evidence of i n f e c t i o n beyond the area of i n j e c t i o n and no i n f e c t i o n or u l c e r a t i o n i n the gut. One patient (patient 19) showed a few. granulomas i n the lung with negative culture for BCG. We have not;:observed the extensive granulomas that have been reported (166,167). The conventional forms of cancer treatment for malignant mela-noma do not as yet o f f e r hope of a 'cure.' Melanoma i s , as a r u l e , 131 r a d i o r e s i s t a n t (168). I r r a d i a t i o n has given a good c l i n i c a l response i n 2/7 patients. These two patients (10 and 11) had a s i n g l e metastatic focus. Unfortunately, multiple metastatic f o c i are not uncommon i n recurring melanoma. Chemotherapeutic e f f e c t s can be achieved more commonly with DIC which i s the single most e f f e c t i v e agent; e i t h e r alone or i n the best combination with bi s c h l o r o e t h y l n i t r o s o u r e a (BCNU) and v i n c r i s t i n e . Again e f f e c t i v e p a l l i a t i o n i s seen i n less than 25% of patients with metastatic malignant melanoma (89). Of the 7 patients receiving DIC during t h i s study, one patient made a complete c l i n i c a l recovery for eleven months but recurred and another patient made a s l i g h t c l i n i c a l response for three weeks. A review of the l i t e r a t u r e shows general agreement that melanoma can be immunologically c o n t r o l l e d when i t i s confined to the s k i n , subcutaneous t i s s u e , and regional lymph nodes i n about twenty-five percent of patients. Shingleton ep a l . (169) have reported objective and subjective improvement i n f o r t y - f i v e percent of t h e i r patients following a four stage immunotherapy program. Patients having v i s c e r a l involvement are generally poorly c o n t r o l l e d by any form of treatment c u r r e n t l y i i n use. In conclusion, BCG immunotherapy, although no panacea, does o f f e r another t o o l to maintain remission i n lymphoma patients and to eradicate or at least control melanomatous neoplasms i n selected patients. The ' i d e a l ' laboratory test to delineate those patients who w i l l present management problems or to i n d i c a t e recurrence e a r l i e r than i s apparent c l i n i c a l l y remains to be found. CONCLUSIONS CLINICAL 1. Oral BCG i s an e f f e c t i v e , although perhaps slower, method of immunization and appears to be a s a t i s f a c t o r y mode of therapy f o r metastases l o c a l i z e d to the small bowel. 2. BCG immunotherapy, except f o r a f l u - l i k e i l l n e s s l a s t i n g one or two days and the occasional draining sinus that developed following BCG i n j e c t i o n , has had no serious deleterious side e f f e c t s . 3. Maintenance of remission s o l e l y by repeated BCG vaccinations i n seven patients with non-Hodgkin's lymphoma who had achieved a complete c l i n i c a l remission with i n i t i a l standard therapy has provided s u f f i c i e n t encouragement to begin a randomized c l i n i -c a l t r i a l . 4. As the incidence of spontaneous regression i n malignant melanoma i s 3/1,000 and as our melanoma patients c l a s s i f i e d as responders had no treatment other than immunotherapy i t i s f e l t that BCG immunotherapy can be successful i n eradicating and/or c o n t r o l l i n g metastatic malignant melanoma p a r t i c u l a r l y when the tumour has not involved the v i s c e r a . 5. Vac c i n i a virus immunotherapy i s s i m i l a r l y e f f e c t i v e i n malig-nant melanoma although resistance to th i s form of therapy can develop. 133 LABORATORY 1. Mitogen and antigen i n v i t r o responses are not of prognostic value and do not allow the c l i n i c i a n to f o r e t e l l impending re-currences. The problem i s compounded by the wide monthly f l u c -tuations that occur as w e l l as the inconsistent recovery of responses following chemo- or radiotherapy. 2. In melanoma patients, PHA responses of less than 40% of normal usually meant an i n a b i l i t y to respond c l i n i c a l l y to immunotherapy. 3. Lymphocytopenia, i n lymphoma pati e n t s , was not n e c e s s a r i l y asso-ciated with those patients having early recurrence. 4. Although a high l e v e l of s e n s i t i z a t i o n i s not necessary, patients f a i l i n g to show e i t h e r skin s e n s i t i v i t y or an i n v i t r o PPD re-sponse f a i l e d to respond c l i n i c a l l y . 5. The presence of c e l l s i n the peripheral blood a c t i v e l y synthe-s i z i n g DNA was equated to a c l i n i c a l response where they were found following BCG stimulation i n vivo. Where t h i s baseline transformation remained high i r r e s p e c t i v e of any stimulation, the patient was seen to deteriorate f a i r l y quickly. 6. BCG immunotherapy had no e f f e c t on serum immunoglobulin l e v e l s . 7. 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