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Freeman, C. B., Harris, R. 1973. Brit. J. Med. (in press). Harris, R., Wentzel, J., Carroll, C. A., Garrett, J. V., Jackson, S. M. and Dodge, O. G. 1972. Histocompatability Testing. Munsgaard, Copenhagen. Harris, R. 1973. Nature (in press). McDevitt, H. O. and Landy, M. 1972. Academic Press. Metcalf, D. 1971. Adv. Cancer Res. 14, 181. Morris, P. J., Lawler Sylvia and Oliver, R. T. 1972. Histocompatability Testing. Munksgaard, Copenhagen. p. 669. Ziegler, H. F., Ward, F. E., Amos, D. B. and Stickel, D. L. 1973. Surgery 74, 190. Zervas, J. D., Delamore, I. W. and Israels, M. C. O. 1970. Lancet ii, 634. Immunological Survellance against Neoplasia. Transplantation Review 7, 1971.

IMMUNOTHERAPY FOR ACUTE MYELOGENOUS LEUKAEMIA Ray Powles

Department of Medical Oncology, St. Bartholomew's Hospital, London, and the Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey. Introduction N spite of immunological procedures having been used for three quarters a century for attempts at treating malignant disease in man 1 there has as yet, been no adequate study to show significant benefit, although Math6 ') in a partially controlled trial of BCG for childhood leukaemia found encouraging results.

i of

This present study was designed to determine if immunotherapy given to patients with acute myelogenous leukaemia (A.M.L.) during remission and receiving chemotherapy was superior to patients receiving chemotherapy alone. The essential feature of this study was to show a significant difference between the two comparable groups of patients because such a result might support the enormous drive for immunotherapy now being encouraged by many countries. The exact immunotherapy used in this study wa ~, weekly percutaneous B.C.G. and irradiated allogeneic A.M.L. c e l l s - - t h e rationale for adopting this regime and choosing A.M.L. as the disease to treat have been reported previously:'.

Patients This study included all patients with acute myeloblastic leukaemia who passed into full remission at St. Bartholomew's Hospital between October 1970 and 1st February 1973, all of whom had remission induced with daunorubicin and cytosine arabinoside. There were three subgroups (Trials II, III and IV) because of slight differences in the schedule of the drugs used. Trial IV is at present still accepting new patients, but II and III are complete. The essential part of this study was to determine the effect of immunotherapy on the maintenance of remission in these patients. All remission patients received identical maintenance chemotherapy consisting of pulses of five-day courses of cytosine arabinoside and dauno-

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rubicin, alternating with five days of cytosine arabinoside and ~Thioguanine. between every five days of treatment there was a 23 day gap. These 45 patients were randomized into two groups, one received maintenance chemotherapy, and the other immunotherapy in addition to the chemotherapy.

Immunotherapy As soon as full remission was obtained, all immunotherapy patients received weekly BCG, and irradiated allogeneic myeloblastic leukaemia cells. The injections were timed deliberately to straddle the five days of chemotherapy given every month.

BCG The BCG given was a freeze-dried percutaneous preparation obtained from Glaxo and when reconstituted contained 10 mg of organisms per ml, of which approximately 20 per cent were viable, Forty needle punctures in the skin to a depth of 2 mm using a Heaf gun gave an approximate dose of 1 x 10~ live organisms, The limb that received the BCG vaccine varied from week to week, Cells The cells used for immunotherapy were irradiated allogeneic myeloblastic leukaemia cells and were injected into the three limbs not receiving the BCG. These cells were collected from the peripheral blood of all suitable patients with acute myeloblastic leukaemia using a NCI/IBM cell separator. The leukaemia cells are mixed with 10 per cent dimethylsulphoxide, frozen slowly at l ~ to - 3 0 ~ using a Planer Ltd. Gas Phase Programmed Freezer and stored in liquid nitrogen~',. Each ampoule contains approximately 1 x 10~ cells. When required the cells are rapidly thawed at 37~ washed, and resuspended in medium 199 at 4~ This process is found to damage only a small fraction of the calls'. The cells are then thawed, washed, and irradiated to 10,000 rads using a .,/-source, then immediately injected into the three limbs, both intradermally and subcutaneously. Approximately 1 x 109 cells are injected each week.

Results The total entry of new patients with myeloblastic leukaemia to Trials II, III and IV was 107 (1 February 1973) and 45 of these have so far passed into full remission. They were then included in the controlled immunotherapy section of the trial. Nineteen of these patients received only maintenance chemotherapy and 23 received immunotherapy also. Three immunotherapy patients have been excluded--two because they have only just passed into remission, and one because he died of infection immediately following remission induction, before immunotherapy was started.

Remiss/on Length In the two groups that are now accepting no new patients (Trials II and Ill), only three of the 15 patients receiving chemotherapy alone remain in

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remission and the median remission length is 30 weeks, whereas in the chemotherapy plus immunotherapy group four of the 15 remain in remission with a median remission length of 53 weeks. Actuarial analysis of all three trials (including Trial IV which still takes new patients) shows the median remission length for the 19 chemotherapy patients will be 23 weeks and for the 23 chemotherapy plus immunotherapy patients 45 weeks. At present, in these groups eight of the 23 immunotherapy patients remain in remission, compared with five of the 19 chemotherapy patients. Survival Similar analysis for the survival of the same group of patients in Trials II and III shows nine of the 15 chemotherapy plus immunotherapy patients remain alive, so a median has not yet been reached. However, only four of the 15 chemotherapy alone patients remain alive, with the median, at present for this group is 35 weeks. Actuarial analysis (including Trial IV) gives a median survival time, after attaining remission, of 78 weeks for the immunotherapy patients (16 of 23), compared with 43 weeks for the chemotherapy alone group (7 of 19 survive). Discussion The results of this study show a significant lengthening of both remission duration and survival for patients with A.M.L. given immunotherapy in the way we describe. This is not to say that we feel this method of treatment is the best at present available, as one must consider with care the recent reports of the benefits obtained using intensive maintenance chemotherapy 5,'. This, however, was not the purpose of this present study. We were more concerned with showing a definite significant effect of immunotherapy to give factual encouragement to the present tremendous drive to use these procedures for treating cancer. But it so happens that as things stand (at present) our groups of patients receiving a combination of chemotherapy and immunotherapy are faring as well as any group treated by other c~ntres, and it is our impression the quality of life of our patients is superior to those receiving intensive chemotherapy. A comparison of our results with those of Math~ is difficult because he treated a different disease and gave different immunotherapy, but this should not detract from the results of either. There are no other instances to my knowledge of immunotherapy giving a positive result in man. The future of immunotherapy for cancer in man is uncertain; clearly our patients are not cured, as most relapse and many have already died, and so better methods must be found for using these agents, and this must depend upon the accurate measurement of specific host responses to both the disease and treatment, and there is now some evidence these tests are already becoming avaitabte '.s,~. However, the tong-term future of immunotherapy for treating cancer should be regarded with caution and it is reasonable to suppose the final answer will be of a chemotherapeutic nature. Perhaps the final role of immunotherapy will be the same as occurred with tubercle, whereby treatment is of a chemotherapeutic nature, but the less glamorous control of the disease is in the hands of the immunologist in the form of immunodiagnosis and immunoprophylaxis.

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This study was conducted by the following workers: P. Alexander, D. Crowther, T. McEIwain, G. Hamilton Fairiey, H. E. M. Kay, and many others. We were indebted for financial support to the Imperial Cancer Research Fund, the Leukaemia Research Fund and the Medical Research Council, and we thank Sir Ronald Bodley Scott and Dr. P. E. Thompson Hancock for their advice and help.

References 1. Currie, G. A. 1972. Eighty Years of Immunotherapy : A review of immunological methods used for the treatment of human cancer. Br. J. Cancer. 26, 141. 2. Math6, G. 1969. Approaches to the immunological treatment of cancer in man. Br. Med. J. iv, 7. 3. Powles, R., Crowther, D., Bateman, C. J. T., Beard, M. E. J., McEIwain, T. J., Russell, J., Lister, T. A., Whitehouse, J. M. A., Wrigley, P. F. M., Pike, M., Alexander, P. and Hamilton Fairley, G. 1973. Brit. J. Cancer. In press. 4. Powles, R. L. and Grant, C. 1973. Some properties of cryopreserved acute leukaemia cells cryobiology. In press. 5. Children's Cancer Study Group A. 1973. B.C.G. in the treatment of acute lymphoblastic leukaemia. Proc. Am. Assoc. Cancer Res. 14, 45. 6. Whitecar, J. P., Bodey, G. P., Freireich, E. J., McCredie, K. B. and Hart, J. S. 1972. Cyclophosphamide (NSC-26271), vincristine (NSC-67574), cytosine arabinoside (NSC63878) and prednisone (NSC-10023). (COAP) Combination Chemotherapy for Acute Leukaemia in Adults. Cancer Chemotherapy Reports, 56, 543. 7. Alexander, P. and Powles, R. 1973. The possible occurrence in vivo of the autostimulating factor (A.S.F.) for lymphocytes. Published in Birth Defects, Original Article Series Lon-term Lymphocyte Cultures in Human Genetics. Voi. ix, No. 1. p. III. Published by The National Foundation--March of Dimes. 8. Gutterman, J. U., Mavligit, G., McCredie, K. B., Freireich, E. J. and Hersh, E. M. 1973. Auto-immunization with acute leukaemia cells: demonstration of increased lymphocyte responsiveness. Int. J. Cancer ii, 3, 521. 9. Heberman, R. B. 1973. Immune responses to virus induced experimental leukaemia and to human leukaemia. Proceedings of the Miles Seventh International Symposium, "The Role of Immunological Factors in Virus and Oncogeneic Process". In press.

CHAIRMAN I'd just like to discuss a few points with bearing on both papers. We had a family in which three members developed Hodgkin's disease. There are five sisters, the oldest developed the disease in 1967, the second in 1969, and the third in 1973 (Fig. 1). With this unique occurrence we thought we should look at these cases from the immunological, genetic and viral aspect. I am indebted to Dr. Aldona McBride who is responsible for these results. The three patients with Hodgkin's disease showed negative PPD and DNCB reactions. The youngest with infectious mononucleosis, and the mother, had negative PPD and DNCB tests. Tests on K., the uninvolved sister, and the father were strongly positive. As regards the lymphocyte transformation test, all of the family showed a depression except for K, the uninvolved sister, and the baby of one of the patients, indicating a further immunological depletion. The main depletion, therefore, was in the mother who was PPD and DNCB negative and had completely flat lymphocyte transformation indicating depressed T lymphocyte function. We wonder if the inheritance of this is an important factor; possibly Dr. Harris could comment on that.

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HD'67

H D ' 6 9 HD'73

24yrs

20yrs

21yrs

Herpes 18yrs

i.mono'73 15yrs

Fig. 1--Family tree of family with Hodgkin's Disease. H.D.=Hodgkin's Disease. members shown in shaded circles.

Involved

In reference to Dr. Powles' paper in acute lymphatic leukaemias, we find about 85 per cent of these go into remission and that about 80 per cent of these will go on for twelve months. This is a study only going on two years so that there is quite a number of them continuing in remission. We actually haven't had a patient go into a blast crisis now for over one year and all of those continue in remission on chemotherapy alone. Chemotherapy alone in acute myeloid leukaemia produces remission in about 50 per cent of cases and many of these are going over a year and a few of them over two years (these patients are treated in St. Vincent's Hospital and Temple Street Children's Hospital in association with Dr. L. O'Connell and Dr. S. Cahalane). Sometimes when I look at the chemotherapy figures in the immunotherapy studies I wonder if the chemotherapy figures might be improved on. At present I feel that these figures would bear up fairly well with your immunotherapy figures but I'd like comments maybe later from Dr. Powles. However, now I would like to ask Professor Douglas Thornes from the Colman Byrnes Research Institute of the Richmond Hospital to open the discussion. PROFESSOR THORNES Well, Dr. Powell and Dr. Harris have shown the importance of immunotherapy in the field of leukaemia but what about other forms of cancer. I feel that it is essential, for immunotherapy to be effective, to have an immunocompetent host. There is no gain in using B.C.G. and other stimulants, however specific, if the cellular immune mechanism does not function. It must be shown that the therapy used affects the immune mechanism before it can be called immunotherapy. Now, we have been trying to study the possibilities of stimulating the cellular immune mechanism in patients with cancer at the Richmond. There 82 out of 100 patients recently tested by skin tests for delayed hypersensitivity gave no reaction or, in other words were anergic. Fortutously, a previously anergic patient with carcinoma of the lung was given a fibrinolytic

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agent called Brinase when his skin tests were repeated. The tests were strongly positive within 24 hours. Repeated again, a week later, the skin tests were negative but thereafter Brinase weekly lead to continued positive tests. Brinase is a protease from Aspergillus Oryzae which we use to lower the inhibitors of plasmin in patients. Plasmin is a naturally occuring fibrinolytic enzyme found in human blood and serum. Inhibitors to, called antiplasmins, are raised in patients with leukaemia and cancer. The phenomena observed in patients treated with Brinase were initially attributed to direct proteolytic action but, probably, more properly should be considered due tO activation or unblocking of the immune mechanism. When Brinase is used to lower antiplasmin levels below 50 per cent of normal blast cells are removed from the circulation in patients with acute leukaemia within one hour. Leukaemic cells can be shown to break up within 15 minutes of an infusion to a patient with chronic lymphatic leukaemia. Leukaemic cell counts have been lowered by up to 70,000 c.mm in chronic lymphatic leukaemia and 40,000 c.mm in chronic myeloid leukaemia during a one hour infusion of Brinase. In the same patients Brinase added in vitro to a blood samp!e did not lyse or break down any leukaemic cells even though the concentration was 10 times higher than could have been expected in vivo following the infusion. It was also found that patients with acute leukaemias developed transient autocytotoxic antibodies against lymphocytes, platelets and leukaemic cells 10 to 28 days after the start of Brinase infusions. The induction of fibrinolysis by either Streptokinase or Brinase can be shown to activate the cellular immune mechanism when measured by : 1. Conversion to positive of delayed hypersensitivity skin tests using P.H.A., P.P.D. and Streptokinase. 2. Increased leucocyte migration rate. 3. Increased percentage of spontaneous rosette formation by sheep red blood cells around lymphocytes from 36.1% • 4.4 to 49.5% -+ 4.9. 4. Patients previously insensitive to D.N.C.B. could be sentitized. Britanse added in vitro to lymphocytes increased rosette formation with sheep red cells from 36.1% -+ 4.4 to 67.7% -+ 3.6. To the best of my knowledge these observations are original and are the result of co-operative study with the Medical Staff of Our Lady's Hospital for Sick Children in Crumlin, The Richmond Hospital and the Immunology Division of Pathology at the Royal College of Surgeons in Ireland. At present we are using these findings to treat patients with various malignancies who are found to be anergic. Their cellular immune mechanism is first activated with Brinase before immunotherapy with B.C.G. or specific antigens is commenced. Their immune mechanism is checked monthly to ensure continued activation. Most cases with residual tumour revert rapidly to the anergic state while those with minimal tumour remain converted with an active immune system. It is still to early to assess our results for the clinical regression of cancer but I am satisfied that immunotherapy requires an immunologically competent host.

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CHAIRMAN Thank you very much Professor Thornes. Dr. Harris would like to say a few words in relation to Dr. Powles' paper, but we might just have some questions from the floor while we are waiting. There is a question here from Dr. Brendan Duffy : What would your approach be to a patient with Hodgkin's lymphocyte predominance histology and generalised immunological deficiency with frequent infection. Is there a place for radiation, gammaglobulin infection. or some other magic cocktail ? DR. HARRIS Well, it is unusual to have lymphocyte predominant disease and severe immunological deficiency. Quite often it is the other way round with the lymphocytes depleted and immunoglobuin deficiency and then, of course, there is nothing you can do. In this situation you describe I think that I would ignore immunoglobulins and treat this patient with quadruple chemotherapy in an attempt just to put them into remission. I think if one attempts immunotherapy with this patient you'll do more harm than good. CHAIRMAN Thank you very much Dr. Harris. A question here from Dr. Donald Weir. Can we assess the genetic immune tolerance of people to pick those who are reliable to develop leukaemia ? DR. HARRIS That is an extremely important question. It turns upon the excellence or otherwise of tests of immune competence and we have a number of things we can measure, for example, immunoglobulins, levels of isoantibody, the number of T cell functions, but when it comes down to reassessing for each of these tests what's normal and what's abnormal, we find that our quantitation isn't terribly good at the moment. And every time what we are really up against is not searching for a general depression but a depression of the immune response. We are seeking an immune response against a particular antigen but those antigens are really not very often recognised in man, so we are up against a very difficult situation. This leads to that really most interesting family reported by Dr. Fennelly and Dr. McBride with three people with Hodgkin's in a fatuity. You do get, of course, very rare families where you find the same type of cancer. One cannot but feel that a common disease will occur by chance in some families more than once but I find your family to be a bit beyond that sort of chance phenomenon. I honestly don't know at the present what one can do to investigate these in these specific terms. I suspect that next year one will know, I mean within 12 or 24 months. I suspect rather strongly that what one will do here is to take the HLA types of these patients, to discover that a normal sibling and a Hodgkin's sibling were HLA identical and therefore should not stimulate their lymphocyte in a two-way or a double one-way reaction. And then find that that Hodgkin'S stimulates the normal HLA identical sibling. CHAIRMAN Now, Dr. Harris, I know, has a few observations to make but before he

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does there are two questions on Hodgkin's which I would like to link together; first Dr. Michael Whelton raises the very pertinent point: In view of the reports of increased incidence of Hodgkin's in some institutions is there any evidence of an increased incidence of Hodgkin's in medical and para-medical staff treating such patients ? Now Dr. Dermot Hourihane has asked a question also to to Dr. Harris. Have there been any studies of antigens which included people who later developed Hodgkin's disease. Is it possible just to link these two questions ? DR. HARRIS Well, the second question, I don't know of any such studies at all, no. We are however doing a prospective survey of patients at presentation with Hodgkin's and we are seeing whether those patients with certain HLA types live longer than those with other HLA types. In other words, does the survival of the patient, once he develops Hodgkin's depend upon his HLA type. As far as the epidemiology of Hodgkin's is concerned I don't know, in fact I have never heard of increased incidence in laboratory or workers or doctors treating Hodgkin's patients. I do know however that the serum of patients working with animal or human leukaemia is said frequently to contain antibodies against leukaemia, animal leukaemia viruses. There is an excellent annotation in the Lancet in the last six weeks or so on the epidemiology of Hodgkin's which does in fact give all the up-to-date information that I know about. Ray, I am sure you have more experience of this than I have. DR. RAY POWLES Hodgkin's disease actually worries me as a separate disease because I think it is so atypical of all the other malignant diseases that I know that I think to apply any of the general rules to it is actually impossible. For instance, we have no idea in Hodgkin's disease what even the malignant cell is. I am quite sure that if the Albany work was substantiated we would have a few less people specialising in the disease. CHAIRMAN I have a question from Dr. Brian Lemass : How do you manage lymphoma or CLL patients with haemolytic anaemia without antibodies. Would you manage them with chemotherapy and predisone, predisone alone or would you suggest any other treatment ? DR. HARRIS I think actually that again this becomes a clinical question and not a scientific question and I think that if you have evidence in a non-Hodgkin's lymphoma of a haemolytic anaemia then in our present knowledge you should give that patient steroids. Now it just so happens that if you treat that patient also specifically against this disease more often than not you control the disease, the haemolytic anaemia will also become controlled but this is not always the case. In fact there are, certainly I know of two, lymphosarcoma patients who, even though they went into full remission on chemotherapy as far as their clinically detectable disease was concerned, still had haemolytic anaemia. Under those circumstances actually I think that I only know of steroids that can control that.

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CHAIRMAN I know that Dr. Harris has a few questions or points that he'd like to make to Dr. Powles' paper and slides. DR. HARRIS ! want to say how much we owe to Dr. Pow~es for introducing immunotherapy to acute myeloid leukaemia. This really has been an important step I believe in cancer therapy. We in Manchester have followed very closely on the protocol that Dr. Powles was associated with, we just simply copied that protocol. Except that in our first series of patients we used a slight reduction course of chemotherapy. I think looking at the results of the MRC trial with which we are associated I suspect rather strongly that our first series is going to turn out better. The method of immunotherapy is exactly the same as Dr. Powles described and there are no controls in this case series at all. These are simply patients who were put into remission who were then given immunotherapy. The second series, a much bigger one, a joint one with the MRC and there are controls of exactly the sort that Dr. Powles has mentioned, i.e. chemotherapy compared with chemotherapy plus immunotherapy. We only have a 25 per cent remission rate at present time in Manchester area I think with a slightly different group of patients to the ones people in London seem to have, that is, they tend to be older, to have higher blasts per capita, perhaps they take longer to get to us, I don't know. Of patients who were treated with immunotherapy only two have died, one of them has massive infiltration of the testicles right from the start, I don't think he ever went into remission. The other patient who died had some form of platelet abnormality with spontaneous clumping of the platelets; I am afraid we simply missed the fact that she was relapsing and we didn't treat her quickly at all when she eventually relapsed. The remaining patients are in excellent health, none have had a clinical relapse or a haematological relapse although the marrow blast count indicates the onset of marrow relapse. In Manchester we do monthly bone marrows and as soon as there is any sort of relapse then we resume reinduction chemotherapy. And I suspect this may be very important. All of the work in Manchester is done by my colleagues who look at every patient individually and again I suspect that the intense attention they get is also helpful. Nevertheless one has got to say that these patients are much happier and healthier on immunotherapy alone. They are extremely easy to re-induce as soon as the bone marrow shows signs of relapse and they always go back into remission very very quickly indeed. So in conclusion, and this wants saying, I support everything Dr. Powles has said and I want to say again how much I think we owe to Dr. Powles for introducing this form of treatment. CHAIRMAN Thank you very much Dr. Harris. One of the interesting things coming from this paper is the variation in remission rate that occurs from year to year in acute myeloid leukaemias. Always the first reports are best, there is no doubt that acute mye~oid leukaemias were going into remission two years ago in 60 per cent of cases but numbers get bigger somehow or other the figure dropped to 50 per cent and then below 50 per cent. You may get a run of 6 patients, none of whom go into remission which of course this will really upset the figures. We use

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a three monthly re-induction with cytosine arabinoside, daunorubicin, vincristine just for five days. Of the patients that go into a smooth remission about half stay so for a year and a number for on to two years. I think, that the question of immunotherapy is certainly stimulating and interesting and I would like just to ask you both, "Would you feel that it is essential to have an IBM separator, would you go so far as to say immunotherapy in acute myeloid leukaemia is essential ?" DR. POWLES At a glance at your figures and I could only do it quite quickly, your median survival I calculate to be 11 months which is exactly par for the course; it is exactly what White Carr's group and the South West Group in the States have found, and also Clarkson - - they are about 11-12 months median survival. Our median survival actually as the figures stand are 550 days which is about 18 months. This is apart from the controlled part of our trial which of course is the essential part, I think one would have to say as it stands that immunotherapy is probably playing some part, but the point that I'd really like to stress is the point that Dr. Harris mentioned which is that the quality of life of these immunotherapy patients, if this is an alternative method of treatment, is far far better than the quality of life of even moderately intense maintenance chemotherapy. I can't answer for Dr. Fennelly's patients but the South West group of patients who are receiving extremely intensive chemotherapy are so ill they can't work even though they are in remission. DR. HARRIS I really think that immunotherapy as a way of life for the patients is so immeasurably superior to the maintenance chemotherapy that is necessary even to get approaching the same results that this is of over-riding importance. I am not 100 per cent convinced that this is due to the development of specific immunity. In fact we don't have any concrete evidence that immunotherapy done this way is producing a specific immune response. I agree that this is the next phase and has been the next phase for two years, we tried to find evidence that these patients were in fact mounting immune response against the cells we are injecting into them and we cannot find this at the moment because we don't have sufficiently subtle techniques. I think this is the answer to this phenomenon. But you know this all pales into scientific insignificance when it comes to the way these patients are living. They are immeasurably better off. CHAIRMAN Before concluding I would like on your behalf to thank Dr. Rodney Harris, Dr. Ray Powles and Professor Douglas Thornes for their contributions this morning and I think you will all agree it has been a very stimulating morning. Thank you.