Immunological aspects of acute leukemia in man

Europ. J. Cancer Vol. 12, pp. 511-517. Pergamon Press 1976. Printed in Great Britain

Perspectives in Cancer Research

Immunological Aspects of Acute Leukemia in Man WILLY F. PIESSENS* and W I L L I A M C. MOLONEY Departments of Medicine, Harvard Medical School and Peter Bent Brigham Hospital, Boston, Massachusetts 02115, U.S.A. Abstract During recentyears the immunological aspects of acute leukemia in man have been the subject of an ever increasing number of studies. Our purpose is to review progress made in four specific areas of research: the relationship between immune competence and susceptibility to leukemia, the role of immune competence during overt leukemia and its modification by chemotherapy, the characterization of leukemia antigens, and the effect of active immunotherapy on acute leukemia in man.

from genetic family studies, and additional antigens found on cells obtained during active disease but not during remission. The exact nature of these " n e w " antigens is not clear but similar anomalies of H L - A typing occur after exposure of normal lymphocytes to phytohemagglutinin [8] or proteolytic enzymes [9], indicating that this phenomenon is not specific for leukemia and m a y represent phenotypic rather than genotypic alterations. Thus, unequivocal evidence for a relationship between the histocompatibility type and the incidence of leukemia in man is not available at the present time. Other evidence that immune competence correlates with the incidence of leukemia in man is also lacking. Despite some early reports to the contrary [10] it is clear that "immunoprophylaxis" with BCG vaccination at an early age neither increases nor decreases the incidence of acute leukemia in children [11, 12]. Primary immunodeficiency diseases and chronic immunosuppression to prevent the rejection of organ grafts are associated with an increased incidence of tumors. However, most of these are lymphomas and solid tumors rather than leukemias [13, 14]. There is an increased incidence of leukemias following exposure to high doses of radiation, but whether this is due to the leukemogenic effect of irradiation per se or to its immunosuppressive effects remains unknown [15].

1. I M M U N E C O M P E T E N C E A N D SUSCEPTIBILITY TO LEUKEMIA THE SUSCEPTIBILITYof inbred strains of mice to viral leukemias is linked to the major histocompatibility (H-2 locus). The same chromosomal region appears to contain genes controlling the immune responsiveness to various antigens, the susceptibility to leukemia and the major histocompatibility antigens [1, 2]. Whether a similar relationship exists in man between the incidence of leukemia and the histocompatibility (HL-A) type has been the subject of several studies. H L - A antigens can be detected on leukemia cells by conventional tissue typing methods [3]. Whereas several studies failed to reveal any differences in the number and distribution of H L - A antigens on leukemic cells [3, 4], other studies suggest an increased incidence of certain H L - A antigens or haplotypes in leukemia, in comparison to their incidence in the normal population [5, 6]. In addition, anomalies in the H L - A typing of leukemic cells occur rather regularly [7]. These include the finding of extra H L - A antigens which should not be present on the basis of what can be predicted *Cancer Research Scholar of the American Cancer Society, Massachusetts Division Inc. Reprint requests to Willy F. Piessens, M.D., Peter Bent Brigham Hospital, 721 Huntington Ave., Boston, MA 02115, U.S.A. 511

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Wil~ F. Piessens and William C. Moloney IMMUNE COMPETENCE DURING O V E R T LEUKEMIA

Studies on the immune competence of patients with overt leukemia and on the effects of chemotherapy on the immune responsiveness are important for several reasons. Infections continue to be the leading cause of death in the leukemias; therefore, how the modification of the host's immune responses by chemotherapy alters the patient's resistance to infections should be of p r i m a r y concern to everyone treating this disease. Furthermore, as immunotherapy is rapidly becoming an acceptable treatment modality, it is important to determine whether the patient's immunity to his own leukemia has any bearing at all on long-term prognosis. The effects of immunosuppressive chemotherapy and of immunotherapeutic manipulations have to be examined with regard to these basic questions. Most studies reveal a qualitatively normal but a quantitatively decreased general immune competence in patients with acute leukemia [16-20]. In addition, specific immunological reactions against leukemia-associated antigens can be detected in over half of all patients (see below). It has been claimed that those patients with an intact immune system at the time of diagnosis are more likely to obtain a remission and thus have a better prognosis, presumably because leukemic cells remaining after a drug induced remission are further eliminated by the host's immune defense against "foreign" tumor cells [207]. However, a correlation between immune competence and prognosis can not always be demonstrated [18]. This could be explained by the variable effects of different drug regimens on the immune competence and on the leukemia itself. It is obvious that if a chemotherapeutic regimen that would effectively eradicate leukemia were available, the effect of the host's immune competence would be largely irrelevant. O n the other hand, even the strongest expression of cellular immunity may be without detectable effect on the prognosis following treatment with ineffective drugs that are also immunosuppressive. Thus, the prognostic significance of the patient's immune competence may depend in part on the nature of the chemotherapy, but this possibility has not been adequately studied. An interesting finding in these studies is the demonstration that immunosuppression by chemotherapeutic drugs is to a large extent

reversible, not only after cessation of treatment [21], but also with continued intermittent chemotherapy [22]. Long-term survivors of acute leukemia have high avidity antibody to leukemia-associated antigens in their serum, but this may result from prolonged exposure to low doses of antigen rather than being the cause of high intrinsic resistance to this disease [23]. Thus, the hypothesis that the immunological reactivity of leukemic patients towards their own tumor contributes to the overall prognosis in this disease is an attractive one, but many more studies are needed to settle this problem. 3.

LEUKEMIA ANTIGENS

Attempts to demonstrate leukemia-associated antigens have been numerous, have employed a variety of immunological methods, and have met with encouraging albeit variable success [24]. Autoantibodies against leukemic cells have been demonstrated in the sera of untreated patients with several forms of leukemia. Dor~ obtained positive results in 12 of 51 patients and Yoshida in 15 of 30 patients tested [25, 26]. More recently, antibody dependent lymphocyte killing of myeloblasts has also been reported [27]. The failure to demonstrate autoantibodies in the majority of patients has resulted in numerous attempts to produce leukemia specific heteroantisera by immunizing a variety of animal species with human leukemic cells. The prevalence of positive results varies considerably, although surprisingly less than one might expect from the differences in the clinical material and the immunization procedures used. It is unlikely that all antisera detect the same antigen(s); nevertheless, some generalizations can be made. Following the appropriate absorptions with normal leukocytes, most antisera detect antigens more or less specific for either the myeloid or the lymphoid leukemias. In general, the same antigens appear to be shared by the acute and chronic forms of the same cell type, myeloid or lymphoid. Although most leukemias of a given cell type have common antigens, antigens unique for each individual leukemia can also be detected following the appropriate absorptions of the antisera [28, 29]. By analogy with the situation in murine leukemias, the finding of common leukemia antigens has been interpreted as evidence for a viral origin of human leukemia. In contrast to the antisera mentioned above, the antibodies developed by Mann and Herbermann react with A M L and ALL cells but not

Immunological Aspects of Acute Leukemia in Man with leukocytes from normal donors or from patients with CML, CLL or Hodgkin's disease [30]. Antisera with similar specificity were developed by Billing and Terasaki using either Raji cells or fresh lymphosarcoma cells as the source of the antigen, which appears to be a glycoprotein [31, 32]. In a long-term study of 10 patients with acute leukemia (8 ALL, 2 A M L ) , reactivity of the antisera with peripheral blood cells correlated well with the amount of tumor in the marrow, as reactivity disappeared during remission [33]. Hence, the use of such antisera m a y be useful for immunodiagnosis of early relapse. T h e major problems and pitfalls in the characterization of leukemia antigens with xenogeneic antisera have been summarized [24]. Whether the antigens that are good immunogens for animals play any role in the i m m u n e response of the cancer host to his own tumor remains to be determined. Cellular i m m u n e reactions to leukemia antigens were first studied with the mixed lymphocyte culture test, in which remission lymphocytes are exposed in vitro to autologous blasts. Stimulation of remission lymphocytes by autologous blasts has been reported by several investigators, indicating the presence of " n e w " antigens on the leukemic cells [34-38]. I n some studies the degree of stimulation is less when the culture m e d i u m is supplemented with autologous rather than allogeneic serum. This has been attributed to the presence of"blocking factors" in these sera; in view of recent animal studies this should be detrimental to the patient; however, the contrary seems to be true [38]. It is of interest that the cells of 7 of 8 patients with A M L and serum inhibition in the M L C had IgG on their membrane detectable by direct immunofluorence. Perhaps the lymphocytes in this case were stimulated not by antigen(s) present on the blasts, but by antigen-antibody complexes, as noted in other systems [39]. A short-lived increase in the reactivity of lymphocytes to autologous blasts occurs following immunotherapy. Enhanced lymphocyte reactivity can also be observed as a rebound phenomenon after the cessation of chemotherapy [22, 37]. The mixed lymphocyte reaction probablyrepresents an in vitro expression of delayed hypersensitivity to a new antigen present on the leukemic cells. However, other possible mechanisms for lymphocyte stimulation in mixed cell cultures, such as the formation of mitogenic factors by leukemic cells, have never been adequately studied. The antigens are

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certainly tumor-associated, but proof that they are true tumor specific antigens is still lacking. The issue has become more confusing by the demonstration that normal lymphocytes react in the MLC to autologous cultured lymphoblastoid cell lines or to mitogen induced autologous lymphoblasts [40, 41]. The hypothesis that during the process of transformation by mitogens or by long-term culture " h i d d e n " (fetal?, viral?) antigenic components become exposed is an attractive one, but the relationship, if any, of these antigens to leukemia antigens is a matter of speculation. Because these cells clearly are not leukemic but nevertheless are capable of stimulating autologous lymphocytes in the MLC, caution should be used to attribute the lymphocyte stimulation by leukemic cells to the presence of leukemia specific antigens. Lymphocytes from histocompatible, identical siblings also react in the M L C to blast cells from the leukemic sibling, but not to remission leukocytes [42, 43]. This again suggests the appearance of a new antigen on the leukemic cells, as cells from normal identical siblings do not interact in the MLC. However, equally convincing negative findings have been reported in four sets of identical siblings (2 ALL, 2 AML) [44].

4.

IMMUNOTHERAPY

Since the turn of the century, numerous attempts have been made to immunize cancer patients with tumor cells or tumor extracts but until recently the results have been consistently disappointing. Based on the results from extensive studies with the murine leukemias (summarized in reference [45]) the first successful clinical trial of immunotherapy for A L L was reported by Math6 [46]. T w e n t y patients with ALL in complete remission induced by chemotherapy were treated with irradiated leukemia cells, BCG or both. Ten control patients were left untreated. All 10 controls relapsed within 130 days after the chemotherapy was stopped, but only 9 of the 20 patients given immunotherapy had relapsed by that time. These preliminary studies have since been extended to include over 100 patients treated with a variety of experimental protocols, continuously modified to incorporate new concepts derived from results obtained in animal models or the preceding clinical trials [47]. The results continue to support the original conclusions that under well defined circumstances certain modalities of active

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i m m u n o t h e r a p y increase the duration of remissions in ALL. However, in trials of similar design by the Medical Research Council and by the Children's Cancer Study Group A, BCG alone did not prolong remissions in A L L [48, 49]. Trials o f i m m u n o t h e r a p y for A M L have met with more consistent success. Powles et al. [50] have shown that the duration of remissions induced by chemotherapy can be significantly prolonged by treatment with weekly injections of irradiated allogeneic leukemia cells and BCG, in addition to monthly courses of maintenance chemotherapy. The median duration of remissions was 188 days with chemotherapy and 312 days with the combination of chemotherapy and immunotherapy. It is not clear whether the prolongation of remissions was due to the BCG or to the tumor cells. In a pilot study by the same group, immunotherapy without maintenance chemotherapy appeared equally effective as the combination of both for the maintenance of remission of A M L [51], but this was not corroborated in another study of similar design [52]. When relapses occurred, successful reinduction of remissions was easier in the group treated with i m m u n o t h e r a p y alone than in those patients receiving maintenance chemotherapy as well; this was attributed to a greater bone marrow reserve in those patients not receiving chronic maintenance chemotherapy. Gutterman et al. studied the effect ofimmunotherapy with BCG alone on the length of remission in A M L maintained with intermittent chemotherapy [53]. Median duration of remission was 72 + weeks on the combined treatment as compared to 60 weeks on chemotherapy alone. Curiously, they could not demonstrate a beneficial effect on the length of remissions in patients with A L L treated with the same regimen. Even a less intensive course of immunotherapy (with BCG given twice a week for a total of 4 weeks) prior to the start of maintenance chemotherapy prolonged remissions in A M L from 26 to 39.4 weeks [54]. Several interesting features emerge from these studies. In most trials the initial relapse rates for patients in the control and the immunotherapy groups are very similar. However, while the control group continues its downward trend, the relapse rate for patients on immunotherapy levels off. I f the results from animal studies can be extrapolated to man, this would suggest that the tumor burden in those patients who relapse early was not decreased to a number of cells the immune

response can cope with. At the present time no methods are available to differentiate a priori these unfortunate patients from those who will do well. Perhaps the use of immunodiagnostic methods such as the one described by Haltermann may eventually be a major step in this direction. The morbidity associated with long-term immunotherapy with living tubercle bacilli appears to be acceptable but by no means negligible. The side effects of BCG as used for the treatment of malignant diseases have been recently summarized [55]. Fortunately, enhancement of tumor growth has so far not been a major problem in the h u m a n leukemias, although it regularly occurs in some experimental tumor systems [56, 57]. Finally, m a n y different BCG vaccines are available. Most of them will protect against tuberculosis (for which they were developed in the first place), but there are considerable differences among the vaccines, notably in the mycobacterial strains used and the technical procedures employed to deliver the final product. The conflicting results from various trials have been attributed in part to the use of different vaccines and different routes of administration with variable effects on the immune response. This may be true to a certain extent as, for instance, dead mycobacteria favor antibody responses rather than cell mediated immunity which appears to be more important for resistance to tumors [58]. The ratio of living versus dead organisms varies considerably between different BCG vaccines and even from one lot to another of the same vaccine. The many uncertainties of using such a variable material as BCG cannot be overemphasized, especially as the desired immunoprophylactic antileukemic effect is merely a byproduct of the specific immunization (against tuberculosis). It is imperative that standardized extracts of tubercle bacilli with effects comparable to those of intact bacilli be tested in controlled, standardized, and preferably international clinical trials of immunotherapy for acute leukemia in man.

CONCLUSION Although the studies on the immunological aspects of h u m a n acute leukemia have increased our understanding of the biology of this disease, there is no unequivocal evidence to date that immune reactions play a major role in the incidence, the morbidity and the prognosis of acute leukemia in adults. There is some evidence

Immunological Aspects of Acute Leukemia in Man that certain forms of i m m u n o t h e r a p y can prolong remissions in A M L in perhaps as m a n y as one-half of the patients, but the importance of this finding is lessened by the fact that less than 50% of all adults with acute leukemia ever achieve a remission. Because i m m u n o t h e r a p y is ineffective when large numbers of tumor cells are present, the immediate

improvement of the outlook for adults with acute leukemia will result from improvements in chemotherapy and not from the addition of i m m u n o t h e r a p y to our therapeutic arsenal. Nevertheless, further studies on i m m u n i t y in h u m a n leukemia are urgently needed to answer the m a n y unsolved questions discussed in this brief review.

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