Biomed& Pharmacother(1990)44.93-102 Q Elsevier,Paris
K Ota I’, N Ogawa * ’ Nagoya Memorial Hospital, 4-305 Hirabari. Tenpaku-ku.Nagoya 468; ’ Pharmacological ?nstitute, Ehime University School of Medicine, Ehime 791-02. Japan (Received 5 February1990; accepted 12 February1990)
Summary - A new immunomodulating agent, bestatin (INN: Ubenimex has low toxicity even after long-term oral administration and has significant modifications in immunological response. A cooperative randomized controlled study of bestatin immunotherapy in combination with remission maintenance chemotherapy for adult acute noniymphocytic leukemia (ANLL) was performed. After induction of complete remission. patients were randomized to the bestatin group (30 mglbw per OS (PO) daily) and the control group. The 101 eligible cases (best&n: 48, control: 53) were analyzed; the bestatin group achieved longer remission than the control group and a statistically Qnificant longer survival. Though this prolongation of remission was not significant in the bestatin group compared to the cu#rol group in the 15-49 yr age group, in the SO-65 yr age group it was significantly longer. Bestatin is shown to be a clinically zsefui drug for immunotherapy of adult ANLL, since it has prolonged survival and remission especially in elderly patients, wr:h few side-effects. bestatin / immunotherapy / acute leukemia R&urn4 - 6:tude contr6Me et randomisbe de la chimio-immunotherapie de la leuc6mie aigue non-lympbocytaire chez I’adulte par la bestatine. Un nouvel agent immuno-modulant, la bestatine (DCI : Ubenimex) se montre mains toxique que les autres mltme aprPs une administration prolongde per OS.Ce mPdicament provoque des modifications significatives dons la rPponse immunitaire. Une Ctude coop&rative randomisee et contr6lJe a PtPeffect&e sur l’immunoth&apie par la bestatine en association avec une chimiotht+apie d’entretien aprPs remission dans la leu&mie aiguL;non-lymphocytaire de I’adulte (ANLL). Apt& induction Gune remission complPte, les malades ont et&rdpartis par randomisation entre un groupe bezatine (recevant 30 mglkg-t-tj-’ de bestatine per OS) et un groupe Gmoin. Les 101 cas ligibles (48 “bestatine” et 53 Mmoins) ont &! analysps. Le groupe bestatine a Wn@icie’ d’une r&missionplus longue que le groupe tPmoin ainsi que d’une survie significativement plus longue. L’allongement de la &mission n’itait pas statistiquement significatif par rapport au groupe tPmoin dans la tranche IS- 49 ans mais il I’Ptait dans la tranche SO- 65 ans. La bestatine est ainsi prt!sentJe comme un medicament utile en clinique dans l’immunothtrapie de I’ANLL de l’adulte compte-tenu du fait qu’elle prolonge survie et remission, particuli$rement cher les sujets a&S et cela avec peu seffets secondaires. bestatine I immunothdrapic I leuc&tie aigue
Introduction As a result of recent progress in chemotherapy, adult patients with acute nonlymphocytic leukemia (ANLL) can now be expected to show a complete remission rate of over 60% and a long* Correspondenceand reprints
term survival rate of approximately 30%. In order to eradicate minimal residual leukemic cells after z.chievement of complete remission, various methods for enhancing cure rate in adult ANLL have been attempted (eg, immunotherapy and bone marrow transplantation). This immunotherapy has most commonly consisted of bacterial preparations such as bacillus Calmette-Guerin (BCG), or homologous leukemic cells. There has
94
K Ota, N Ogawa
been much debate concerning the efficacy of these forms of immunotherapy. Bestatin (INN: ubenimex) is a small molecular weight dipeptide. N-[(2S, 3R)-3-amino-2-2hydroxy_4_phenylbutanoyl] L-leucine, discovered by Umezawa et 01 [12] in the culture filtrate of Streptomyces ofivoreticufi (fig 1). Fundamental studies have revealed that bestatin has low toxicity even after long-term oral administration, with the modulating activity of cellular immunity possibly via its binding to aminopeptidases located on the cell surface of immune cells, and host-mediated antitumor activity against murine tumors [4]. In a Phase I study, the safety of orally administered besiatin was confirmed, and it was suggested that the appropriate dosage level of this substance was in the range of 30-100 mg/day [5, 111. The present cooperative randomized controlled study of immunotherapy with bestatin in combination with chemotherapy in adult patients with ANLL was conducted at 16 collaborating institutions [9].
Stratified random allocation Following the induction of complete remission and consolidation, at each institution patients were randomly allocated to the bestatin group or the control group after classifying patients on the basis of disease type (my elogenous or monocytic) and age (1549 or 50-64 yr). which can be considered factors relating to prognosis of ANLL [I, 61. Cases of myeloblastic leukemia and promyelocytic leukemia were combined and treated as myelogenous leukemia (AML) cases. Maintenance chemotherapy For remission maintenance chemotherapy, alternating therapy was carried out every 5 weeks with VEMP (1.4 mglm’ vincristine per week iv (days 1, 8, 15). 550 mg/m* cyclophosphamide per week iv (days 1, 8, 15), 70 mg/m* 6-mercaptopurine per day and 20 mg/m’ prednisolone per day) for 2 weeks and BHAC.DMP therapy for 2 weeks, or some other combination chemotherapy with similar activity. In addition, as a rule, 10 mg/m* methotrexate was injected twice intrathecally at the time of consolidation chemotherapy, and also once every 4 months thereafter. The neutrophil count did not drop below 500 mm3 and the nucleated bone marrOwcell count did not fall below 5 x 104/mm3. Immunotherapy with bestatin The patients allocated to the bestatin group were given bestatin concomitantly with the maintenance chemotherapy. One 30-mg bestatin capsule (Nippon Kayaku Co, Ltd) was administered orally each day before breakfast, and this regimen was generally continued for as long as possible, even after recurrence of leukemia (fig 2).
(_)-N-[(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl] L.-leucioe (M.W.: 308.38) Fig 1. Chemical structure of bestatin.
Materials and Methods Study group The study group consisted of adult patients aged between 15-65 yr who were diagnosed as ANLL and registered at the 16 institutions participating in this study during the period from July 1. 1980 to January 31, 1983. Remission induction therapy was given with combination chemotherapy, principally BHAGDMP, consisting of 170 mg/m2 per day, behenoyl cytosine arabinoside, 25 mg/m* per day daunorubicin (on days 1 and 2). 70 mg/m* 6-mercaptopurine per day and 20 mg/m* prednisolone per day for IO-15 days, or a similar active combination of BHAC DMP, etc. After complete remission was achieved, remission consolidation therapy consisting of 3 administrations of BHAC.DMP was given for 6 days.
Dropout uiteria The following criteria were established as the basis for defining dropout or exclusion cases; I), cases in which recurrence of the disease or death occurred, or followup study became impossible 2 months after initiating the study; 2). cases in which it was not possible to carry out the stipulated therapy: 3). cases judged to be unsuitable by the Evaluation Committee; and 4). cases in which death from reasons other than leukemia occurred. These cases were treated as discontinued cases, but were included in analyses. Following completion of the follow-up survey of aii patients, the Evaluation Committee, consisting of the principal investigators, the Controller statistical reviewer and 2 members, studied the eligibility of each patient. Analyses Based on the results of the follow-up survey carried out until March 31, 1985. the Kaplanmeier method was employed to calculate the remission and the survival rate for both treatment groups. The duration of remission was defined as the interval from the first day of induction of complete remission to the day when recurrence was diagnosed, or the day when observation of the case
Bestatin treatment in acute nonlymphocytic leukemia
Adult acute non-lymphocytic leukemia
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.Bestatingroup (Chemotherapy + Bestatin)
Combination
chemotherapy
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chemotherapy +
/
(3 courses>
(ho prior therapy)
Control group (Chemotherapy hl"W) ho response off study
Fig 2. Basic schema of protocol of randomized controlled study on chemoimmunotherapy for ANLL with bestatin. was discontinued. Similarly, the survival period was defined as the interval from the first day of therapy for induction of remission to the day on which the patient died or the day when observation of the case was discontinued. The survival period following recurrence was regarded as the period from the day on which recurrence was noted until the day on which the patient died, or the day when observation of the case was discontinued. Analyses of the remission rate and survival rate curves were carried out by the generalized Wilcoxon method (GW) and the Cox-Mantel method (CM). In addition, the distribution of the background factors was analyzed for biases by the 2’ test.
Results Composition of cases A total of 115 patients were registered in this study: 58 in the bestatin group and 57 in the control group. Of these, 7 patients (5 from the bestatin group and 2 from the control group) were excluded from the analysis because of non-compliance with the subject selection criteria: 1 case of lymphocytic leukemia; 2 cases re&tered after the reinduction of complete remission: 2 cases in which complete remission was not achieved; and 2 cases in which bestatin had been administered prior to registration. In addition, 7 cases (4 in the bestatin group and 3 in the control group) were judged to be non-evaluable dropout cases for the following reasons: in 4 cases, recurrence took place within 2 months after induction therapy (since this study was designed to compare only those cases with complete remission, cases of such early relapse were excluded, in 1 case follow-up could not be-performed during the early
period; and in 2 cases, maintenance therapy was not carried out as stipulated. In addition, although the subjects of this study were supposed to be under 65 yr of age, a 65yr-old patient was also included in the analysis. The subjects analyzed therefore totalled 101 patients: 48 in the bestatin group and 53 in the control group. Four patients requiring bone marrow transplantation or some other special form of therapy were treated as discontinued cases effective on the day the said therapy was begun and included in the analyses. Table I shows the various background factors for patients in the bestatin group and the control group: sex, age, type of leukemia, period of registration. and method of induction chemotherapy, consolidation and maintenance chemotherapy. No significant biases were detected between the 2 groups in terms of these background factors. Overall analysis The 50% remission duration was 21 months in the bestatin group and 12 months in the control group and long-term remission rate at 4 yr was 36.7% for the bestatin group and 24.1% for the control group. The bestatin group achieved prolongation of the remission duration compared with the control group (at P = 0. I 13 by GW and 0.060 by CM). The 50% survival time in the bestatin group was 33 months and 19 months in the control group, and long-term survival rate at 4 yr was 46.1% for the bestatin group and 25.9% for the control group. The bestatin group achieved a statistically significant prolongation of survival compared with the control group (at P = 0.020 by GW and 0.007 by CM) (fig 3).
K
96
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Ota, N Opawa
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Stratified analysis By leukemia type. The most common type of leukemia in these patients was AML; there were also a few cases of monocytic leukemia: 7 cases in the bestatin group and 13 cases in the control group. Therefore only the AML cases analyzed here. The 50% remission duration for the AML cases was 22 months in the bestatin group and 12 months in the control group. Long-term remission rate at 4 yr was 39.5% for the bestatin group and 25.1% for the control group. The bestatin group achieved prolongation of remission duration compared with the control group (P = 0.158 by GW and 0.072 by CM). The 50% survival time was over 55 months (did not reach 50%) for the bestatin group. and 18 months for the control group; long-term survival rate at 4 yr was 55.1% for the bestatin group compared with 24.4% for the control group. The bestatin group achieved a statistically significant prolongation of survival compared with the control group (P = 0.021 by GW and 0.003 by CM). By age. Patients were analyzed in relation to age group: 1549 yr and 50-65 yr. In the 15-49 yr
group, the 50% remission duration was 21 months for the bestatin group and 15 months for the cnntrol group, and long-term remission rate at 3.5 yr was 37.7% for the bestatin group and 27.0% for the control group; 50% survival time was 40 months for the bestatin group and 24 months for the control group, while the long-term survival rate at 3.5 yr was 46.9% for the bestatin group and 31.5% for the control group. There was no significant difference in remission duratioc (P = 0.489 by GW and 0. I39 by CM), but the difference in. survival time was significant (P = 0.265 by GW and 0.049 by CM) (fig 4). In the 50-65 yr group, the 50% remission duration was 22 months for the bestatin group, 10 months for the control group: the long-term remission rate at 4 yr was 35.6% for the bestatin group and 18.7% for the control group. The bestatin group achieved a significantly longer remission duration than the control group (P = 0.094 by GW and 0.044 by CM). The 50% survival time was 33 months for the bestatin group and 15 months for the control group. The long-term survival rate at 4 yr was 44.6% for the bestatin group and 15.8% for the control group. The bestatin group achieved a significantly longer survival than the control group (P = 0.017 by GW and 0.003 by CM) (fig 5).
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Remission duration and survival time in the chemoimmunotbapy ANLL with bcstatin.
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LL (age ; 50-65
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Bestatin treatmentin acute nonlymphocytic leukemia Remission rate and survival time after recurrence As of the follow-up survey day, recurrence was observed in 28 (58.3%) bestatin group cases and 39 (73.6%) control group cases. Remission was again induced in 15 (53.6%) of the bestatin group and 12 (30.8%) of the control group. The bestatin group tended to have a higher rate of reinduction of remission (P < 0.1). The 50% survival time after recurrence was 9 months for the bestatin group and 8 months for the control group. No statistically significant difference was found between the 2 groups in terms of survival curves after relapse (P = 0.259 by GW and 0.068 by CM).
An investigation into the safety of bestatin was carried out in 52 patients, including the cases treated as dropouts. Side-effects which could be attributed to bestatin were recorded in 5 of these patients, with an incidence of 9.6%. These sideeffects consisted of 2 cases (3.8%) of rash and itchiness, and 1 case of numbness (1.9%). However, itchiness disappeared while bestatin administration was continued in both cases, and the cause-effect relationship with the drug was not clear. All of the other symptoms disappeared when the bestatin therapy was di~ontinued. In addition, there were no abnormalities in the laboratory test values which could be considered as caused by the bestatin administration. Analysis ajfer bond-term~oliow-up After a follow-up period of 5 yr and 10 months following completion of the study, the 50% survival time in the bestatin group was 35 months and 19 months in the control group, and long-term survival rate of 5 yr was 36.1% for the bestatin group and 24.3% for the control group. The bestatin group achieved a statistically significant p~longation of survival compared with the control group (P = 0.034 by GW) (fig 6). The remission duration, however, was not significantly longer in the bestatin group than the control group (P = 0.150 by GW).
iscussion There have been a number of reports [3,7,8, 101 concerning the use of immunotherapy after attaining complete remission in cases of adult ANLL. However, no consensus has yet been reached with
99
regard to the efficacy of these immunotherapeutic approaches. Poweles et af [lo] carried out a study om the efficacy of immunotherapy consisting of administration of BCG and leukemic cells during remission maintenance therapy subsequent to induction of complete remission in cases of AML. They reported a prolongation of survival time, but not of remission duration. However, this was accompanied by an extension of survival time after recurrence, and the same authors speculated that immunotherapy must be able to stimulate bone marrow function surviving the chemotherapy administered following recurrence. In addition, Foon et al [3], analyzed 24 published reports involving use of immunotherapy with BCG, Corynebucterium, or leukemic cells as an adjuvant therapy in cases of AML. They found that patients who received a combination of immunotherapy with maintenance chemotherapy had no significant prolongation of remission duration over those who received only maintenance chemotherapy. But a significant survival advantage was detected in patients receiving immunotherapy and chemotherapy for maintenance therapy. The same investigators noted that immunotherapy did not lead to a prolongation of disease-free survival, and had no impact on the cure of patients with AML. Two similar studies of immunotherapy with a protein-bound polysaccharide preparation (PSK) and nocardia rubra cell-wall skeleton (N-CWS) for ANLL in adults were investigated in Japan [7, 81. These 3 studies were performed in similar institutions and for an almost similar period of time. The median duration of remission and survival of the control group was quite similar between PSK, N-CWS and the bestatin study group. This means that the credibility of these studies was extremely high. PSK and N-CUTS, however, did not achieve statistically significant prolongation of remission duration and survival compared with the control group (table II). It is known that the remission rate with chemotherapy and the prognosis of adults patients with ANLL differ according to the type of leukemia and the age of patients. The remission rate is lower and prognosis poorer in cases Of monocytic leukemia than in myelogenous leukemia, and in elderly patients as opposed to young patients. This was why the patients in the present study were analyzed for bestatin efficacy by stratifying them on the basis of type of
K Gta, N Ggawa
100 T&e
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Investigat4x Institmion No Eligible cases Duratiml of study ~Uow-up Remission induction
Remission maintenance Dropout cases
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study of chemoimmunotherapy of ANLL with PSK, N-CWS and beatatin. m = months. Resratin
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Ota 16
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Kiiura 12 67-36 (control)
31 (PSK) 3yr 1978.1~1981.9
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3yr f978.12-1981.12
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1984.6
1985.1
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BHAC.DMP
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DCMP + VEMP
BHAGDMP + VEMP
2i69 (3 96)
6l72 05 %)
7/108 (6 %)
Remission dfuWic3n {mm){medianj
Contml Immunotherapy Ratio Difference
10.1
14.5 x 1.4 NS
10.5
14.5 x 1.4 P = 0.08 (GW) Swvivaf
Control Immunotherapy Ratio Difference
22.7 20.2 x 0.9 NS
leukemia and age. There was nothing particular of note in the results in terms of the type of leukemia, but those in terms of the patient’s age were quite interesting. Thus, in the 15-49 yr age bracket the bestatin group and the control group showed no statistically significant differences in remission duration, whereas in the 50-65 yr age bracket the bestatin group showed a significant prolongation of both remission duration and survival time compared with the control group. These results can be inte~reted as indicating that in patients under 50 yr of age chemotherapy was effective, and the effect of concomitant use of immuno~er~py with bestatin was not clearly recognized, whereas in the 50-6s yr age group chemotherapy had a comparatively weak effect and the effect of immunotherapy with bestatin was thus more clearly recognized. It is generally agreed that immune activity is suppressed in elderly patients, and a recovery of suppressed immune activity has been reported in aged animals following bestatin treatment [2]. It is therefore
11.3 20.4 x 1.8 P = 0.06 (CM)
13.7 21.5 x 1.6 P=O.15 (GW)
18.1 33.0 x 1.8 P < 0.05 (CM, GW)
18.9 35.8 x 19 P = 0.03 (GW)
Im j (median j
19.4 25.0 x 1.3 NS
reasonable to assume that the effects of bestatin would be more clearly manifested in elderly compared with younger patients. In the present study, survival time was significantly longer in the bestatin group than in the control group in overall ANLL and AML. A prolongation of remission duration was also observed in the bestatin group, but the differences were borderline in nature. The differences in survival were more convincing than the differences in remission duration. The reason might be the enhancement of defence mechanisms by bestatin against infections or other complications, but there is still no such evidence. However, the present investigators consider that there was a dilution in the overall effect of bestatin on the prolongation of remission duration in the SO65 yr age group, because of the small effect in the 15-49 age group for the above-mentioned reason. Another ~sibili~ might be the tendency towards a higher mmission reinduction rate after recurrence in the bestatin group than in the control group.
Bestatin treatment in acute nonlymphocytic leukemia The prolongation of remission duration and survival-time seen in the bestatin group, especially in the etderly patients, suggest that bestatin is effective in suppressing the small number of leukemic cells which remain even after complete remission induction and subsequent consolidation chemotherapy. On the basis of the above findings, it is concluded that bestatin is a clinically useful drug for the t~atment of adult patients with ANLL, since it has been shown to prolong survival time and remission duration especially in elderly patients and has few side-effects.
5 Majima H, Suzuki M, Nakano M (1980) Clinical experience of bestatin-preliminary report. Jpn J Cancer Chemother 7, 89 6
7
8
References 1 Brandman J. Bulowski RM. Greenstreet R. Hewlett JS, Hoffman GC (197b) Prognostic faci tars affecting remission, remission duration, and survival in adult acute nonlymphocytic leukemia. Cancer 44, 1062 Bruley-Rosset M, Frorentin I, Kiger N, Schulz J, Math6 G (1979) Resoration of impaired immune functions of aged animals by chronic bestatin treatment. immttnoiog~ 38, 75 Fosn KA, Smalley RV, Riggs CW, Gale RP (19831 The role of immunotherapy in acute myelogenous leukemia. Arch Intern Med 143, 1726 Ishizuka M, Masuda T, Kanbayashi N. Fukasawa S, Takeuchi T, Aoyagi T, Umezawa H (1980) Effect of bestatin on mouse immune system and ex~rimenta1 murine tumors. J A~rj~iur (Tokyo) 33, 642
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Me~elsm~n R, Thaler HT. To L. Gee TS, McKenzie S, Schauer P, Friedman A, Arlin 2. Cirrincione C, Clarkson B (1980) Morphological classification, response to therapy, and survival in 263 adult patients with acute nonlymphoblastic leukemia. Blood 56, 773 Ohno R. Yamada K, Masaoka T, ~~hirna T, Amaki I (f984) A randomized trial of chemoimmunotherapy of acute nonlymphocytic leukemia in adults using a protein-bound polysaccharide preparation. Cancer Immrmol Immunother 18, 149 Ohno R, NakamuraH, Kodera Y, Ezaki K. Yokomaku S (1986) Randomized controlled study of chemoimmunother~y of acute myelog~ous leukemia (AML.1 in adults with Nocardia rubra celt-wall skeleton and irradiated allogeneic AML cells. Comer 57. 1483 Ota K, Kurita S, Yamada K, Masaoka T. Uzuka Y, Ogawa N (1986) Immunotherapy with bestatin for acute nonlymphocytic leukemia in adult. Cancer Inz#~;tnoiZmrn~in~t~er23, 5 Powles RL, Russell J, Lister TA, Oliver T. Whitehouse JM, Malpas J, Chapuis B, Crowther D, Atexander P (1977) Immunotherapyfor acute myelogenous leukemia: a controlled clinical study two and a half years after entry of the last patient. Br J Cuneer 35,
265 Saito K, Miyasato H, Tajima K, Ikeda S (1983) Phase I study of bestatin (1) A clinical study on determination of an optimal dose of bestatin. Jpr! I Cancer Chemother 10 (2) Part I, 21 I 12 Umezawa H, Aoyagi T, Suda H, Hamada M. Takeu-
11
chi T (1976) Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes. J A~tjb~ot {Tokyo) 29, 97
K Ota I’, N Ogawa * ’ Nagoya Memorial Hospital, 4-305 Hirabari. Tenpaku-ku.Nagoya 468; ’ Pharmacological ?nstitute, Ehime University School of Medicine, Ehime 791-02. Japan (Received 5 February1990; accepted 12 February1990)
Summary - A new immunomodulating agent, bestatin (INN: Ubenimex has low toxicity even after long-term oral administration and has significant modifications in immunological response. A cooperative randomized controlled study of bestatin immunotherapy in combination with remission maintenance chemotherapy for adult acute noniymphocytic leukemia (ANLL) was performed. After induction of complete remission. patients were randomized to the bestatin group (30 mglbw per OS (PO) daily) and the control group. The 101 eligible cases (best&n: 48, control: 53) were analyzed; the bestatin group achieved longer remission than the control group and a statistically Qnificant longer survival. Though this prolongation of remission was not significant in the bestatin group compared to the cu#rol group in the 15-49 yr age group, in the SO-65 yr age group it was significantly longer. Bestatin is shown to be a clinically zsefui drug for immunotherapy of adult ANLL, since it has prolonged survival and remission especially in elderly patients, wr:h few side-effects. bestatin / immunotherapy / acute leukemia R&urn4 - 6:tude contr6Me et randomisbe de la chimio-immunotherapie de la leuc6mie aigue non-lympbocytaire chez I’adulte par la bestatine. Un nouvel agent immuno-modulant, la bestatine (DCI : Ubenimex) se montre mains toxique que les autres mltme aprPs une administration prolongde per OS.Ce mPdicament provoque des modifications significatives dons la rPponse immunitaire. Une Ctude coop&rative randomisee et contr6lJe a PtPeffect&e sur l’immunoth&apie par la bestatine en association avec une chimiotht+apie d’entretien aprPs remission dans la leu&mie aiguL;non-lymphocytaire de I’adulte (ANLL). Apt& induction Gune remission complPte, les malades ont et&rdpartis par randomisation entre un groupe bezatine (recevant 30 mglkg-t-tj-’ de bestatine per OS) et un groupe Gmoin. Les 101 cas ligibles (48 “bestatine” et 53 Mmoins) ont &! analysps. Le groupe bestatine a Wn@icie’ d’une r&missionplus longue que le groupe tPmoin ainsi que d’une survie significativement plus longue. L’allongement de la &mission n’itait pas statistiquement significatif par rapport au groupe tPmoin dans la tranche IS- 49 ans mais il I’Ptait dans la tranche SO- 65 ans. La bestatine est ainsi prt!sentJe comme un medicament utile en clinique dans l’immunothtrapie de I’ANLL de l’adulte compte-tenu du fait qu’elle prolonge survie et remission, particuli$rement cher les sujets a&S et cela avec peu seffets secondaires. bestatine I immunothdrapic I leuc&tie aigue
Introduction As a result of recent progress in chemotherapy, adult patients with acute nonlymphocytic leukemia (ANLL) can now be expected to show a complete remission rate of over 60% and a long* Correspondenceand reprints
term survival rate of approximately 30%. In order to eradicate minimal residual leukemic cells after z.chievement of complete remission, various methods for enhancing cure rate in adult ANLL have been attempted (eg, immunotherapy and bone marrow transplantation). This immunotherapy has most commonly consisted of bacterial preparations such as bacillus Calmette-Guerin (BCG), or homologous leukemic cells. There has
94
K Ota, N Ogawa
been much debate concerning the efficacy of these forms of immunotherapy. Bestatin (INN: ubenimex) is a small molecular weight dipeptide. N-[(2S, 3R)-3-amino-2-2hydroxy_4_phenylbutanoyl] L-leucine, discovered by Umezawa et 01 [12] in the culture filtrate of Streptomyces ofivoreticufi (fig 1). Fundamental studies have revealed that bestatin has low toxicity even after long-term oral administration, with the modulating activity of cellular immunity possibly via its binding to aminopeptidases located on the cell surface of immune cells, and host-mediated antitumor activity against murine tumors [4]. In a Phase I study, the safety of orally administered besiatin was confirmed, and it was suggested that the appropriate dosage level of this substance was in the range of 30-100 mg/day [5, 111. The present cooperative randomized controlled study of immunotherapy with bestatin in combination with chemotherapy in adult patients with ANLL was conducted at 16 collaborating institutions [9].
Stratified random allocation Following the induction of complete remission and consolidation, at each institution patients were randomly allocated to the bestatin group or the control group after classifying patients on the basis of disease type (my elogenous or monocytic) and age (1549 or 50-64 yr). which can be considered factors relating to prognosis of ANLL [I, 61. Cases of myeloblastic leukemia and promyelocytic leukemia were combined and treated as myelogenous leukemia (AML) cases. Maintenance chemotherapy For remission maintenance chemotherapy, alternating therapy was carried out every 5 weeks with VEMP (1.4 mglm’ vincristine per week iv (days 1, 8, 15). 550 mg/m* cyclophosphamide per week iv (days 1, 8, 15), 70 mg/m* 6-mercaptopurine per day and 20 mg/m’ prednisolone per day) for 2 weeks and BHAC.DMP therapy for 2 weeks, or some other combination chemotherapy with similar activity. In addition, as a rule, 10 mg/m* methotrexate was injected twice intrathecally at the time of consolidation chemotherapy, and also once every 4 months thereafter. The neutrophil count did not drop below 500 mm3 and the nucleated bone marrOwcell count did not fall below 5 x 104/mm3. Immunotherapy with bestatin The patients allocated to the bestatin group were given bestatin concomitantly with the maintenance chemotherapy. One 30-mg bestatin capsule (Nippon Kayaku Co, Ltd) was administered orally each day before breakfast, and this regimen was generally continued for as long as possible, even after recurrence of leukemia (fig 2).
(_)-N-[(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl] L.-leucioe (M.W.: 308.38) Fig 1. Chemical structure of bestatin.
Materials and Methods Study group The study group consisted of adult patients aged between 15-65 yr who were diagnosed as ANLL and registered at the 16 institutions participating in this study during the period from July 1. 1980 to January 31, 1983. Remission induction therapy was given with combination chemotherapy, principally BHAGDMP, consisting of 170 mg/m2 per day, behenoyl cytosine arabinoside, 25 mg/m* per day daunorubicin (on days 1 and 2). 70 mg/m* 6-mercaptopurine per day and 20 mg/m* prednisolone per day for IO-15 days, or a similar active combination of BHAC DMP, etc. After complete remission was achieved, remission consolidation therapy consisting of 3 administrations of BHAC.DMP was given for 6 days.
Dropout uiteria The following criteria were established as the basis for defining dropout or exclusion cases; I), cases in which recurrence of the disease or death occurred, or followup study became impossible 2 months after initiating the study; 2). cases in which it was not possible to carry out the stipulated therapy: 3). cases judged to be unsuitable by the Evaluation Committee; and 4). cases in which death from reasons other than leukemia occurred. These cases were treated as discontinued cases, but were included in analyses. Following completion of the follow-up survey of aii patients, the Evaluation Committee, consisting of the principal investigators, the Controller statistical reviewer and 2 members, studied the eligibility of each patient. Analyses Based on the results of the follow-up survey carried out until March 31, 1985. the Kaplanmeier method was employed to calculate the remission and the survival rate for both treatment groups. The duration of remission was defined as the interval from the first day of induction of complete remission to the day when recurrence was diagnosed, or the day when observation of the case
Bestatin treatment in acute nonlymphocytic leukemia
Adult acute non-lymphocytic leukemia
Combination --)
.Bestatingroup (Chemotherapy + Bestatin)
Combination
chemotherapy
+
(2 or 3 courses>
chemotherapy +
/
(3 courses>
(ho prior therapy)
Control group (Chemotherapy hl"W) ho response off study
Fig 2. Basic schema of protocol of randomized controlled study on chemoimmunotherapy for ANLL with bestatin. was discontinued. Similarly, the survival period was defined as the interval from the first day of therapy for induction of remission to the day on which the patient died or the day when observation of the case was discontinued. The survival period following recurrence was regarded as the period from the day on which recurrence was noted until the day on which the patient died, or the day when observation of the case was discontinued. Analyses of the remission rate and survival rate curves were carried out by the generalized Wilcoxon method (GW) and the Cox-Mantel method (CM). In addition, the distribution of the background factors was analyzed for biases by the 2’ test.
Results Composition of cases A total of 115 patients were registered in this study: 58 in the bestatin group and 57 in the control group. Of these, 7 patients (5 from the bestatin group and 2 from the control group) were excluded from the analysis because of non-compliance with the subject selection criteria: 1 case of lymphocytic leukemia; 2 cases re&tered after the reinduction of complete remission: 2 cases in which complete remission was not achieved; and 2 cases in which bestatin had been administered prior to registration. In addition, 7 cases (4 in the bestatin group and 3 in the control group) were judged to be non-evaluable dropout cases for the following reasons: in 4 cases, recurrence took place within 2 months after induction therapy (since this study was designed to compare only those cases with complete remission, cases of such early relapse were excluded, in 1 case follow-up could not be-performed during the early
period; and in 2 cases, maintenance therapy was not carried out as stipulated. In addition, although the subjects of this study were supposed to be under 65 yr of age, a 65yr-old patient was also included in the analysis. The subjects analyzed therefore totalled 101 patients: 48 in the bestatin group and 53 in the control group. Four patients requiring bone marrow transplantation or some other special form of therapy were treated as discontinued cases effective on the day the said therapy was begun and included in the analyses. Table I shows the various background factors for patients in the bestatin group and the control group: sex, age, type of leukemia, period of registration. and method of induction chemotherapy, consolidation and maintenance chemotherapy. No significant biases were detected between the 2 groups in terms of these background factors. Overall analysis The 50% remission duration was 21 months in the bestatin group and 12 months in the control group and long-term remission rate at 4 yr was 36.7% for the bestatin group and 24.1% for the control group. The bestatin group achieved prolongation of the remission duration compared with the control group (at P = 0. I 13 by GW and 0.060 by CM). The 50% survival time in the bestatin group was 33 months and 19 months in the control group, and long-term survival rate at 4 yr was 46.1% for the bestatin group and 25.9% for the control group. The bestatin group achieved a statistically significant prolongation of survival compared with the control group (at P = 0.020 by GW and 0.007 by CM) (fig 3).
K
96
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I
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23
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Ota, N Opawa
23
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laintenance
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1
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12
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1
Stratified analysis By leukemia type. The most common type of leukemia in these patients was AML; there were also a few cases of monocytic leukemia: 7 cases in the bestatin group and 13 cases in the control group. Therefore only the AML cases analyzed here. The 50% remission duration for the AML cases was 22 months in the bestatin group and 12 months in the control group. Long-term remission rate at 4 yr was 39.5% for the bestatin group and 25.1% for the control group. The bestatin group achieved prolongation of remission duration compared with the control group (P = 0.158 by GW and 0.072 by CM). The 50% survival time was over 55 months (did not reach 50%) for the bestatin group. and 18 months for the control group; long-term survival rate at 4 yr was 55.1% for the bestatin group compared with 24.4% for the control group. The bestatin group achieved a statistically significant prolongation of survival compared with the control group (P = 0.021 by GW and 0.003 by CM). By age. Patients were analyzed in relation to age group: 1549 yr and 50-65 yr. In the 15-49 yr
group, the 50% remission duration was 21 months for the bestatin group and 15 months for the cnntrol group, and long-term remission rate at 3.5 yr was 37.7% for the bestatin group and 27.0% for the control group; 50% survival time was 40 months for the bestatin group and 24 months for the control group, while the long-term survival rate at 3.5 yr was 46.9% for the bestatin group and 31.5% for the control group. There was no significant difference in remission duratioc (P = 0.489 by GW and 0. I39 by CM), but the difference in. survival time was significant (P = 0.265 by GW and 0.049 by CM) (fig 4). In the 50-65 yr group, the 50% remission duration was 22 months for the bestatin group, 10 months for the control group: the long-term remission rate at 4 yr was 35.6% for the bestatin group and 18.7% for the control group. The bestatin group achieved a significantly longer remission duration than the control group (P = 0.094 by GW and 0.044 by CM). The 50% survival time was 33 months for the bestatin group and 15 months for the control group. The long-term survival rate at 4 yr was 44.6% for the bestatin group and 15.8% for the control group. The bestatin group achieved a significantly longer survival than the control group (P = 0.017 by GW and 0.003 by CM) (fig 5).
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Remission duration and survival time in the chemoimmunotherapy of ANLL in the age group IS-49 years with bestatin.
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Remission duration and survival time in the chemoimmunotbapy ANLL with bcstatin.
ti
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Remission duration and survival time in the chemoimmuaotherapy of ANLL in the age group SO-65 years with bestatin.
12
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6
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LL (age ; 50-65
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Survival time in the chemoimmunotherapy of ANLL with bestatin after a fol-
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low-up period of 5 years and IO months following completion of the study.
Fig 6.
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Bestatin treatmentin acute nonlymphocytic leukemia Remission rate and survival time after recurrence As of the follow-up survey day, recurrence was observed in 28 (58.3%) bestatin group cases and 39 (73.6%) control group cases. Remission was again induced in 15 (53.6%) of the bestatin group and 12 (30.8%) of the control group. The bestatin group tended to have a higher rate of reinduction of remission (P < 0.1). The 50% survival time after recurrence was 9 months for the bestatin group and 8 months for the control group. No statistically significant difference was found between the 2 groups in terms of survival curves after relapse (P = 0.259 by GW and 0.068 by CM).
An investigation into the safety of bestatin was carried out in 52 patients, including the cases treated as dropouts. Side-effects which could be attributed to bestatin were recorded in 5 of these patients, with an incidence of 9.6%. These sideeffects consisted of 2 cases (3.8%) of rash and itchiness, and 1 case of numbness (1.9%). However, itchiness disappeared while bestatin administration was continued in both cases, and the cause-effect relationship with the drug was not clear. All of the other symptoms disappeared when the bestatin therapy was di~ontinued. In addition, there were no abnormalities in the laboratory test values which could be considered as caused by the bestatin administration. Analysis ajfer bond-term~oliow-up After a follow-up period of 5 yr and 10 months following completion of the study, the 50% survival time in the bestatin group was 35 months and 19 months in the control group, and long-term survival rate of 5 yr was 36.1% for the bestatin group and 24.3% for the control group. The bestatin group achieved a statistically significant p~longation of survival compared with the control group (P = 0.034 by GW) (fig 6). The remission duration, however, was not significantly longer in the bestatin group than the control group (P = 0.150 by GW).
iscussion There have been a number of reports [3,7,8, 101 concerning the use of immunotherapy after attaining complete remission in cases of adult ANLL. However, no consensus has yet been reached with
99
regard to the efficacy of these immunotherapeutic approaches. Poweles et af [lo] carried out a study om the efficacy of immunotherapy consisting of administration of BCG and leukemic cells during remission maintenance therapy subsequent to induction of complete remission in cases of AML. They reported a prolongation of survival time, but not of remission duration. However, this was accompanied by an extension of survival time after recurrence, and the same authors speculated that immunotherapy must be able to stimulate bone marrow function surviving the chemotherapy administered following recurrence. In addition, Foon et al [3], analyzed 24 published reports involving use of immunotherapy with BCG, Corynebucterium, or leukemic cells as an adjuvant therapy in cases of AML. They found that patients who received a combination of immunotherapy with maintenance chemotherapy had no significant prolongation of remission duration over those who received only maintenance chemotherapy. But a significant survival advantage was detected in patients receiving immunotherapy and chemotherapy for maintenance therapy. The same investigators noted that immunotherapy did not lead to a prolongation of disease-free survival, and had no impact on the cure of patients with AML. Two similar studies of immunotherapy with a protein-bound polysaccharide preparation (PSK) and nocardia rubra cell-wall skeleton (N-CWS) for ANLL in adults were investigated in Japan [7, 81. These 3 studies were performed in similar institutions and for an almost similar period of time. The median duration of remission and survival of the control group was quite similar between PSK, N-CWS and the bestatin study group. This means that the credibility of these studies was extremely high. PSK and N-CUTS, however, did not achieve statistically significant prolongation of remission duration and survival compared with the control group (table II). It is known that the remission rate with chemotherapy and the prognosis of adults patients with ANLL differ according to the type of leukemia and the age of patients. The remission rate is lower and prognosis poorer in cases Of monocytic leukemia than in myelogenous leukemia, and in elderly patients as opposed to young patients. This was why the patients in the present study were analyzed for bestatin efficacy by stratifying them on the basis of type of
K Gta, N Ggawa
100 T&e
II. ~~ornizeel fmmrmotherapy
Investigat4x Institmion No Eligible cases Duratiml of study ~Uow-up Remission induction
Remission maintenance Dropout cases
conwilled
study of chemoimmunotherapy of ANLL with PSK, N-CWS and beatatin. m = months. Resratin
N-cws
PX
Ota 16
Yamada 2 66-34 (control) 32 (N-CWS)
Kiiura 12 67-36 (control)
31 (PSK) 3yr 1978.1~1981.9
101-53 (control) 48 (beststin) 2.5 yr 1980.7-1983.1
3yr f978.12-1981.12
1983.9 2-5 yr
1984.6
1985.1
2yr6m-5yr6m
1988.11 5yrlOm-8yr4m
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DCMP~two-stepor BHAC-DMP
DCMP*two-stepor BHAC-DMP
BHAC.DMP
DCMP+VEMP
DCMP + VEMP
BHAGDMP + VEMP
2i69 (3 96)
6l72 05 %)
7/108 (6 %)
Remission dfuWic3n {mm){medianj
Contml Immunotherapy Ratio Difference
10.1
14.5 x 1.4 NS
10.5
14.5 x 1.4 P = 0.08 (GW) Swvivaf
Control Immunotherapy Ratio Difference
22.7 20.2 x 0.9 NS
leukemia and age. There was nothing particular of note in the results in terms of the type of leukemia, but those in terms of the patient’s age were quite interesting. Thus, in the 15-49 yr age bracket the bestatin group and the control group showed no statistically significant differences in remission duration, whereas in the 50-65 yr age bracket the bestatin group showed a significant prolongation of both remission duration and survival time compared with the control group. These results can be inte~reted as indicating that in patients under 50 yr of age chemotherapy was effective, and the effect of concomitant use of immuno~er~py with bestatin was not clearly recognized, whereas in the 50-6s yr age group chemotherapy had a comparatively weak effect and the effect of immunotherapy with bestatin was thus more clearly recognized. It is generally agreed that immune activity is suppressed in elderly patients, and a recovery of suppressed immune activity has been reported in aged animals following bestatin treatment [2]. It is therefore
11.3 20.4 x 1.8 P = 0.06 (CM)
13.7 21.5 x 1.6 P=O.15 (GW)
18.1 33.0 x 1.8 P < 0.05 (CM, GW)
18.9 35.8 x 19 P = 0.03 (GW)
Im j (median j
19.4 25.0 x 1.3 NS
reasonable to assume that the effects of bestatin would be more clearly manifested in elderly compared with younger patients. In the present study, survival time was significantly longer in the bestatin group than in the control group in overall ANLL and AML. A prolongation of remission duration was also observed in the bestatin group, but the differences were borderline in nature. The differences in survival were more convincing than the differences in remission duration. The reason might be the enhancement of defence mechanisms by bestatin against infections or other complications, but there is still no such evidence. However, the present investigators consider that there was a dilution in the overall effect of bestatin on the prolongation of remission duration in the SO65 yr age group, because of the small effect in the 15-49 age group for the above-mentioned reason. Another ~sibili~ might be the tendency towards a higher mmission reinduction rate after recurrence in the bestatin group than in the control group.
Bestatin treatment in acute nonlymphocytic leukemia The prolongation of remission duration and survival-time seen in the bestatin group, especially in the etderly patients, suggest that bestatin is effective in suppressing the small number of leukemic cells which remain even after complete remission induction and subsequent consolidation chemotherapy. On the basis of the above findings, it is concluded that bestatin is a clinically useful drug for the t~atment of adult patients with ANLL, since it has been shown to prolong survival time and remission duration especially in elderly patients and has few side-effects.
5 Majima H, Suzuki M, Nakano M (1980) Clinical experience of bestatin-preliminary report. Jpn J Cancer Chemother 7, 89 6
7
8
References 1 Brandman J. Bulowski RM. Greenstreet R. Hewlett JS, Hoffman GC (197b) Prognostic faci tars affecting remission, remission duration, and survival in adult acute nonlymphocytic leukemia. Cancer 44, 1062 Bruley-Rosset M, Frorentin I, Kiger N, Schulz J, Math6 G (1979) Resoration of impaired immune functions of aged animals by chronic bestatin treatment. immttnoiog~ 38, 75 Fosn KA, Smalley RV, Riggs CW, Gale RP (19831 The role of immunotherapy in acute myelogenous leukemia. Arch Intern Med 143, 1726 Ishizuka M, Masuda T, Kanbayashi N. Fukasawa S, Takeuchi T, Aoyagi T, Umezawa H (1980) Effect of bestatin on mouse immune system and ex~rimenta1 murine tumors. J A~rj~iur (Tokyo) 33, 642
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Me~elsm~n R, Thaler HT. To L. Gee TS, McKenzie S, Schauer P, Friedman A, Arlin 2. Cirrincione C, Clarkson B (1980) Morphological classification, response to therapy, and survival in 263 adult patients with acute nonlymphoblastic leukemia. Blood 56, 773 Ohno R. Yamada K, Masaoka T, ~~hirna T, Amaki I (f984) A randomized trial of chemoimmunotherapy of acute nonlymphocytic leukemia in adults using a protein-bound polysaccharide preparation. Cancer Immrmol Immunother 18, 149 Ohno R, NakamuraH, Kodera Y, Ezaki K. Yokomaku S (1986) Randomized controlled study of chemoimmunother~y of acute myelog~ous leukemia (AML.1 in adults with Nocardia rubra celt-wall skeleton and irradiated allogeneic AML cells. Comer 57. 1483 Ota K, Kurita S, Yamada K, Masaoka T. Uzuka Y, Ogawa N (1986) Immunotherapy with bestatin for acute nonlymphocytic leukemia in adult. Cancer Inz#~;tnoiZmrn~in~t~er23, 5 Powles RL, Russell J, Lister TA, Oliver T. Whitehouse JM, Malpas J, Chapuis B, Crowther D, Atexander P (1977) Immunotherapyfor acute myelogenous leukemia: a controlled clinical study two and a half years after entry of the last patient. Br J Cuneer 35,
265 Saito K, Miyasato H, Tajima K, Ikeda S (1983) Phase I study of bestatin (1) A clinical study on determination of an optimal dose of bestatin. Jpr! I Cancer Chemother 10 (2) Part I, 21 I 12 Umezawa H, Aoyagi T, Suda H, Hamada M. Takeu-
11
chi T (1976) Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes. J A~tjb~ot {Tokyo) 29, 97
