Biomed& Fharmacother (1991) 45.87-93 0 Elsevier,Paris
87
estatin tr~atmeut of mye~ody$p~a§t~c and chronic myelogenou~ leuke Y Usuka, Y Saito The Third department of Internat medicine, Tohoku ~nive~i~ School of ~~i~~ne,
se&i,
Japan
Summary - A high remission rate (56%) was achieved in a preliminary study using Bestatin in patients with myelodysplastic syndromes. In particular, 9 out of 13 patients (69%) in the high blast group achieved hematologic remission. After Bestatin treafment, int~nsic ~e~~to~ietic stem cell ~b~u~aIities as wet1 as hemato~ogic ending were markedty improved. The success of Bestatin therapy in MDS led us to investigate the clinical activity of Bestatin in CML. In the current study the busulfan and Bestatin combination therapy resulted in complete hematologic remission in all of the patients. The most exciting result was the suppression of the Philadelphia chromosomes among the responding patients. Complete cytogenetic response was obtained in 3 patients (21%), partial cytagenetic response in 1 (7%). and minor cytogenetic response in 5 (36%). in particular, the majority of early chronic phase CML patients achieved significant cytogenetic response with sustained Pb’ negativity. The results are very enco~aging and warrant funher studies. myelodysplastic syndromes / chronic myelogenous leukemia I Bestatin biotherapy
R&u?n4 - Traitement par la Bestatlne des syndromes my4lodysplasiques et de la leudmie my4loide chronique. in taux &lev&de r~n~issionsa &e’obtenu dans une &de pr~~i~in~ire utilisant lo Bestatine chez les malades atteints de syndromes my~lodyspiasiques (SMD). En ~rticulier, 9 malades sur les 13 169%) du group li btastes nombreux sunt arriv&s li la r6mission h~matologique. AprPs un traitement par la Eestatine, &esanomalies intrins&ques de la celhde souche h&matopoUtique. ainsi que les rPsultats h&atologiques ont et6 nettement ame’lior&s.L’efficacitci de la Bestatine &ns les SMD a conduit les auteurs ri Qudier en clinique l’activite de la Bestatine au tours de la leuc&mie myt!loiile chronique (CML). Dans la prisente &tude, le traitement par I’association busulfan-Bestatine a eu pour rPsultat une rPmission h&nato~ogique con~p~~techet tous les maiades. Le r&&at le plus re~rq~b~e a it& ia d~s~rition du chromosome Ph~~~elphie chez les malades r&mdeurs. Une r&ponse con&&e sur la cytoge&e a &t! obtenue ckez 3 malades ~~~%), une Gponse partieile chez I seul (7%) et une rhponse mineure chez 5 (36%). En parriculier. la majorit& des malades atteints de LMC en dPbut de phase clrronique ont eu une riponse significative sur la cytogendse avec n&ativation maintenue du Ph’. Ces r&dtats sont tr2s encourageants et mCritent la poursuite des recherches dons ce domaine. syndrome my~lodysplasique I leuc&nie cbronique my&oide I biotitetapie B la Bestrtine
Introduction Myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) are clonal hemopathies that occur as a result of malignant transformation of a pluripotent hematopoietic stem cell. Early in the course of these diseases, the progeny of the malignant clone retains some degree of differentiation and normal function. These “premalignant” characteristics [6] of MDS and CML in the chronic phase impart therapeutic
treatment through biotherapy. These hematologic malignancies involve a variety of different cellular mechanisms including the activation of protooncogene and the release of growth factors. Accordingly, the use of biological ~tigrowth factor such as Bestatin would logically provide a new approach to these diseases. Recently we have obtained significant improvement by using Bestatin in both MDS and CML. We describe here a new app~ach to treating these diseases using this drug.
Y Us&a, Y Saito
88
yelodysplastic syndromes
fective hematopoiesis therefore studied.
Primary myelodysplastic syndrome (MDS) consists of a heterogenous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis with various combinations of anemia, leukopenia and thrombocytopenia. The treatment of MDS is dictated not only by the risk of evolving acute leukemia but also by the risk of infection and bleeding which result in a fatal outcome in the majority of patients. The lesions underlying this defect reside mainly at the level of the hematopoietic stem cells. Furthermore, recent studies have suggested that abnormalities exist in the distribution of T-cell subsets and their functions in MDS [3, 51. As T-cells contribute to the immunoregulation of inducible hematopoiesis, abnormalities in their numbers and functions are believed to play a pathophysiologic role in MDS. Advances in understanding the complexity of factors that govern the process of hematopoiesis have furthered our understanding of the biological abnormalities that underlie MDS and have provided new insight into therapeutic intervention. This report discusses a new attempt to treat MDS utilizing Bestatin (N- ((2s. 3R)-3-amino-2-hydroxy4-phenylbutanoyl J-~leucine), a small molecular immuno-modifier developed by Umezawa et al 1141. The mechanisms of Bestatin action on de-
in this
syndrome
were
Patients and Methods Sixteen patients with MDS included in this study had a median age of 68 yr (range 49-83 yr). They were diagnosed and classified according to the FAB classification [l]. Nine patients had refractory anemia with excess of blasts (RAEB), 4 patients RAEB in transformation (RAEB-t), 2 refractory anemia (RA), and one had chronic myelomonocytic leukemia (CMML). Bestatin was administered orally at a dose of 30 mg per day for 1 month or more. Complete response was defined as the normalization of peripheral and bone marrow findings, and the disappearance of dysplastic changes in hematopoietic cells. Partial response was defined as a reduction in the blast count in the peripheral blood and marrow. and an increase in peripheral blood counts, ie platelets to > 50 x lo9 per liter granulocytes to > 2 x IO9 per liter, and PCV > 30, which could be maintained without transfusion for a period of > 2 months.
Results The results of Bestatin treatment in MDS patients are shown in table I. Two of the 4 patients with
Table 1. Outcomes in Bestatin-treated MDS patients. CR, complete remission; PR, partial remission; F. failure. Bestatin Patients
Age (yr), scs
I
49 M
2 3
65 M 83 M
4 5 6 7 8 9 IO II 12 13 14 15 16
73 61 73 55 67 52 73 73 59 68 68 59 70
M M F M M M F F M M F M M
Diagnosis
RAEB-T RAEB-T RAEB-T - AMoL RAEB-T RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RA RA CMML
Dose
Duration
Imsld)
(dl
Effects
Days required for response
Remission duration W)
Survival (months)
30 30 30
128 273 46
CR F F
12
14
9 12+ 3
30 30 30 30 30 30 30 30 30 30 30 30 30
147+ 518 93 413 174 986+ 379 253 249+ 98 74 1028+ 50
CR CR F PR PR PR PR PR CR F F F F
16 57
ll+ 13+
22 22 65 141 123 121
49+ 8 132+ 7 127a 13+
5+ 18+ 30+ 14+ 22+ 33+ 34+ 34+ lO+ 36+ 28+ 35+ 4
Bestatin treatment of MDS and CML RAEB-t and 2 of 9 patients with RAEB achieved complete response (total complete response rate 4116 = 25%). Five patients with RAEB achieved partial response. The total response rate was 56%. The time required for response was 12 to 141 d with a median of 57 d. The duration of remission ranged from c 1I to + 132 wk. All patients except one whose condition evolved to overt acute leukemia are still alive (median follow-up period of 31 months). No side-effects were observed in any case.
Discussion In the current study, Bestatin therapy produced a complete response rate of 25% and a partial response rate of 31%. To elucidate the mechanisms of Bestatin action on normal and defective hematopoiesis, bone marrow cells from normal volunteers and patients with MDS were cultured by CFU-GEMM assay. The details of the experiments have been reported elsewhere [15] and are not presented here. Clonogenic marrow cell culture studies in patients with MDS have demonstrated intrinsic hematopoietic stem cell abnorerythroid particular, and malities. In erythroid-mixed colony formation were found to be defective in all patients. Moreover, it was suggested that the malignancy involves a variety of different cellular mechanisms, including the activation of protooncogenes and the release of growth factors, After Bestatin therapy, these abnormalities as well as hematologic findings were markedly improved. The use of a biological antigrowth factor such as Bestatin, therefore, is likely to provide a promising approach to MDS. However, the precise mechanisms of action of Bestatin are difficult to determine, particularly in a clinical setting, and further studies are necessary to elucidate its mechanisms of action.
Chronic myelogenous
leukemia
Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation between ch~mosome 9 and 22 { t(9;22)(q3~qll) ], or Philadelphia chromosome (Ph’). Until now a variety of therapeutic trials have been employed without significant success to alter the natural history of the disease and to delay the blastic phase
89
]81. Allogenic bone marrow tr~splantation provided the first breakthrough and is currently the only curative treatment for CML. However, its application is limited to a minority of cases. Since 1981 141, recombinant alpha interferon has been investigated as a possible therapy for CML. Interferon therapy, pioneered by Talpaz et al [2, 7, 9-131, has had the greatest impact on the treatment of CML. Although the effects of recombinant interferon alpha may be of significant clinical importance, its role in the overall treatment of the disease remains to be determined. In this report, we propose a new approach for treating patients with CML using a combination therapy of busulfan and Bestatin, and evaluate the clinical effects and cytogenetic changes associated with the therapy. As it is still too early for the results of the therapy in terms of survival to be estimated, the interim results of ongoing trials and an account of the clinical effects of Bestatin treatment will be presented.
Patients and Methods The clinical characteristics of 14 patients with Phi-positive CML treated with busulfan and Bestatin in combi-
nation and of 25 patients with CML treated with busulfan alone are summarized in table II. Ten patients in the chronic phase and 4 in the accelerated phase with cytogenetic clonal evolution were treated with the combination therapy. All pretreatment was te~inated at least 4 wk before initiating combination therapy. The median age of the patients was 57 yr (range 22-73 yr). The median time from the diagnosis to the commencement of the combination therapy was 13 months (range 2-62 months).
Pretreatment and follow-up studies included physical examinations, complete blood counts (CBC) and differential count, standard blood chemistry analysis, determination of serum vitamin Bl2, lactic dehydrogenase (LDN), leukocyte alkaline phosphatase (LAP) score, bone marrow aspiration, eytogenetic studies with Giemsa banding, and molecular genetic analysis of pe-
ripheral and marrow samples using the Southern blot technique.
Therapeutic protocol Patientswere first given bus&fan to reduce the white blood cell counts to c 10 OOO/pl before Bestatin administration. Busulfan and Bestatin (30 mg daily) were then administered in combination and continued until the
Y Usuka. Y Saito
90 Table U.
Patientcharacteristics. 3~u~~n alone
Patients on b~s~~~~estar~n Total No Age (median;yrj Male Female Diseasestage Chronic Accelerated Tie from dia~osis cytogenetic Fd+ PhtNot determined WB-30 OOWPI P~atelets~~/~I Splenomegaly Previous therapy Busulfan/carboquone None Period
14 22-73(57) 8 6
25 17-71(4S) 16 9
10 4 2-62 rno~~s (13 rnon~s~
25 0 l-2 (1 rnon~~
13 1 (maskedPh’) 0 12 12 10
25 0 0 24 22 19
14 0 Match 19SS- March 1990
0 25 March 19S~~~u~
white blood cell counts were decreased to < 5 OOO6 OOO/ul.After this, busulfan was discontinued and Bestatin alone was continued for 6 months or more. in ail patients, treatment was performed on an outpatient basis.
esponsc criteria Haematologic and cytogenetic responses were defined according to Talpaz’s criteria [7] with minor modi~cations.
Results Induction therapy with busulfan alone resulted in complete hematologic remission in 11 patients (44%), but the malignant clone remained fully predominant in all 25 patients. All 14 patients treated with busulfan/Bestatin were followed up and evaluated for a median duration of 12 months (range 5-33 months}. The combination therapy resulted in complete hematologic remission in all 14 patients. The median time before complete hematologic remission was achieved was 10 wk with a range of 5-20 wk from initiation of Bestatin therapy. Complete cytogenetic remission
19SS
(CCR) was achieved in 3 patients (21%). partial cytogenetic remission (PCR) in 1 (7%), and minor cytogenetic response (MCR) in 5 (36%). The significant cytogenetic response (CR+PR) rate was 28%, and the total cytogenetic response rate 64% (table III). The maximum cytogenetic response was obtained after more than 3 months of Bestatin therapy (table IV). Suppression of the Philadelphia chromosome was observed after a median duration of 21 wk (range 3-47 wk) of Bestatin therapy. The cytogenetic effects of combination therapy in various disease phases are shown in table V. Among the 5 patients in the early chronic phase (within 12 months of diagnosis), 2 achieved CCR, 1 achieved PCR, and 1 MCR. Complete suppression of the Ph’ clone was further confirmed in 3 patients by molecular analysis. The results in the late chronic phase (> 12 months after diagnosis) CML patients were less favorable; two of the 5 patients obtained MCR, but none obtained significant cytogenetic response. Among the 4 patients in the accelerated phase, 1 achieved CCR and 2 obtained MCR. Patient outcome is summarized in table VI. At the time of analysis, 11 out of 14 patients who obtained complete bematologic remission remain in continuous hematologic remission. Three of the 14 patients required continuous busulfan therapy
Bestatin treatment of MDS and CML
91
Table III. Results of busulfan/Bestatin combination chemotherapy vs busulfan alone.
Response category
No of patients in treatment category BusulfanlBestatin
Complete remission Continuous CR (unmaintained) Philadelphia status 100% (NR) 35%-95% (MR) 50/a-34% (PR) 0% (CR) ND Partial hematologic remission No response
Busulfan alone
14 (100%) 11(79%)
1I (44%) (f < 0.01) 5 (20%) (f < 0.01)
5 (36%) 5 (36%) 1(7%) 3 (21%) 0 0 0
21/21 (loo) 0 0 0 4 14 (56%) 0
Table IV. Serial cytogenetic changes with busulfan/Bestatin therapy.
Case
Disease phase
Percentage of Ph’-positive metaphase 0
1-3
4-6
> 7 months
(Months after initiation of Bestatin administration) 1 2 3 4 5
Early Early Early Early Early
6 7 8 9 10 I1 12 13 14
0* 100 100 100 100
0** 35 35 100 100
0** 5** 35** 90
Late chronic Late chronic Late chronic Late chronic Late chronic
100 100 100 100 loo
100 100 100 100 100
83 90
100
Accelerated Accelerated Accelerated Accelerated -BC
100 100 100 100
0* 100 100 100
0* 90 75 100
BC: blastic crisis; * BCR-ABL
chronic chronic chronic chronic chronic
rcarrangcment+: **
BCR-ABL
0** 0**
100 0’
100
rearrangement-.
Table
V. Cytogencticcffectsofbusulfan/Bestatin therapyanddiseasephase. CCR,Completecytogeneticresponse; cytogcnetic response: MCR, Minor cytogenetic response, NCR; No cytogenetic response.
Disease phase
Early chronic Late chronic Accelerated Total
PCR,Partial
No of cases
CCR
PCR
MCR
NCR
Sigu#Zant response (CR+PR %)
Disappearance of BCR-ABL rearrangement (%)
5 5 4 14
2 0 1 3
1 0 0 1
I 2 2 5
1 3 0 4
60 0 25 29
60 0 0 21
Cytogenetic response
Y Usuka. Y Saito
92
Table VI. Busulfan/Bestatin therapy in CML: present status of patients.
Stntlrs
Continuedtherapy with busulfan Off busulfan therapy Continued therapy with Bestatin Off Bestatin therapy
Total
No of total cases
~e~~to~~gie retnissfan
of
Cytageneiic remission
responders
Corrlinuous remission
No of responders
Continuous remission
3
3
1
1
1
11
11
10
8
8
11
II
9
7
7
3
3
2
2
2
14
14
11
9
9
No
for disease control and only one of these 3 patients remains in continuous complete hematologic remission. Busuifan therapy has been discontinued in the remaining 11 patients, 10 of which remain in continuous hematologic remission. Neither hematologic nor cytogenetic relapses were observed in the patients who obtained cytogenetic responses. Nine of the 11 patients who continued Bestatin therapy, and 2 of 3 patients who discontinued Bestatin therapy remain in continuous hematologic remission. All 9 patients who responded cytogenetically manifested evidence of continued suppression of the Philadelphia chromosome in cyogenetic follow-up studies. Cytogenetic conversion to a diploid state has persisted for 6 months or more in all responders. After a median followup of 12 months all patients are alive and no progression of the disease has been observed.
Discussion and Couclusion The success of Bestatin therapy in MDS led us to investigate the clinical activity of Bestatin in CML. In the current study, a busulfan - Bestatin combination therapy resulted in complete hematologic remission in all of the 14 patients treated. The most exciting results were the suppression of the Philadelphia chromosome among the responding patients. The combination therapy strongly suppressed the Philadelphia chromosomes in 2 1% of the patients and achieved partiai cytogenetic response in an additional 7%. In particular, the
majority of early chronic phase CML patients achieved significant cytogenetic response. Moreover, the disappearance of BCR-ABL rearrangement was observed in 3 of 5 patients with early chronic CML. It is still too early to determine whether this therapy will prolong survival time, but the ability to induce sustained Ph’ negativity without any toxicity with treatment toxicity, on an outpatient basis in very encouraging and further studies seem warranted. The results of the current combination therapy are comparable to those obtained by alpha interferon therapy [2, 4, 7-131. However, compared to the serious toxicity encountered with alpha interferon, no serious toxicity was observed in patients treated by busulfan-Bestatin combination therapy. Although the precise mechanism whereby Bestatin has an effect on the bone marrow remains unknown, it may be not merely antiproliferative, but have more complex biological properties, capable of persistently modifying the biological-proliferative disequilibrium caused by the disease. The action of Bestatin therefore seems to be biological, and opens up new and promising prospects in the treatment of CML.
References 1 Bennett JM, Camovsky D, Daniel MT, Frandrin G,
Calton DAG, Gralnick HR, Sultan C (1982) Proposals for the classification of the myelodysplastic syndrome. Br J Haematol 51, 189
Bestatin treatment of MDS and CML 2 Goldman JM, Groveld G, Baltimore D, Gale RP (1990) Chronic myelogenous leukemia. The unfolding saga. Leukemia 4, 163 3 Greenberg PL (1987) Biologic nature of the myelodysplastic syndromes. Acta Haematol78, (suppl 1) 94 4 Koeffler HP, Golde DW (1981) Chronic myelogenous ieukcmia - new concepts. New Engl J Med 304, 1201 5 List AF, Garewaltl HS, Sandbcrg AA (1990) The myelodysplastic syndromes: biology and implications for management. J Clint Oncoi 8, 1424 6 Gzer H (1988) Biotherapy of chronic myelogenous leukemia witb interferon. Semh Otzcol 1% 14 7 Talpaz M, Kantarjian HM, Kurzrock R, Gutterman JU (1988) Therapy of chronic myelogenous leukemia: chemotherapy and interferon. Semin Hematol 25, 62 8 Talpaz M, Kantarjian
H, Kurzlock R, Gutterman JU (1990) Update on therapeutic options for chronic myelogenous leukemia. Semin iiematof 27, (suppl 4) 31 9 Talpaz M, Kantarjian HM, McCredie K. Trujillo JM, Keating MJ, Gutterman JU (1986) Hematologic remission and cytogenetic improvement induced by
93
recombinant human interferon afpha in chronic myelogenous leukemia. N Engi J Med 314, 1065 10 Talpaz M, Kantarjian HM, McCredie KB. Keating MJ, Trujillo JN. Gutterman JU (1987) Clinical investigation of human alpha interferon in chronic myelogenous leukemia. Blood 69. 1280 11 Talpaz M. Kutzrock R, Kantarji~ HM, Gu~e~an JU (1988) Recent advances in the therapy of chronic myelogenous leukemia. fmporr Adv Oncol, p 297 12 Talpaz M, Malvigit GM, Keating M, Watters RS, Gutterman JU (1983) Human leukocyte interferon to control throm~ytosis in chronic mye~ogenous leukemia. Ann Intertt Med 99, 789 13 Talpaz M, McCredie KB, Mavligit GM, Cutterman JU (1983) Leukocyte interferon-induced myeloid cytoreduction in chronic myelogenous leukemia. Blood 62. 689 14 Umezawa H, Aoyagi T, Suda H et al (1984) Bestatin, an inhibitor of aminopept~dase produced by actinomycetes. J Antibiot 29, 197 15 Uzuka Y and Saito Y (1990) Bestatin treatment of myelodysplastic syndromes (MDS) and the effects of Bestatin on hematopoiesis in MDS. Acta Oncol 29, 803
87
estatin tr~atmeut of mye~ody$p~a§t~c and chronic myelogenou~ leuke Y Usuka, Y Saito The Third department of Internat medicine, Tohoku ~nive~i~ School of ~~i~~ne,
se&i,
Japan
Summary - A high remission rate (56%) was achieved in a preliminary study using Bestatin in patients with myelodysplastic syndromes. In particular, 9 out of 13 patients (69%) in the high blast group achieved hematologic remission. After Bestatin treafment, int~nsic ~e~~to~ietic stem cell ~b~u~aIities as wet1 as hemato~ogic ending were markedty improved. The success of Bestatin therapy in MDS led us to investigate the clinical activity of Bestatin in CML. In the current study the busulfan and Bestatin combination therapy resulted in complete hematologic remission in all of the patients. The most exciting result was the suppression of the Philadelphia chromosomes among the responding patients. Complete cytogenetic response was obtained in 3 patients (21%), partial cytagenetic response in 1 (7%). and minor cytogenetic response in 5 (36%). in particular, the majority of early chronic phase CML patients achieved significant cytogenetic response with sustained Pb’ negativity. The results are very enco~aging and warrant funher studies. myelodysplastic syndromes / chronic myelogenous leukemia I Bestatin biotherapy
R&u?n4 - Traitement par la Bestatlne des syndromes my4lodysplasiques et de la leudmie my4loide chronique. in taux &lev&de r~n~issionsa &e’obtenu dans une &de pr~~i~in~ire utilisant lo Bestatine chez les malades atteints de syndromes my~lodyspiasiques (SMD). En ~rticulier, 9 malades sur les 13 169%) du group li btastes nombreux sunt arriv&s li la r6mission h~matologique. AprPs un traitement par la Eestatine, &esanomalies intrins&ques de la celhde souche h&matopoUtique. ainsi que les rPsultats h&atologiques ont et6 nettement ame’lior&s.L’efficacitci de la Bestatine &ns les SMD a conduit les auteurs ri Qudier en clinique l’activite de la Bestatine au tours de la leuc&mie myt!loiile chronique (CML). Dans la prisente &tude, le traitement par I’association busulfan-Bestatine a eu pour rPsultat une rPmission h&nato~ogique con~p~~techet tous les maiades. Le r&&at le plus re~rq~b~e a it& ia d~s~rition du chromosome Ph~~~elphie chez les malades r&mdeurs. Une r&ponse con&&e sur la cytoge&e a &t! obtenue ckez 3 malades ~~~%), une Gponse partieile chez I seul (7%) et une rhponse mineure chez 5 (36%). En parriculier. la majorit& des malades atteints de LMC en dPbut de phase clrronique ont eu une riponse significative sur la cytogendse avec n&ativation maintenue du Ph’. Ces r&dtats sont tr2s encourageants et mCritent la poursuite des recherches dons ce domaine. syndrome my~lodysplasique I leuc&nie cbronique my&oide I biotitetapie B la Bestrtine
Introduction Myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) are clonal hemopathies that occur as a result of malignant transformation of a pluripotent hematopoietic stem cell. Early in the course of these diseases, the progeny of the malignant clone retains some degree of differentiation and normal function. These “premalignant” characteristics [6] of MDS and CML in the chronic phase impart therapeutic
treatment through biotherapy. These hematologic malignancies involve a variety of different cellular mechanisms including the activation of protooncogene and the release of growth factors. Accordingly, the use of biological ~tigrowth factor such as Bestatin would logically provide a new approach to these diseases. Recently we have obtained significant improvement by using Bestatin in both MDS and CML. We describe here a new app~ach to treating these diseases using this drug.
Y Us&a, Y Saito
88
yelodysplastic syndromes
fective hematopoiesis therefore studied.
Primary myelodysplastic syndrome (MDS) consists of a heterogenous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis with various combinations of anemia, leukopenia and thrombocytopenia. The treatment of MDS is dictated not only by the risk of evolving acute leukemia but also by the risk of infection and bleeding which result in a fatal outcome in the majority of patients. The lesions underlying this defect reside mainly at the level of the hematopoietic stem cells. Furthermore, recent studies have suggested that abnormalities exist in the distribution of T-cell subsets and their functions in MDS [3, 51. As T-cells contribute to the immunoregulation of inducible hematopoiesis, abnormalities in their numbers and functions are believed to play a pathophysiologic role in MDS. Advances in understanding the complexity of factors that govern the process of hematopoiesis have furthered our understanding of the biological abnormalities that underlie MDS and have provided new insight into therapeutic intervention. This report discusses a new attempt to treat MDS utilizing Bestatin (N- ((2s. 3R)-3-amino-2-hydroxy4-phenylbutanoyl J-~leucine), a small molecular immuno-modifier developed by Umezawa et al 1141. The mechanisms of Bestatin action on de-
in this
syndrome
were
Patients and Methods Sixteen patients with MDS included in this study had a median age of 68 yr (range 49-83 yr). They were diagnosed and classified according to the FAB classification [l]. Nine patients had refractory anemia with excess of blasts (RAEB), 4 patients RAEB in transformation (RAEB-t), 2 refractory anemia (RA), and one had chronic myelomonocytic leukemia (CMML). Bestatin was administered orally at a dose of 30 mg per day for 1 month or more. Complete response was defined as the normalization of peripheral and bone marrow findings, and the disappearance of dysplastic changes in hematopoietic cells. Partial response was defined as a reduction in the blast count in the peripheral blood and marrow. and an increase in peripheral blood counts, ie platelets to > 50 x lo9 per liter granulocytes to > 2 x IO9 per liter, and PCV > 30, which could be maintained without transfusion for a period of > 2 months.
Results The results of Bestatin treatment in MDS patients are shown in table I. Two of the 4 patients with
Table 1. Outcomes in Bestatin-treated MDS patients. CR, complete remission; PR, partial remission; F. failure. Bestatin Patients
Age (yr), scs
I
49 M
2 3
65 M 83 M
4 5 6 7 8 9 IO II 12 13 14 15 16
73 61 73 55 67 52 73 73 59 68 68 59 70
M M F M M M F F M M F M M
Diagnosis
RAEB-T RAEB-T RAEB-T - AMoL RAEB-T RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RA RA CMML
Dose
Duration
Imsld)
(dl
Effects
Days required for response
Remission duration W)
Survival (months)
30 30 30
128 273 46
CR F F
12
14
9 12+ 3
30 30 30 30 30 30 30 30 30 30 30 30 30
147+ 518 93 413 174 986+ 379 253 249+ 98 74 1028+ 50
CR CR F PR PR PR PR PR CR F F F F
16 57
ll+ 13+
22 22 65 141 123 121
49+ 8 132+ 7 127a 13+
5+ 18+ 30+ 14+ 22+ 33+ 34+ 34+ lO+ 36+ 28+ 35+ 4
Bestatin treatment of MDS and CML RAEB-t and 2 of 9 patients with RAEB achieved complete response (total complete response rate 4116 = 25%). Five patients with RAEB achieved partial response. The total response rate was 56%. The time required for response was 12 to 141 d with a median of 57 d. The duration of remission ranged from c 1I to + 132 wk. All patients except one whose condition evolved to overt acute leukemia are still alive (median follow-up period of 31 months). No side-effects were observed in any case.
Discussion In the current study, Bestatin therapy produced a complete response rate of 25% and a partial response rate of 31%. To elucidate the mechanisms of Bestatin action on normal and defective hematopoiesis, bone marrow cells from normal volunteers and patients with MDS were cultured by CFU-GEMM assay. The details of the experiments have been reported elsewhere [15] and are not presented here. Clonogenic marrow cell culture studies in patients with MDS have demonstrated intrinsic hematopoietic stem cell abnorerythroid particular, and malities. In erythroid-mixed colony formation were found to be defective in all patients. Moreover, it was suggested that the malignancy involves a variety of different cellular mechanisms, including the activation of protooncogenes and the release of growth factors, After Bestatin therapy, these abnormalities as well as hematologic findings were markedly improved. The use of a biological antigrowth factor such as Bestatin, therefore, is likely to provide a promising approach to MDS. However, the precise mechanisms of action of Bestatin are difficult to determine, particularly in a clinical setting, and further studies are necessary to elucidate its mechanisms of action.
Chronic myelogenous
leukemia
Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation between ch~mosome 9 and 22 { t(9;22)(q3~qll) ], or Philadelphia chromosome (Ph’). Until now a variety of therapeutic trials have been employed without significant success to alter the natural history of the disease and to delay the blastic phase
89
]81. Allogenic bone marrow tr~splantation provided the first breakthrough and is currently the only curative treatment for CML. However, its application is limited to a minority of cases. Since 1981 141, recombinant alpha interferon has been investigated as a possible therapy for CML. Interferon therapy, pioneered by Talpaz et al [2, 7, 9-131, has had the greatest impact on the treatment of CML. Although the effects of recombinant interferon alpha may be of significant clinical importance, its role in the overall treatment of the disease remains to be determined. In this report, we propose a new approach for treating patients with CML using a combination therapy of busulfan and Bestatin, and evaluate the clinical effects and cytogenetic changes associated with the therapy. As it is still too early for the results of the therapy in terms of survival to be estimated, the interim results of ongoing trials and an account of the clinical effects of Bestatin treatment will be presented.
Patients and Methods The clinical characteristics of 14 patients with Phi-positive CML treated with busulfan and Bestatin in combi-
nation and of 25 patients with CML treated with busulfan alone are summarized in table II. Ten patients in the chronic phase and 4 in the accelerated phase with cytogenetic clonal evolution were treated with the combination therapy. All pretreatment was te~inated at least 4 wk before initiating combination therapy. The median age of the patients was 57 yr (range 22-73 yr). The median time from the diagnosis to the commencement of the combination therapy was 13 months (range 2-62 months).
Pretreatment and follow-up studies included physical examinations, complete blood counts (CBC) and differential count, standard blood chemistry analysis, determination of serum vitamin Bl2, lactic dehydrogenase (LDN), leukocyte alkaline phosphatase (LAP) score, bone marrow aspiration, eytogenetic studies with Giemsa banding, and molecular genetic analysis of pe-
ripheral and marrow samples using the Southern blot technique.
Therapeutic protocol Patientswere first given bus&fan to reduce the white blood cell counts to c 10 OOO/pl before Bestatin administration. Busulfan and Bestatin (30 mg daily) were then administered in combination and continued until the
Y Usuka. Y Saito
90 Table U.
Patientcharacteristics. 3~u~~n alone
Patients on b~s~~~~estar~n Total No Age (median;yrj Male Female Diseasestage Chronic Accelerated Tie from dia~osis cytogenetic Fd+ PhtNot determined WB-30 OOWPI P~atelets~~/~I Splenomegaly Previous therapy Busulfan/carboquone None Period
14 22-73(57) 8 6
25 17-71(4S) 16 9
10 4 2-62 rno~~s (13 rnon~s~
25 0 l-2 (1 rnon~~
13 1 (maskedPh’) 0 12 12 10
25 0 0 24 22 19
14 0 Match 19SS- March 1990
0 25 March 19S~~~u~
white blood cell counts were decreased to < 5 OOO6 OOO/ul.After this, busulfan was discontinued and Bestatin alone was continued for 6 months or more. in ail patients, treatment was performed on an outpatient basis.
esponsc criteria Haematologic and cytogenetic responses were defined according to Talpaz’s criteria [7] with minor modi~cations.
Results Induction therapy with busulfan alone resulted in complete hematologic remission in 11 patients (44%), but the malignant clone remained fully predominant in all 25 patients. All 14 patients treated with busulfan/Bestatin were followed up and evaluated for a median duration of 12 months (range 5-33 months}. The combination therapy resulted in complete hematologic remission in all 14 patients. The median time before complete hematologic remission was achieved was 10 wk with a range of 5-20 wk from initiation of Bestatin therapy. Complete cytogenetic remission
19SS
(CCR) was achieved in 3 patients (21%). partial cytogenetic remission (PCR) in 1 (7%), and minor cytogenetic response (MCR) in 5 (36%). The significant cytogenetic response (CR+PR) rate was 28%, and the total cytogenetic response rate 64% (table III). The maximum cytogenetic response was obtained after more than 3 months of Bestatin therapy (table IV). Suppression of the Philadelphia chromosome was observed after a median duration of 21 wk (range 3-47 wk) of Bestatin therapy. The cytogenetic effects of combination therapy in various disease phases are shown in table V. Among the 5 patients in the early chronic phase (within 12 months of diagnosis), 2 achieved CCR, 1 achieved PCR, and 1 MCR. Complete suppression of the Ph’ clone was further confirmed in 3 patients by molecular analysis. The results in the late chronic phase (> 12 months after diagnosis) CML patients were less favorable; two of the 5 patients obtained MCR, but none obtained significant cytogenetic response. Among the 4 patients in the accelerated phase, 1 achieved CCR and 2 obtained MCR. Patient outcome is summarized in table VI. At the time of analysis, 11 out of 14 patients who obtained complete bematologic remission remain in continuous hematologic remission. Three of the 14 patients required continuous busulfan therapy
Bestatin treatment of MDS and CML
91
Table III. Results of busulfan/Bestatin combination chemotherapy vs busulfan alone.
Response category
No of patients in treatment category BusulfanlBestatin
Complete remission Continuous CR (unmaintained) Philadelphia status 100% (NR) 35%-95% (MR) 50/a-34% (PR) 0% (CR) ND Partial hematologic remission No response
Busulfan alone
14 (100%) 11(79%)
1I (44%) (f < 0.01) 5 (20%) (f < 0.01)
5 (36%) 5 (36%) 1(7%) 3 (21%) 0 0 0
21/21 (loo) 0 0 0 4 14 (56%) 0
Table IV. Serial cytogenetic changes with busulfan/Bestatin therapy.
Case
Disease phase
Percentage of Ph’-positive metaphase 0
1-3
4-6
> 7 months
(Months after initiation of Bestatin administration) 1 2 3 4 5
Early Early Early Early Early
6 7 8 9 10 I1 12 13 14
0* 100 100 100 100
0** 35 35 100 100
0** 5** 35** 90
Late chronic Late chronic Late chronic Late chronic Late chronic
100 100 100 100 loo
100 100 100 100 100
83 90
100
Accelerated Accelerated Accelerated Accelerated -BC
100 100 100 100
0* 100 100 100
0* 90 75 100
BC: blastic crisis; * BCR-ABL
chronic chronic chronic chronic chronic
rcarrangcment+: **
BCR-ABL
0** 0**
100 0’
100
rearrangement-.
Table
V. Cytogencticcffectsofbusulfan/Bestatin therapyanddiseasephase. CCR,Completecytogeneticresponse; cytogcnetic response: MCR, Minor cytogenetic response, NCR; No cytogenetic response.
Disease phase
Early chronic Late chronic Accelerated Total
PCR,Partial
No of cases
CCR
PCR
MCR
NCR
Sigu#Zant response (CR+PR %)
Disappearance of BCR-ABL rearrangement (%)
5 5 4 14
2 0 1 3
1 0 0 1
I 2 2 5
1 3 0 4
60 0 25 29
60 0 0 21
Cytogenetic response
Y Usuka. Y Saito
92
Table VI. Busulfan/Bestatin therapy in CML: present status of patients.
Stntlrs
Continuedtherapy with busulfan Off busulfan therapy Continued therapy with Bestatin Off Bestatin therapy
Total
No of total cases
~e~~to~~gie retnissfan
of
Cytageneiic remission
responders
Corrlinuous remission
No of responders
Continuous remission
3
3
1
1
1
11
11
10
8
8
11
II
9
7
7
3
3
2
2
2
14
14
11
9
9
No
for disease control and only one of these 3 patients remains in continuous complete hematologic remission. Busuifan therapy has been discontinued in the remaining 11 patients, 10 of which remain in continuous hematologic remission. Neither hematologic nor cytogenetic relapses were observed in the patients who obtained cytogenetic responses. Nine of the 11 patients who continued Bestatin therapy, and 2 of 3 patients who discontinued Bestatin therapy remain in continuous hematologic remission. All 9 patients who responded cytogenetically manifested evidence of continued suppression of the Philadelphia chromosome in cyogenetic follow-up studies. Cytogenetic conversion to a diploid state has persisted for 6 months or more in all responders. After a median followup of 12 months all patients are alive and no progression of the disease has been observed.
Discussion and Couclusion The success of Bestatin therapy in MDS led us to investigate the clinical activity of Bestatin in CML. In the current study, a busulfan - Bestatin combination therapy resulted in complete hematologic remission in all of the 14 patients treated. The most exciting results were the suppression of the Philadelphia chromosome among the responding patients. The combination therapy strongly suppressed the Philadelphia chromosomes in 2 1% of the patients and achieved partiai cytogenetic response in an additional 7%. In particular, the
majority of early chronic phase CML patients achieved significant cytogenetic response. Moreover, the disappearance of BCR-ABL rearrangement was observed in 3 of 5 patients with early chronic CML. It is still too early to determine whether this therapy will prolong survival time, but the ability to induce sustained Ph’ negativity without any toxicity with treatment toxicity, on an outpatient basis in very encouraging and further studies seem warranted. The results of the current combination therapy are comparable to those obtained by alpha interferon therapy [2, 4, 7-131. However, compared to the serious toxicity encountered with alpha interferon, no serious toxicity was observed in patients treated by busulfan-Bestatin combination therapy. Although the precise mechanism whereby Bestatin has an effect on the bone marrow remains unknown, it may be not merely antiproliferative, but have more complex biological properties, capable of persistently modifying the biological-proliferative disequilibrium caused by the disease. The action of Bestatin therefore seems to be biological, and opens up new and promising prospects in the treatment of CML.
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