Leukemia Research Vol. 14, No. 7, pp. 595-600. 1990. Printed in Great Britain.
0145-2126/90 $3.00 + .00 Pergamon Press plc
R E D U C T I O N OF L E U K E M I A CELL G R O W T H IN A PATIENT WITH A C U T E P R O M Y E L O C Y T I C L E U K E M I A T R E A T E D BY R E T I N O L PALMITATE HIROSHI TSUTANI, HIROMICHI IWASAKI, YASUKAZU KAWAI, TSUNEO TANAKA, TAKANORI UEDA, MICHIHIKO UCHIDA and TORU NAKAMURA The First Department of Internal Medicine, Fukui Medical School, 23 Shimoaizuki, Matsuoka, Fukui, 910-11, Japan (Received 12 September 1989. Revision accepted 3 February 1990) Abstract--A 67-year-old woman with acute promyelocytic leukemia (APL) showed a marked decrease
in leukemic promyelocytes with concomitant maturation of leukemic cells during treatment with retinol palmitate. A culture study in vitro revealed that retinol, which is the main metabolite of retinol palmitate detected in plasma, induced morphological and functional maturation of leukemic promyelocytes. These findings may indicate that retinol palmitate induces cell differentiation and slows proliferation of leukemic cells in vivo, and that the reduction in cell growth is the key phenomenon in the clearing of leukemic cells, rather than the maturation phenomenon itself. Key words: Retinol palmitate, acute promyelocytic leukemia, cell differentiation, antiproliferative effect.
INTRODUCTION
CASE R E P O R T A 67-year-old housewife with a 15-year history of bronchial asthma was admitted to our hospital because of progressive dyspnea, fever, and perianal pain. Physical examination on admission revealed an acutely ill-appearing woman in distress. A tender tumor was felt upon digital rectal examination, and mild stridor was heard on chest auscultation. Laboratory examination revealed a leukocyte count of 3.5 x 109/1 with 92% leukemic cells, a hemoglobin level of 7.7 g/dl, and a platelet count of 42 x 109/1. Coagulation parameters revealed a prothrombin time of 17.6 s, a fibrinogen level of 408 mg/dl, and a F D P level of 80 ~tg/ml. The marrow nucleated cell count was 190 x 109/1 and included 79.6% leukemic cells which were rich in azure granules and stained positive for peroxidase. Occasional leukemic cells had a mass of Auer's body showing the features of a faggot cell. A surface marker analysis using a fluorescence activated cell sorter analyzer showed that the mononuclear cells from the peripheral blood were positive for CD33 (My9), CD15 (LeuM1) and CD13 (MCS2), but negative for C D l l (OKM1), CD14 (My4) and H L A - D R ( O K I a l ) . A marrow cytogenetic study revealed 46, XX, t(15;17) in all 20 cells examined. The patient was diagnosed as having A P L (FAB M3) and a perianal abscess. The peripheral leukocyte count increased to 19.5 x 109/1 on day three of the patient's hospital
differentiation in vitro of some human myeloid cell lines seems to open the way for an alternative approach to the treatment of acute myeloid leukemia [1-3]. Retinoids, a family of molecules comprising both natural and synthetic analogues of retinol, are potent agents which induce terminal differentiation [4]. The dramatic results observed by Huang and colleagues in A P L patients treated with all-trans retinoic acid [5] implied a new approach for the treatment of these patients: an approach in which clinical remission may be obtained without peripheral cytoreduction or much bleeding by the induction of leukemic promyelocyte maturation. However, a somewhat greater emphasis on cell differentiation appears to exist in recent trials with differentiation inducers. Retinol palmitate is a natural analogue of retinol which can induce terminal differentiation of leukemic blast cells [6]. We describe here a patient with A P L in whom leukemia cell growth was inhibited significantly during treatment with retinol palmitate due to its maturation effect. INDUCTION of
Abbreviations: APL; acute promyelocytic leukemia; DIC, disseminated intravascular coagulopathy; TPA, phorbol myristate acetate; NBT, nitro blue tetrazolium. Correspondence to: Dr Hiroshi Tsutani, The First Department of Internal Medicine, Fukui Medical School, 23 Shimoaizuki, Matsuoka, Fukui, 910-11, Japan. 595
596
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FIG. 1. Clinical course. RP: retinol palmitate, BM: bone marrow, NCC: nucleated cell count (x 109/1), PMN: polymorphonuclear leukoeytes, PRC: packed red cells PC: platelet concentrate I1: erythrob|asts, g]: neutrophilsmyelocytes, ~: leukemic promyelocytes
stay. Since the performance status of the patient was extremely poor and was assessed at 20 according to the Karnofsky performance status index [7], it was thought that she would not be able to tolerate agressire combination chemotherapy. Then, treatment with retinol palmitate at a dosage of 210 x 106 IU daily was initiated (Fig. 1). Shortly after the initiation of treatment, a marked decrease in the number of leukemic promyelocytes with a concomitant initial transient neutrophilia and the occasional appearance of dysplastic promyelocyte were observed on the blood smear. Bone marrow cellularity was judged from marrow aspirates performed every 4-7 days instead of bone marrow biopsy to prevent frequent operative maneuver under hemorrhagic tendency. After the ninth day of treatment, the leukemic cell count was 120 x 106/1 in blood and 2.6 x 109/1 in bone marrow, yet both anemia and thrombocytopenia failed to be improved. The marrow aspirate picture showed a decreased number of leukemic promyelocytes along with a slight increase in the number of mature myeloid cellular elements. The appearance of these differentiated cells was bizarre since the lobulated nucleus contained condensed chromatin in spite of the purplish color in the cytoplasm (Fig. 2). Transmission electronmicroscopic examination also confirmed that leukemic promyelocytes from the bone marrow had differentiated to mature granulocytes. Condensation of the heterochromatin became evident, and the shape of the nucleus had often observed as having changed to
beanshaped. The number of azurophilic granules was markedly decreased, and development and dilatation of the rough endoplasmic reticulum became prominent (Fig. 3). The administration of retinol palmitate was discontinued after 6 weeks because of drug intolerance, during which the number of peripheral and bone marrow leukemic promyelocytes had remained low, but anemia and thrombocytopenia had persisted. No aggravation of DIC and pneumonia was observed, However, the proliferation of leukemic cells appeared shortly after the cessation of the treatment. The patient died of septic shock following pneumonia and a perianal abscess. METHODS A modification of the method of Flynn and colleagues [3] was used for short-term suspension culture. Two milliliters of heparinized bone marrow aspirate was obtained 1 day prior to oral retinol palmitate. Cells were diluted with RPMI-1640 supplemented with 20% heat-inactivated fetal calf serum and then overlaid at room temperature onto 5 ml of Ficoll-Hypaque solution and centrifuged at 800 g for 30 rain. The bone marrow leukemic cells and mononuclear cells (leukemic promyelocytes > 95%) were suspended in the same medium, plated in Falcon dishes at 5 × 105 cells/ml and incubated at 37°C in a humid atmosphere of 5 % CO ~in air for 4 days. A l l - t r a n s retinol (Nakarai Chemicals, Kyoto), which is the major metabolite of retinol palmitate in plasma [6], was used in the experiment. Morphological examination of the cell suspensions was performed on a centrifuged preparation stained with MayGriinwald-Giemsa to estimate the grade of maturation.
FIG. 2. Morphological maturation of leukemic promyelocytes in vivo. (a) Bone marrow before treatment with retinol palmitate. (b) Bone marrow after 9 days of treatment. May-Griinwald-Giemsa stain, 1,000×.
597
;;i
¸ i¸
FIG. 3. Electron micrographs of bone marrow leukemic promyelocytes. The leukemic promyelocytes before treatment with retinol palmitate (a), those after 9 days of treatment (b), 9,000×.
598
Reduction of leukemic promyelocytes growth by retinol palmitate
To determine the ability of individual cells to generate superoxide after activation by TPA (Sigma Chemicals, St Louis), a slide test using NBT (Sigma Chemicals, St Louis) dye was performed [8]. Cells (1.0 x 106/ml) in Hank's balanced salt solution and 0.5 mg of NBT/ml were stimulated with TPA (100 ng/ml). At least 100 cells were scored as formazan positive or negative. RESULTS Bone marrow leukemic cells incubated for 4 days in suspension culture, with or without retinol, showed little change in cell density. The viability of both control and retinol-treated cells was consistently >70%. Leukemic promyelocytes showed morphological and functional maturation when cultured with 20 ~tM retinol. The percentage of promyelocytes in the control group and in the retinol-treated group was 91 --- 4% (mean --- S.E.) and 58 ± 10%, the percentage of myelocytes and metamyelocytes in the control group and in the treated group was 8 -+ 4% and 20-+ 4%, and the percentage of polymorphonuclear leukocytes in the groups was 2 - 1% and 22 -+ 4%, respectively. The rate of NBT reduction in retinol-treated cells was 65 ± 5 %, significantly higher than that in the control group (4 ± 3%). DISCUSSION The patient studied had typical clinical features of APL including a 15;17 translocation, leukemic cells negative for H L A - D R , presence of faggot cells, and associated disseminated intravascular coagulation. The number of peripheral leukemic promyelocytes significantly increased shortly after admission, which may have indicated the presence of a new leukemic subclone having a highly malignant and self-renewing potential. This is the first report in which maturation of leukemic cells occurred during retinol palmitate treatment of a patient with APL. Induction of terminal differentiation caused by the administration of retinol palmitate was suspected, since there were apparent findings which indicated cell differentiation: (1) initial transient neutrophilia in the peripheral blood; (2) decreased number of leukemic promyelocytes without hypoplasia of normal elements, as evidenced by the peripheral blood values; (3) bizarre myeloid cellular elements on light- and electronmicrographs indicating a matured form of leukemic cells; and (4) tests showing functional and morphological maturation of retinol-treated leukemic promyelocytes in vitro. The most interesting point in this case is that the population of leukemic promyelocytes greatly decreased during the administration of retinol palmitate with concomitant cell differentiation in both
599
the peripheral blood and bone marrow. We determined marrow cellularity by counting nucleatedcells in marrow aspirates instead of performing bone marrow biopsy. This is a routine method in Japan for the determination of marrow cellularity during combination chemotherapy, and the results have been shown to roughly correlate with those of threpine biopsy with respect to marrow cellularity [9]. Differentiation inducers including retinoids decrease the number of dividing cells and commit them into maturation, which leads to suppressed proliferation. The fact that all-trans retinoic acid inhibits the clonal growth of leukemic cells at concentrations between 10 -6 M and 10-9M should be noted [10]. However, this phenomenon has been observed mainly in vitro, and most previous clinical studies [1-4] have mentioned an increase in the number of matured cells rather than an antiproliferative effect on the leukemic cells. Furthermore, the treatment with retinoids has the striking advantage of suppressing hemorrhagic aggravation or the appearance of coagulation parameter abnormalities suggesting DIC [4]. During the treatment with retinoid, leukemic cells may not be destroyed, but instead may differentiate which is associated with the disappearance of azurophilic granules and the loss of the capacity to release coagulation factors [11]. In our patient, no aggravation of coagulation parameters was observed in spite of the remarkable decrease in leukemic population, which supports the possibility that retinol palmitate suppresses the proliferation of leukemic cells by its maturation effect instead of by a cytocidal effect. The exact mechanism by which retinoids induce cell differentiation and lead finally to remission is unknown. Some studies [12] in patients with myelodysplastic syndrome have revealed that response to differentiation inducer were usually partial and shortlived. In the patient studied, no normal population of hematopoietic cells in erythropoiesis and thrombopoiesis was observed as had been shown in previous studies [5, 10]. The action of these agents may be limited to inducing leukemic blasts to differentiating to mature cells, rather than eliminating leukemic blasts. From the studies by Fearon and colleagues [13] in some leukemic patients receiving combination chemotherapy, the presence of residual mature leukemic cells is a sign of poor prognosis to indicate relapse. However, it is notable that treatment with retinoids has the advantages of good sensitivity for leukemic promyelocytes and less adverse effects as compared to other differentiation inducers [1-4]. Therefore, a long period of treatment with retinoids, although not achieved in this patient, may lead to the clearing of the leukemic clone [5] and the recovery of normal hematopoiesis. It might be
600
H. TSUTANIet al,
strongly suggested that retinol palmitate is an effective agent for A P L due to its maturation-induction and subsequent antiproliferative effect, and that the reduction in cell growth is the key p h e n o m e n o n in the clearing of leukemic cells, rather than the maturation p h e n o m e n o n itself. However, a method for obtaining and maintaining complete remission requires further study.
REFERENCES 1. Housset M., Daniel M. T. & Degas L. (1982) Small dose of ara-C in the treatment of acute myeloid leukemia: differentiation of myeloid leukemia cell. Br. J. Haemat. 51, 125. 2. Nilsson B. (1984) Probable in-vivo induction of differentiation by retinoic acid of promyelocytes in acute promyelocytic leukemia. Br. J. Hemat. 57, 365. 3. Flynn P. J., Miller W. J. & Weisdorf D, J. (1983) Retinoic acid treatment of acute promyelocytic leukemia: in-vitro and in-vivo observations. Blood 62, 1211. 4. Breitman T. R., Selonick S. E, & Collins S. J. (1980) Induction of differentiation of the human promyelocytie leukemia cell line (HL-60) by retinoic acid. Proc. natn. Acad. Sci. U.S.A. 77, 2936. 5. Huang M. E., Ye Y. C., Chen S. R., Chai J. R., Lu J. X., Zhoa L., Gu L. J. & Wang Z. Y. (1988) Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72, 567.
6. Tsutani H., Urasaki Y., Kamiya K., Sasaki K., Yoshimura T., Ando S., Kagawa D., Ueda T., Uchida M. & Nakamura T. (1988) Retinoid in the treatment of atypical leukemia. J. Jpn. Soc. Cancer. Ther. 23, 332. 7. Mor V., Laliberte L., Morris J. N. & Wiemann M. (1984) The Karnofsky performance status scale: an examination of its reliability and validity in a research setting. Cancer 53, 2002. 8. Weening R. S., Roos D. & Loos J. A. (1974) Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. J. Lab. clin. Med. 83, 570, 9. Uzuka Y., Liong S. K. & Yamagata S. (1976) Treatment of adult acute non-lymphoblastic leukemia using intermittent combination chemotherapy with daunomycin, cytosine arabinoside, 6-mercaptopurine and predonisolone: DCMP two step therapy. Tohoku J. exp. Med. 118, 217. 10. Douer D. & Koeffler H. P. (1982) Retinoic acid: inhibition of the clonal growth of human myeloid leukemia cells. J. clin. Invest. 69, 277. 11. Wijermans P. W., Rebel V. I., Ossenkoppele G. J., Huijgens P. C. & Langenbuijsen M. M. A. C. (1989) Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemia cells: reversal of the bleeding disorder by cell differentiation. Blood 73, 800. 12. Buzaid A. C., Garewal H. S. & Greenberg B. R. (1986) Management of myelodysplastic syndrome. Am. J. Med. 80, 1149. 13. Fearon E. R., Burke P. J., Schiffer C. A., Zehnbauer B. A. & Valstein B. (1986) Differentiation of leukemia cells to polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia. New Engl. J. Med. 315, 15.
0145-2126/90 $3.00 + .00 Pergamon Press plc
R E D U C T I O N OF L E U K E M I A CELL G R O W T H IN A PATIENT WITH A C U T E P R O M Y E L O C Y T I C L E U K E M I A T R E A T E D BY R E T I N O L PALMITATE HIROSHI TSUTANI, HIROMICHI IWASAKI, YASUKAZU KAWAI, TSUNEO TANAKA, TAKANORI UEDA, MICHIHIKO UCHIDA and TORU NAKAMURA The First Department of Internal Medicine, Fukui Medical School, 23 Shimoaizuki, Matsuoka, Fukui, 910-11, Japan (Received 12 September 1989. Revision accepted 3 February 1990) Abstract--A 67-year-old woman with acute promyelocytic leukemia (APL) showed a marked decrease
in leukemic promyelocytes with concomitant maturation of leukemic cells during treatment with retinol palmitate. A culture study in vitro revealed that retinol, which is the main metabolite of retinol palmitate detected in plasma, induced morphological and functional maturation of leukemic promyelocytes. These findings may indicate that retinol palmitate induces cell differentiation and slows proliferation of leukemic cells in vivo, and that the reduction in cell growth is the key phenomenon in the clearing of leukemic cells, rather than the maturation phenomenon itself. Key words: Retinol palmitate, acute promyelocytic leukemia, cell differentiation, antiproliferative effect.
INTRODUCTION
CASE R E P O R T A 67-year-old housewife with a 15-year history of bronchial asthma was admitted to our hospital because of progressive dyspnea, fever, and perianal pain. Physical examination on admission revealed an acutely ill-appearing woman in distress. A tender tumor was felt upon digital rectal examination, and mild stridor was heard on chest auscultation. Laboratory examination revealed a leukocyte count of 3.5 x 109/1 with 92% leukemic cells, a hemoglobin level of 7.7 g/dl, and a platelet count of 42 x 109/1. Coagulation parameters revealed a prothrombin time of 17.6 s, a fibrinogen level of 408 mg/dl, and a F D P level of 80 ~tg/ml. The marrow nucleated cell count was 190 x 109/1 and included 79.6% leukemic cells which were rich in azure granules and stained positive for peroxidase. Occasional leukemic cells had a mass of Auer's body showing the features of a faggot cell. A surface marker analysis using a fluorescence activated cell sorter analyzer showed that the mononuclear cells from the peripheral blood were positive for CD33 (My9), CD15 (LeuM1) and CD13 (MCS2), but negative for C D l l (OKM1), CD14 (My4) and H L A - D R ( O K I a l ) . A marrow cytogenetic study revealed 46, XX, t(15;17) in all 20 cells examined. The patient was diagnosed as having A P L (FAB M3) and a perianal abscess. The peripheral leukocyte count increased to 19.5 x 109/1 on day three of the patient's hospital
differentiation in vitro of some human myeloid cell lines seems to open the way for an alternative approach to the treatment of acute myeloid leukemia [1-3]. Retinoids, a family of molecules comprising both natural and synthetic analogues of retinol, are potent agents which induce terminal differentiation [4]. The dramatic results observed by Huang and colleagues in A P L patients treated with all-trans retinoic acid [5] implied a new approach for the treatment of these patients: an approach in which clinical remission may be obtained without peripheral cytoreduction or much bleeding by the induction of leukemic promyelocyte maturation. However, a somewhat greater emphasis on cell differentiation appears to exist in recent trials with differentiation inducers. Retinol palmitate is a natural analogue of retinol which can induce terminal differentiation of leukemic blast cells [6]. We describe here a patient with A P L in whom leukemia cell growth was inhibited significantly during treatment with retinol palmitate due to its maturation effect. INDUCTION of
Abbreviations: APL; acute promyelocytic leukemia; DIC, disseminated intravascular coagulopathy; TPA, phorbol myristate acetate; NBT, nitro blue tetrazolium. Correspondence to: Dr Hiroshi Tsutani, The First Department of Internal Medicine, Fukui Medical School, 23 Shimoaizuki, Matsuoka, Fukui, 910-11, Japan. 595
596
H. TSUTANI IRP 2)0 X l O ~ l U / d a y ~
.co,90 of
-
,,
]
.
.....
,,0
,0/--f
2O
PRC I] pcD U EEl
1°1#2°1 ~._;~:/~., .~ --
i
. . . .
et al.
~
~
~
I] El
~
~
I]
I]
i]
.
0
~
E]
~ .
4'0
2'0 Hospital
60
Days
FIG. 1. Clinical course. RP: retinol palmitate, BM: bone marrow, NCC: nucleated cell count (x 109/1), PMN: polymorphonuclear leukoeytes, PRC: packed red cells PC: platelet concentrate I1: erythrob|asts, g]: neutrophilsmyelocytes, ~: leukemic promyelocytes
stay. Since the performance status of the patient was extremely poor and was assessed at 20 according to the Karnofsky performance status index [7], it was thought that she would not be able to tolerate agressire combination chemotherapy. Then, treatment with retinol palmitate at a dosage of 210 x 106 IU daily was initiated (Fig. 1). Shortly after the initiation of treatment, a marked decrease in the number of leukemic promyelocytes with a concomitant initial transient neutrophilia and the occasional appearance of dysplastic promyelocyte were observed on the blood smear. Bone marrow cellularity was judged from marrow aspirates performed every 4-7 days instead of bone marrow biopsy to prevent frequent operative maneuver under hemorrhagic tendency. After the ninth day of treatment, the leukemic cell count was 120 x 106/1 in blood and 2.6 x 109/1 in bone marrow, yet both anemia and thrombocytopenia failed to be improved. The marrow aspirate picture showed a decreased number of leukemic promyelocytes along with a slight increase in the number of mature myeloid cellular elements. The appearance of these differentiated cells was bizarre since the lobulated nucleus contained condensed chromatin in spite of the purplish color in the cytoplasm (Fig. 2). Transmission electronmicroscopic examination also confirmed that leukemic promyelocytes from the bone marrow had differentiated to mature granulocytes. Condensation of the heterochromatin became evident, and the shape of the nucleus had often observed as having changed to
beanshaped. The number of azurophilic granules was markedly decreased, and development and dilatation of the rough endoplasmic reticulum became prominent (Fig. 3). The administration of retinol palmitate was discontinued after 6 weeks because of drug intolerance, during which the number of peripheral and bone marrow leukemic promyelocytes had remained low, but anemia and thrombocytopenia had persisted. No aggravation of DIC and pneumonia was observed, However, the proliferation of leukemic cells appeared shortly after the cessation of the treatment. The patient died of septic shock following pneumonia and a perianal abscess. METHODS A modification of the method of Flynn and colleagues [3] was used for short-term suspension culture. Two milliliters of heparinized bone marrow aspirate was obtained 1 day prior to oral retinol palmitate. Cells were diluted with RPMI-1640 supplemented with 20% heat-inactivated fetal calf serum and then overlaid at room temperature onto 5 ml of Ficoll-Hypaque solution and centrifuged at 800 g for 30 rain. The bone marrow leukemic cells and mononuclear cells (leukemic promyelocytes > 95%) were suspended in the same medium, plated in Falcon dishes at 5 × 105 cells/ml and incubated at 37°C in a humid atmosphere of 5 % CO ~in air for 4 days. A l l - t r a n s retinol (Nakarai Chemicals, Kyoto), which is the major metabolite of retinol palmitate in plasma [6], was used in the experiment. Morphological examination of the cell suspensions was performed on a centrifuged preparation stained with MayGriinwald-Giemsa to estimate the grade of maturation.
FIG. 2. Morphological maturation of leukemic promyelocytes in vivo. (a) Bone marrow before treatment with retinol palmitate. (b) Bone marrow after 9 days of treatment. May-Griinwald-Giemsa stain, 1,000×.
597
;;i
¸ i¸
FIG. 3. Electron micrographs of bone marrow leukemic promyelocytes. The leukemic promyelocytes before treatment with retinol palmitate (a), those after 9 days of treatment (b), 9,000×.
598
Reduction of leukemic promyelocytes growth by retinol palmitate
To determine the ability of individual cells to generate superoxide after activation by TPA (Sigma Chemicals, St Louis), a slide test using NBT (Sigma Chemicals, St Louis) dye was performed [8]. Cells (1.0 x 106/ml) in Hank's balanced salt solution and 0.5 mg of NBT/ml were stimulated with TPA (100 ng/ml). At least 100 cells were scored as formazan positive or negative. RESULTS Bone marrow leukemic cells incubated for 4 days in suspension culture, with or without retinol, showed little change in cell density. The viability of both control and retinol-treated cells was consistently >70%. Leukemic promyelocytes showed morphological and functional maturation when cultured with 20 ~tM retinol. The percentage of promyelocytes in the control group and in the retinol-treated group was 91 --- 4% (mean --- S.E.) and 58 ± 10%, the percentage of myelocytes and metamyelocytes in the control group and in the treated group was 8 -+ 4% and 20-+ 4%, and the percentage of polymorphonuclear leukocytes in the groups was 2 - 1% and 22 -+ 4%, respectively. The rate of NBT reduction in retinol-treated cells was 65 ± 5 %, significantly higher than that in the control group (4 ± 3%). DISCUSSION The patient studied had typical clinical features of APL including a 15;17 translocation, leukemic cells negative for H L A - D R , presence of faggot cells, and associated disseminated intravascular coagulation. The number of peripheral leukemic promyelocytes significantly increased shortly after admission, which may have indicated the presence of a new leukemic subclone having a highly malignant and self-renewing potential. This is the first report in which maturation of leukemic cells occurred during retinol palmitate treatment of a patient with APL. Induction of terminal differentiation caused by the administration of retinol palmitate was suspected, since there were apparent findings which indicated cell differentiation: (1) initial transient neutrophilia in the peripheral blood; (2) decreased number of leukemic promyelocytes without hypoplasia of normal elements, as evidenced by the peripheral blood values; (3) bizarre myeloid cellular elements on light- and electronmicrographs indicating a matured form of leukemic cells; and (4) tests showing functional and morphological maturation of retinol-treated leukemic promyelocytes in vitro. The most interesting point in this case is that the population of leukemic promyelocytes greatly decreased during the administration of retinol palmitate with concomitant cell differentiation in both
599
the peripheral blood and bone marrow. We determined marrow cellularity by counting nucleatedcells in marrow aspirates instead of performing bone marrow biopsy. This is a routine method in Japan for the determination of marrow cellularity during combination chemotherapy, and the results have been shown to roughly correlate with those of threpine biopsy with respect to marrow cellularity [9]. Differentiation inducers including retinoids decrease the number of dividing cells and commit them into maturation, which leads to suppressed proliferation. The fact that all-trans retinoic acid inhibits the clonal growth of leukemic cells at concentrations between 10 -6 M and 10-9M should be noted [10]. However, this phenomenon has been observed mainly in vitro, and most previous clinical studies [1-4] have mentioned an increase in the number of matured cells rather than an antiproliferative effect on the leukemic cells. Furthermore, the treatment with retinoids has the striking advantage of suppressing hemorrhagic aggravation or the appearance of coagulation parameter abnormalities suggesting DIC [4]. During the treatment with retinoid, leukemic cells may not be destroyed, but instead may differentiate which is associated with the disappearance of azurophilic granules and the loss of the capacity to release coagulation factors [11]. In our patient, no aggravation of coagulation parameters was observed in spite of the remarkable decrease in leukemic population, which supports the possibility that retinol palmitate suppresses the proliferation of leukemic cells by its maturation effect instead of by a cytocidal effect. The exact mechanism by which retinoids induce cell differentiation and lead finally to remission is unknown. Some studies [12] in patients with myelodysplastic syndrome have revealed that response to differentiation inducer were usually partial and shortlived. In the patient studied, no normal population of hematopoietic cells in erythropoiesis and thrombopoiesis was observed as had been shown in previous studies [5, 10]. The action of these agents may be limited to inducing leukemic blasts to differentiating to mature cells, rather than eliminating leukemic blasts. From the studies by Fearon and colleagues [13] in some leukemic patients receiving combination chemotherapy, the presence of residual mature leukemic cells is a sign of poor prognosis to indicate relapse. However, it is notable that treatment with retinoids has the advantages of good sensitivity for leukemic promyelocytes and less adverse effects as compared to other differentiation inducers [1-4]. Therefore, a long period of treatment with retinoids, although not achieved in this patient, may lead to the clearing of the leukemic clone [5] and the recovery of normal hematopoiesis. It might be
600
H. TSUTANIet al,
strongly suggested that retinol palmitate is an effective agent for A P L due to its maturation-induction and subsequent antiproliferative effect, and that the reduction in cell growth is the key p h e n o m e n o n in the clearing of leukemic cells, rather than the maturation p h e n o m e n o n itself. However, a method for obtaining and maintaining complete remission requires further study.
REFERENCES 1. Housset M., Daniel M. T. & Degas L. (1982) Small dose of ara-C in the treatment of acute myeloid leukemia: differentiation of myeloid leukemia cell. Br. J. Haemat. 51, 125. 2. Nilsson B. (1984) Probable in-vivo induction of differentiation by retinoic acid of promyelocytes in acute promyelocytic leukemia. Br. J. Hemat. 57, 365. 3. Flynn P. J., Miller W. J. & Weisdorf D, J. (1983) Retinoic acid treatment of acute promyelocytic leukemia: in-vitro and in-vivo observations. Blood 62, 1211. 4. Breitman T. R., Selonick S. E, & Collins S. J. (1980) Induction of differentiation of the human promyelocytie leukemia cell line (HL-60) by retinoic acid. Proc. natn. Acad. Sci. U.S.A. 77, 2936. 5. Huang M. E., Ye Y. C., Chen S. R., Chai J. R., Lu J. X., Zhoa L., Gu L. J. & Wang Z. Y. (1988) Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72, 567.
6. Tsutani H., Urasaki Y., Kamiya K., Sasaki K., Yoshimura T., Ando S., Kagawa D., Ueda T., Uchida M. & Nakamura T. (1988) Retinoid in the treatment of atypical leukemia. J. Jpn. Soc. Cancer. Ther. 23, 332. 7. Mor V., Laliberte L., Morris J. N. & Wiemann M. (1984) The Karnofsky performance status scale: an examination of its reliability and validity in a research setting. Cancer 53, 2002. 8. Weening R. S., Roos D. & Loos J. A. (1974) Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. J. Lab. clin. Med. 83, 570, 9. Uzuka Y., Liong S. K. & Yamagata S. (1976) Treatment of adult acute non-lymphoblastic leukemia using intermittent combination chemotherapy with daunomycin, cytosine arabinoside, 6-mercaptopurine and predonisolone: DCMP two step therapy. Tohoku J. exp. Med. 118, 217. 10. Douer D. & Koeffler H. P. (1982) Retinoic acid: inhibition of the clonal growth of human myeloid leukemia cells. J. clin. Invest. 69, 277. 11. Wijermans P. W., Rebel V. I., Ossenkoppele G. J., Huijgens P. C. & Langenbuijsen M. M. A. C. (1989) Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemia cells: reversal of the bleeding disorder by cell differentiation. Blood 73, 800. 12. Buzaid A. C., Garewal H. S. & Greenberg B. R. (1986) Management of myelodysplastic syndrome. Am. J. Med. 80, 1149. 13. Fearon E. R., Burke P. J., Schiffer C. A., Zehnbauer B. A. & Valstein B. (1986) Differentiation of leukemia cells to polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia. New Engl. J. Med. 315, 15.
