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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3315-3324
Studies on Treatment of Acute Promyelocytic Leukemia With Arsenic
Trioxide: Remission Induction, Follow-Up, and Molecular Monitoring in
11 Newly Diagnosed and 47 Relapsed Acute Promyelocytic Leukemia
Patients
By
Chao Niu,
Hua Yan,
Ting Yu,
Hui-Ping Sun,
Jian-Xiang Liu,
Xiu-Song Li,
Wen Wu,
Fen-Qin Zhang,
Yu Chen,
Li Zhou,
Jun-Min Li,
Xiao-Ying Zeng,
Ren-Rong Ou Yang,
Mi-Man Yuan,
Mei-Yu Ren,
Feng-Ying Gu,
Qi Cao,
Bo-Wei Gu,
Xin-Ying Su,
Guo-Qiang Chen,
Shu-Min Xiong,
Ting-dong Zhang,
Samuel Waxman,
Zhen-Yi Wang,
Zhu Chen,
Jiong Hu,
Zhi-Xiang Shen, and
Sai-Juan Chen
From Shanghai Institute of Hematology, Department of
Hematology/Oncology, Rui Jin Hospital/Samuel Waxman Cancer Research
Foundation Joint Center for Cancer Differentiation Therapy Sponsored by
Reliance Group Holdings Inc, Rui-Jin Hospital, Ren-Ji Hospital; Xin-Hua
Hospital, Shanghai Second Medical University, Shanghai, China;
Zhong-Shan Hospital, Shanghai, China; Gan-Quan Hospital, Shanghai,
China; and First Hospital Affiliated with Harbin Medical University,
Harbin, China.
 |
ABSTRACT |
Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly
diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission
(CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However,
As2O3 treatment resulted in hepatic toxicity in
7 cases including 2 deaths, in contrast to the mild liver dysfunction
in one third of the relapsed patients. Forty of forty-seven (85.1%)
relapsed patients achieved CR. Two of three nonresponders showed clonal
evolution at relapse, with disappearance of t(15;17) and PML-RAR
fusion gene in 1 and shift to a dominant AML-1-ETO population in
another, suggesting a correlation between PML-RAR expression and
therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS
was 17 months. Patients with white blood cell (WBC) count below 10 × 109/L at relapse had better survival than those with WBC
count over 10 × 109/L (P = .038). The duration
of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in
patients treated with As2O3 combined with
chemotherapy, compared with 12 of 18 relapses with
As2O3 alone (P = .01). Reverse
transcription polymerase chain reaction (RT-PCR) analysis in both newly
diagnosed and relapsed groups showed long-term use of
As2O3 could lead to a molecular remission in
some patients. We thus recommend that ATRA be used as first choice for
remission induction in newly diagnosed APL cases, whereas
As2O3 can be either used as a rescue for
relapsed cases or included into multidrug consolidation/maintenance clinical trials.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MOST ACUTE PROMYELOCYTIC leukemia (APL)
patients have the characteristic chromosome translocation t(15;17) that
juxtaposes PML gene on chromosome 15 and RAR gene on chromosome 17, forming a PML-RAR fusion gene.1-3 The PML-RAR protein
encoded by the fusion gene plays an important role in the pathogenesis
of APL.4 Although 70% to 85% of APL patients achieved
complete remission (CR) by cytotoxic chemotherapy,4 a
significant part of patients experienced severe complications,
necessitating intensive supportive care, which is hardly available in
most developing countries. The use of differentiation therapy with
all-trans retinoic acid (ATRA) has not only opened a new
approach in cancer treatment, but also rendered remission induction
relatively easy in most of the APL patients.5-6 Moreover,
the overall disease-free survival in the patients with CR achieved by
ATRA and consolidated as well as maintained by chemotherapy has been
significantly higher than those treated with chemotherapy alone for
remission induction, consolidation, and maintenance treatment. In spite
of these advances, 30% to 40% of patients would relapse within 5 years after CR. The majority of these patients lost sensitivity to ATRA
and chemotherapy and died shortly thereafter.
In early 1970s, Zhang et al7-9, from Harbin Medical
University in Northeast China, found that intravenous administration of
arsenic trioxide (As2O3 ) with relatively small
doses (10 mg/d) was effective in treating patients with APL, lymphoma,
and liver cancer. However, it was only recently that the therapeutic
effect of As2O3 was proven in APL patients by
several groups in China.10-13 Recently, Soignet et
al14 confirmed in Western population that As2O3 treatment achieved CR in 11 out of 12 relapsed APL patients.
In vitro studies indicate that As2O3 may exert
biphasic action on APL cells, induction of apoptosis at higher
concentrations (0.5 to 2 µmol/L), and partial differentiation at
lower concentrations (0.1 to 0.5µmol/L).
As2O3, at both high and low concentrations, is
able to trigger the degradation of PML-RAR fusion
protein.15 Interestingly, the drug was equally effective in
inducing apoptosis in ATRA-sensitive and -resistant APL
cells.16-17
Several important issues, nevertheless, remain to be addressed for
As2O3 treatment in APL. Is
As2O3 treatment equally effective for newly
diagnosed APL patients? Could the drug induce a molecular remission in
these patients as well as in relapsed cases? What could be the possible
prognostic parameters and suitable postremission treatment in relapsed
APL patients rescued with As2O3? In this report, we describe the results of a comprehensive study on 11 previously untreated and 47 relapsed APL cases.
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PATIENTS AND METHODS |
Patients.
The diagnosis of APL was established on the basis of clinical
presentation, morphological criteria of the French-American-British (FAB) classification, cytogenetic evaluation for t(15;17), and reverse
transcription polymerase chain reaction (RT-PCR) analysis for
PML-RAR transcripts. Two groups of patients, newly diagnosed and
relapsed APL, entered into this As2O3 treatment
multicenter study including five hospitals in Shanghai (Rui-Jin
Hospital, Ren-Ji Hospital, Xin-Hua Hospital, Zhong-Shan Hospital, and
Gan-Quan Hospital). Between July 1996 and April 1998, 11 cases of newly diagnosed APL without exposure to any antileukemia treatment were included into this study. Between December 1994 and February 1998, 47 relapsed patients (2 at the second relapse, 2 at the third relapse, and
43 cases at the first relapse) who had received ATRA for the remission
induction and chemotherapy/ATRA in the following consolidation and
maintenance therapy were enrolled. However, none of them were still
taking ATRA at the time of relapse. Informed consent was obtained for
every patient entering into this study. The main clinical and
hematological characteristics of the evaluated patients are shown in
Table 1. Of note, part of the data
concerning As2O3 induction therapy in 15 cases
in the relapsed group was presented in our previous
report.12
Induction therapy.
As2O3 solution was prepared by the Pharmacy of
Traditional Chinese Medicine in the First Hospital affiliated with
Harbin Medical University of China. The following protocol was used: 10 mg As2O3 (10 mL, 0.1% aqua solution) was
diluted in 500 mL of 5% glucose-normal saline solution for intravenous
drip over 2 to 3 hours per day, for 6-weeks duration. If necessary, a
second course was performed after an interval of 7 days. Those patients
who failed to reach CR after 2 courses were considered as nonresponders
(NR) and were treated with chemotherapy.
Supportive care.
Sequential measurements of complete blood cell count (every other day),
bone marrow (BM) cytology (every 10 days), renal functions, and hepatic
functions (every 1 to 2 weeks) were performed during As2O3 remission induction treatment.
Measurement of coagulation and fibrinolysis parameters, including
fibrinogen, DD dimers, fibrin degradation product (FDP), prothrombin
time, and activated partial thromboplastin time was performed by
standard methods for each patient before and during the
As2O3 treatment. Coagulopathy was treated at
the physician's discretion using low-dose heparin, platelet
transfusion, and fresh plasma. Patients were administered hydroxyurea
or moderate chemotherapy (Daunarubicin: 40 mg/m2/d × 3 d; Ara-C: 100 mg/m2/d × 3 to 5 d) when their white
blood cell (WBC) counts were over 30 to 40 × 109/L,
based on the observation that there seems to be less clinical syndrome
associated with As2O3-induced hyperleukocytosis
as compared with ATRA-induced ones. Symptomatic therapy was performed
without discontinuation of As2O3 when moderate
side effects occurred while As2O3 was withdrawn
in the case of serious toxic effects.
Definition of outcomes.
Achievement of CR required patients to have no clinical evidence of
APL, untransfused hemoglobin greater than 10g/dL, neutrophils greater
than 1.5×109/L, platelets greater than
100×109/L, BM to be normocellular or moderately
hypocellular with less than 5% promyelocytes, and absence of leukemic
cells with cytoplasmic Auer rods. Disease-free survival (DFS) was
defined as the time from CR to relapse, death from any cause, or
censoring of the data on the patients.
Follow-up.
After CR was achieved with As2O3, the patients
were treated by three different therapeutic protocols for
consolidation: (1) chemotherapy group: continuous treatment with
chemotherapy DA/MA monthly [Daunorubicin(D): 45mg/m2/d on
day 1 to 3 or Mitoxantrone(M) 8 mg/d on day 1 to 3, and Ara-C(A) 100 to
200 mg/d on day 1 to 7]. One course every 2 months in the first year,
every 3 months in the second year, and every 4 months in the third
year. (2) As2O3 group: 10 mg
As2O3 daily continuing 28 to 30 days as a
course with approximately 30 to 60 days interval between two cycles
within the first year, approximately 7 to 14 days as a course every 2 months over the second and third year. (3) Chemotherapy and
As2O3 combination group: chemotherapy was
administered as group (1) while As2O3 was used
as group (2), but during the interval of chemotherapy. No randomization
was performed due to the short supply of As2O3
for some of the patients and the refusal of some patients to further
use of chemotherapy or As2O3. Follow-up was
terminated on March 31, 1999.
Statistical analysis.
Association between pairs of patients' covariates, including
individual characteristics and the treatment indicator, was evaluated using Fisher's exact test and generalized exact test. Analysis of DFS
and overall survival were performed with Kaplan-Meier product-limit estimation.
Cytogenetic studies.
Metaphase chromosomes were prepared from BM cells after short-term
culture (24 hours). RHG-banding technique was used and karyotype
analysis was performed according to International System for Human
Cytogenic Nomenclature (ISCN).18
RT-PCR.
RT-PCR analysis for PML-RAR transcripts was performed according to
our previously described methods.19
Fluorescence in situ hybridization (FISH).
Dual-color FISH was performed with the probe YAC 185B2, P1 164 (biotinylated), and YAC 417D9, Co664 (digoxigenin-labeled) prepared by
nick translation for PML-RAR and AML1-ETO detection, respectively.
Chromosome painting was performed employing whole chromosome painting
(WCP) probes for chromosome 5,15,17, and 22 (Cambio, Cambridge, UK, & Oncor, Gaithersburg, MD). The procedure was as recommended
by the manufacturer. In brief, slides were denatured in 70%
formamide/2 × SSC (1 × SSC: 0.15 mol/L NaCl and 0.015 mol/L
natriumcitrate, pH 7.0) at 70°C. Then, the probes were applied to
denatured slides and hybridized at 37°C overnight. After
posthybridization washing, probes were detected using avidin conjugated
Texas Red and fluorescein isothiocyanate (FITC)-labeled antibodies.
DAPI was used as a counterstain.
Combination of Wright's staining and dual-color FISH.
BM samples collected before and 20 days after
As2O3 treatment from five patients (case
21,22,29,35, and 37) were subjected to simultaneous morphological and
FISH analysis. The procedures described by Haferlach et
al20 were used with modification. Briefly, mononuclear cell
fractions were isolated from BM aspirate by Ficoll-Hypaque density
gradient centrifugation, and were spun onto slides by cytospin
(Shandon, Runcorn, UK; 800 rpm, 4 minutes). For morphological
observation, Wright's staining was performed. Leukemic promyelocytes
or myelocyte-like cells were photographed by conventional
microphotography (Olympus, Tokyo, Japan), and the location
of the cells was documented. Slides were incubated in xylene for 5 minutes to remove the cedar wood oil, fixed in Carnoy's fixation
(methanol: acetic acid, 3:1) for 15 minutes, washed in phosphate-buffer
solution for 1 minute, and fixed again in paraformaldehyde for 1 minute. The PML-RAR fusion gene was detected by using t(15;17)
translocation DNA probe (Oncor, Gaithersburg, MD). Dual-color FISH was
performed following the manufacturer's instructions. The results were
observed through a triple-bandpass filter (Olympus) equipped on an
Olympus microscope and pictured with Kodak 400 film (Eastman Kodak,
Rochester, NY).
| |
RESULTS |
Newly Diagnosed Patient Group
CR.
There were 11 newly diagnosed APL patients entered into this study, 7 cases were treated with As2O3 and 4 with
combined As2O3 and chemotherapy. Eight (72.7%)
entered into CR, and the median time to obtain CR was 35 days (range,
30 to 36 days) with a median dosage of 295 mg
(Table 2). One patient died of cerebral
hemorrhage on day 1 of As2O3 treatment. The
other 2 patients (cases 2 and 8) died on day 15 after
As2O3 treatment.
Hyperleukocytosis.
Hyperleukocytosis, as defined by WBC count superior to 10 × 109/L, developed in 8 of the 11 (72.7%) newly diagnosed
patients with WBC counts from 26 to 183 × 109/L
(median, 41.5 × 109/L), before, or 5 to 20 days after
receiving As2O3
(Fig 1). WBC counts declined with moderate
chemotherapy in 2 cases and spontaneously in 4 cases, without
occurrence of the related adult respiratory distress syndrome (ARDS)
clinical syndromes. Among the others, 2 cases died, hyperleukocytosis
may be one of the causes leading to treatment failure in 1 patient.
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| Fig 1.
Hyperleukocytosis developed in newly diagnosed patients
during As2O3 treatment. * represents that
patient died on day 15 after As2O3 treatment.
Arrow indicates the time when chemotherapy was administered. Case 11 is
not shown here because the patient died on day 1 of
As2O3 treatment.
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Toxic effects.
The major As2O3-related toxicities, as listed
in Table 3, were skin reactions (rash,
itching, erythema) (3 of 11), gastrointestinal reactions (vomiting,
nausea, and diarrhea) (4 of 11), cardiac dysfunction (1 of 11), similar
to those in the relapsed group. However, hepatic damage occurred in 7 of 11 patients with elevation of the serum glutamic pyruvic
transaminase (SGPT) ranging from 82 to 918 IU/L (median, 266 IU/L;
normal range, 10 to 64 IU/L) and serum glutamic oxaloacetic
transaminase (SGOT) 58 to 934 IU /L (median, 114 IU/L; normal range, 10 to 42 IU/L). Among these 7 patients, symptomatic medication was
administered and withdrawal of As2O3 was
indicated when severe liver dysfunction occurred. Five patients
recovered and the other 2 failed. These 2 patients are worth particular
attention because of the development of lethal hepatic damage as
described below.
Case 2: a 33-year-old woman with previously untreated APL was treated
with 10 mg As2O3 daily and continued for 10 days. No previous history of hepatitis was noted and the tests for
hepatitis B virus (HBV) and hepatitis C virus (HCV) were negative. The
percentage of promyelocytes in BM decreased from 78.5% to 40%, WBC
count in peripheral blood increased from 1.1 × 109/L
to 9.5 × 109/L, whereas sequential measurement showed
that SGPT and SGOT increased from 52 IU/L and 53 IU/L to 633 IU/L and
576 IU/L, respectively. This patient died on day 15 with liver failure:
SGPT 918 IU/L, SGOT 934 IU/L, bilirubin 20.6µmol/L, and alkaline
phosphatase 65 IU/L in spite of intensive supportive care. Liver biopsy
was not performed because of patient and relatives' refusal. Renal function test was normal during the treatment. RT-PCR showed positive results of S-type PML-RAR transcript.
Case 8: a 34-year-old woman, with no history of liver dysfunction (SGPT
54 IU/L, SGOT 49 IU/L, bilirubin 4.7 µmol/L, alkaline phosphatase 76 IU/L), received As2O3 as induction therapy. One week later, liver toxicity occurred with SGPT (255 IU/L) and SGOT (305 IU/L) increased significantly, the WBC count and percentage of APL
cells in PB reached the highest level (50×109/L and
93%, respectively). She died of cerebral hemorrhage accompanied with
severe liver impairment (SGPT 900 IU/L; SGOT 905 IU/L bilirubin 14.1 µmol/L, alkaline phosphatase 73 IU/L) and hyperleukocytosis (50×109/L), despite timely withdrawal of
As2O3 and hepatic supportive treatments. Renal
function index was normal during the whole
As2O3 treatment. She had L-type fusion gene by
RT-PCR.
Disease-free survival.
After CR, five patients received chemotherapy for maintenance
whereas three patients received As2O3, one
(case 9) of the three latter cases being shifted to chemotherapy after
two courses of As2O3. With a median follow-up
of 12 months, all these eight patients are still in CR (range: 8 to 20 months) (Fig 2A).
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| Fig 2.
(A) Kaplan-Meier product-limit estimate of DFS from the
time of CR for newly diagnosed patients. (B) Kaplan-Meier product-limit
estimate of DFS and overall survival from the time of CR for relapsed
patients. (C) DFS in relapsed APL patients with regard to WBC count at
relapse. (D) DFS in relapsed APL patients between arsenic group and
combination group.
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Cytogenetics and molecular genetics data.
t(15;17) was found in cases 2 to 9 and 11 when diagnosed, whereas
cytogenetic analysis failed in cases 1 and 10 due to lack of metaphase
cells. All of the 11 newly diagnosed APL patients were PML-RAR
positive by RT-PCR at diagnosis. RT-PCR remained positive in four of
five patients when CR was obtained, but became negative after 1 to 3 months of consolidation with chemotherapy in three cases
(Fig 3).
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| Fig 3.
RT-PCR and follow-up data of 11 newly diagnosed patients.
D, at diagnosis; CR, complete remission; L/S, long/short-type isoform
of PML-RAR transcripts. Arrows indicate treatment protocol,
As2O3 or chemotherapy, as postremission
therapy.
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RELAPSED PATIENT GROUP |
Remission induction.
Among the 47 patients treated with As2O3, 4 died of cerebral hemorrhage at early days of treatment (one on day 7 and three on day 8) due to low platelet and low fibrinogen (3 cases) or tumor cell infiltration into the central nervous system (1 case). A
total of 31 were treated with As2O3 alone, 11 with combination of As2O3 and moderate
chemotherapy, and 5 with As2O3 and ATRA. A
total of 40 of 47 (85.1%) patients went into CR, whereas the CR rate
was 83.9% (26 of 31) when 31 patients receiving
As2O3 alone were analyzed (Table 2). The
overall median time for getting CR was 31 days, with a median dosage,
correspondingly, of 310 mg. In the 3 resistant patients (cases 10, 27, and 33), case 10 had 85% APL cells at the onset of relapse and the
leukemic cells in BM rose to 71.5% after a transient drop to 38% with
initial As2O3 treatment, losing response to
As2O3 or chemotherapy. There was no decrease in
blast percentage in case 27 after two courses of
As2O3 treatment, then the patient was treated
with chemotherapy but failed to respond to either. In case 33, the
percentage of promyelocytes in the BM increased from 19.5% to 94%
after nearly 2 months of treatment with As2O3
and the patient finally died.
Hyperleukocytosis.
Hyperleukocytosis developed during As2O3
treatment in 26 of the 47 relapsed patients (55%) with the WBC counts
ranging from 11.9 × 109/L to 167 × 109/L (median, 38 × 109/L) after 1 to 43 days (median, 17 days) of treatment. The WBC counts in 11 of 26 patients returned to normal after chemotherapy, including 1 patient who
developed ARDS on day 22 of As2O3 treatment when the WBC count was 67.0 × 109/L, whereas those in
the other 14 cases fell to normal spontaneously. One patient presenting
hyperleukocytosis died of cerebral hemorrhage with low platelet count
and low fibrinogen on day 7.
Side effects.
As2O3-related toxicities occurred in 12 of 47 with skin reactions, 10 of 47 with gastrointestinal reactions
(vomiting, nausea, diarrhea), 15 of 47 with liver dysfunction, 8 of 47 with cardiac dysfunction, and 5 of 47 with facial edema and neuropathy
(Table 3). Most of the side effects were modest and responded to
symptomatic treatment, further confirming our previous
report.12 There was no difference in frequency or in extent
of side effects between patients treated with
As2O3 alone and those with combination therapy (As2O3 + chemotherapy or
As2O3 + ATRA).
Disease-free survival.
The follow-up data were available in 33 patients, the other 7 were out
of follow-up. As shown in Fig 2B, the estimated DFS rates at 1 and 2 years of the 33 patients followed were 63.6% and 41.6%, respectively,
and the median DFS was 17 months. The estimated 1 and 2 year overall
survival rates were 72.1% and 50.2%, respectively, whereas the actual
median overall survival was 25 months (Fig 2B).
A number of factors with possible influence on the DFS were analyzed.
The disease status before As2O3-induced CR was
at first relapse in 29, at second relapse in 2, and at third relapse in 2 cases. Of note, 3 of 4 patients treated with
As2O3 at advanced stage (the second or third
relapse) relapsed again, compared with 14 of 29 treated at the first
relapse. More importantly, patients presenting for
As2O3 treatment with WBC lower than 10 × 109/L had DFS significantly better than those with WBC
higher than 10 × 109/L (P = .038)(Fig 2C).
For postremission therapy after As2O3-induced CR, 4 were treated with chemotherapy alone (from 8 to 17 months), 18 with As2O3 alone (from 7 to 48 months), and 11 with combination therapy (from 11 to 44 months). Disease recurrence
developed in 3 of 4 cases treated with chemotherapy alone, 12 of 18 with As2O3 alone, and 2 of 11 with the
combination, respectively. Therefore, the duration of CR also tended to
be related to postremission treatment protocols, with combination of
As2O3 and chemotherapy giving better DFS
compared with As2O3 alone (P = .01)
(Fig 2D).
Table 4 shows the outcomes of the 17 cases
that relapsed again after As2O3 treatment. Only
one (case 17) patient has regained durable CR for 20 months with
chemotherapy and ATRA as the maintenance treatment, whereas others
unfortunately died, although a short CR was obtained among 4 cases.
Cytogenetics and molecular genetics.
Karyotyping was performed successfully in 22 cases at diagnosis of
relapse. A total of 19 of 22 had t(15;17) and 3 cases had not. In
addition, a complex karyotype, 46,XY, t(5;15)(q14;q22), t(15;17)(q22;q11-21), ins(16;17)(p11p12;q?), was observed in case 11 when relapse occurred after As2O3-induced CR,
which was confirmed by dual-color painting with WCP probes
for chromosome 5, 15, 16, and 17.
RT-PCR results were obtained in 29 patients at the time of relapse
before As2O3 treatment. Among them, case 10 was
PCR negative for PML-RAR in spite of the fact that fusion gene
transcript was positive at first disease presentation. In case 27, both
PML-RAR and AML1-ETO were amplified by RT-PCR though only AML1-ETO
was detected by FISH. These two patients showed no response to
As2O3 induction as described above. In another
nonresponder (case 33), RT-PCR analysis was not obtained due to lack of material.
In the other patients, four cases (cases 4, 5, 25, and 38) had S-type
fusion genes, one of whom remained in CR until now, one died, and the
other two were lost to follow-up. The remaining 23 cases all showed
RT-PCR positivity for L-type transcript, among whom 9 remained in CR, 9 died. Follow-up data were not available in the remaining 5 cases.
RT-PCR data immediately after As2O3-induced hematological CR were available only in 15 cases. Positive PML-RAR fusion transcripts were detected in 14 of 15 cases, indicating that
As2O3 induction of clinical CR was not
associated with molecular CR in the majority of patients
(Fig 4). It is worth noting, however, that
RT-PCR negative results were obtained in two patients (cases 2 and 3, Fig 4) after long-time maintenance therapy (41 and 37 months,
respectively) with As2O3 alone.
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| Fig 4.
RT-PCR and follow-up data after postremission treatment
with As2O3 alone (A), or chemotherapy alone, or
chemotherapy/As2O3 combination (B) in 43 relapsed APL patients. D, at diagnosis; CR, complete remission; L/S,
long/short-type isoform of PML-RAR transcripts. * Indicates each
time of relapse.
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Presence of t (15;17) in partially differentiated APL cells occurs
during As2O3 treatment.
To evaluate the possible in vivo partial differentiation of APL cells
induced by As2O3, as suggested by our previous
report,12 fresh APL cells in BM were obtained from five
cases during remission induction and analyzed by morphological
examination and combination of Wright's staining and dual-color FISH.
These patients all had t(15;17) on chromosome karyotyping and
PML-RAR transcripts by RT-PCR at diagnosis of relapse. The
percentages of leukemic promyelocytes in BM was over 64.5% in four
cases, whereas the remaining one had only 11% promyelocytes. However,
the percentage of promyelocytes in BM decreased gradually in all the
five cases during As2O3 treatment. In contrast,
after 15 to 20 days of treatment, increased number of myelocyte-like
cells and many degenerating cells with condensed or coarse nuclei with
scanty cytoplasm ("nude" nucleus) was observed in both BM and
peripheral blood. The percentage of myelocyte-like cells in BM was
highest 20 to 25 days after the initiation of As2O3 treatment
(Table 5), however, terminally
differentiated elements such as polynucleated granulocytes did not
increase with As2O3 treatment.
Dual-color FISH generated two red and two green spots in normal
interphase cells, corresponding to PML and RAR genes, respectively. It can be expected that in APL cells with t(15;17) have a yellow signal
due to the fusion of one PML and one RAR allele in addition to one
red and one green signal (Fig 5A). This
three-color signal complex was observed not only in typical leukemic
promyelocytes before As2O3 (Fig 5B), but also
in more differentiated elements, such as myelocyte-like cells (Fig 5C),
during the treatment (Fig 5D), confirming that the partially
differentiated granulocytes were indeed derived from the leukemia clone
and not from the residual normal hematopoietic precursors.
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| Fig 5.
BM samples before (A and B) and during (C and D)
As2O3 treatment were collected for analyzing
the origin of differentiated myeloid cells. Morphological examination
(A and C) showed promyelocytes (A) and myelocyte-like cells (C), in
which one red, one green, and one yellow fusion signal could be
observed in the same cells (B and D). Arrows pointed yellow signals
that represented PML- RAR fusion gene.
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| |
DISCUSSION |
In the present study, we were able to conduct a multicenter clinical
research on 11 previously untreated and 47 relapsed patients. Eight of
eleven (72.7%) newly diagnosed patients achieved CR with the median
time of 35 days, similar to a previous report in China with a CR rate
of 73% in newly diagnosed APL patients.10 These results
are comparable to that obtained by ATRA in newly diagnosed patients. On
the other hand, 40 of 47 (85.1%) relapsed APL patients achieved CR.
Hence, As2O3 is able to induce high CR rate in
both newly diagnosed and relapsed APL patients. Among the 51 patients (8 primary and 43 relapsed) who received enough long
As2O3 remission induction, 3 cases should be
considered as nonresponders because they failed to achieve CR even
after two courses of the drug. Among these 3 patients, 1 had no
available cytogenetic and molecular data, the other 2 (cases 10 and
27), although presenting with t(15;17) and PML-RAR at first
diagnosis, had altered genotype of leukemic cells at relapse. Case 10 was negative for PML-RAR by RT-PCR, whereas case 27 developed a new
malignant clone with AML1-ETO fusion gene, which is characteristic of
AML-M2, in addition to PML-RAR. However, the clone with AML1-ETO
transcript was dominant because cytogenetically only t(8;21), but not
t(15;17), was detectable using the FISH method. The in vivo sensitivity
to As2O3 appears to require the expression of
PML-RAR, although a recent in vitro study showed that
As2O3- induced apoptosis of APL cells was
not dependent on PML or PML-RAR expression.21 In APL
cells, PML and/or nuclear body (NB) functions are lost because
PML-RAR displaces PML and other NB components to nuclear
microspeckles, whereas As2O3 appears to target
PML and PML-RAR onto NB and induce their degradation.22
Though As2O3 at relatively high concentrations (1 to 2 µmol/L) exerts apoptotic effect on a wide range of cell lines
including lymphoid lineage, in vitro differentiation-inducing effects
at low dose (0.1 to 0.5 µmol/L) were observed selectively in APL
cells (unpublished data). In accordance with our findings, partially
differentiated myeloid cells occurred in 8 of 11 newly diagnosed and 26 of 47 relapsed patients with hyperleukocytosis, and a large number of
myelocyte-like cells containing PML-RAR fusion gene emerged after 15 to 20 days of in vivo treatment with As2O3 (Fig
5). Notably, our previous pharmacokinetic data showed that except for a
short period (2 to 3 hours) due to intravenous drip, the in vivo plasma
concentration of As2O3 was low (<0.5 to 1 µmol/L) most of the time over the treatment course.12
One sensible issue on As2O3 treatment is the
adverse effects. In this study, when the newly diagnosed and relapsed
APL groups were compared, they showed almost similar incidence for
hyperleukocytosis, skin reaction, gastrointestinal reaction, and
cardiovascular system dysfunction. However, an unexpected finding was
that the hepatotoxicity was much higher in newly diagnosed patients
than in relapsed ones, because 7 of 11 (63.6%) (2 cases in grade 1, 3 cases in grade 2, and 2 cases in grade 3) newly diagnosed APL patients
developed hepatic dysfunction in contrast to 15 of 47 (31.9%) (14 cases in grade 1 and 1 case in grade 2) in relapsed cases (P = .001). None of the 7 newly diagnosed patients with liver toxicity
presented abnormal liver function tests or HBV or HBC antigens and
antibodies before As2O3 treatment. More
importantly, among these patients, 2 died with highest SGPT and SGOT
levels, 918 IU/L and 934 IU/L (case 2) and 900 IU/L and 905 IU/L (case
8), respectively. Because both patients died on day 15 of
As2O3 induction, a period not long enough to
achieve CR, it is difficult to evaluate the response with regard to the
reduction of leukemia cell population, though hyperleukocytosis
appeared in one case, suggesting a possible differentiation-inducing
effect of As2O3. Meanwhile, we did not find
severe hepatic damage happening in a group of relapsed APL patients
treated during the same period with the same batch of As2O3, eliminating the possibility of variation
in drug quality.
The significant difference between the two groups could be ascribed to
their distinct sensitivity towards the toxic effects of the drug.
Recent data suggested that intracellular antioxidant levels may be
involved in the defense of cells against arsenite genotoxicity.23 It was found that the activities of two
antioxidant enzymes, catalase and glutathione peroxidase, were 5.4-fold
and 5.8-fold lower in xrs-5 cells than those in Chinese hamster ovary (CHO)-k1 cells. The xrs-5 cells are x-ray hypersensitive CHO subclone with higher sensitivity to sodium arsenite inhibition of cell growth
and micronuclei induction compared with CHO-k1 cells.24 Therefore, we postulate that in relapsed patients,
long-term treatment with ATRA and/or chemotherapeutic drugs could
induce or modify some antioxidant enzymatic system and enhance the
antioxidant ability, so they had better tolerance to arsenic than newly
diagnosed patients. Another possibility is that patients with higher
susceptibility to As2O3-induced damage may
belong to a special group with reduced capacity of drug detoxication
and could be already selected out through previous ATRA/chemotherapy.
The shortage of materials from our newly diagnosed patient group,
unfortunately, did not allow us to perform further study on the enzymes
related to the toxicity of As2O3. Because
remission induction with ATRA in newly diagnosed APL patients never
gives rise to such a severe liver toxicity and the retinoic acid
syndrome now can be easily handled, we believe that ATRA should be used
as the first-line drug for remission induction, whereas
As2O3, until further evaluation of its
toxicity, should be incorporated into a multidrug
consolidation/maintenance therapy during remission or as a rescue in
relapsed patients. Additionally, drugs can be used to relieve the side
effects of As2O3 in cases of severe
intoxication. As Moore et al25 reported, 2,3-dimercaptopropanesulphonate (DMPS) was able to reduce toxicity of
As2O3. Dimercaptosuccinic acid (DMSA) analogues
were also put forward to decreasing the tissue content of arsenic in
acute As2O3 poisoning in NMRI male
mice,26 suggesting that potentially protective measures are
available while using As2O3 to treat the malignancies.
ATRA has been proven to have a highly specific effect on the newly
diagnosed APL patients with t(15;17) and PML-RAR expression. Previous studies showed, however, when ATRA was used as the maintenance treatment alone after CR achieved with the same drug, most patients relapsed within 6 months, mainly because of the development of drug
resistance. On the contrary, long-time remission was reported in a
series of 32 APL patients treated with As2O3 as
single therapeutic agent, among whom one fourth had a survival time of
more than 10 years,9 suggesting long-term treatment with
As2O3 may induce molecular remission. In the
present work, we analyzed this issue by using RT-PCR before and after
As2O3-induced CR. It was found that immediately
after CR, the leukemic clone persisted in 4 of 5 newly diagnosed
patients and 14 of 15 relapsed patients investigated. Therefore,
As2O3 induction is not sufficient to induce a
molecular remission. Nevertheless, a relatively long DFS (48 and 44 months in cases 2 and 3, respectively) with negative RT-PCR was
observed in 2 cases in the relapsed group, indicating that long-term
use of As2O3 alone could indeed lead to a
molecular remission in some patients. This result suggests that
As2O3 may be more potent than ATRA in terms of
maintaining molecular/clinical remission and justifies inclusion of
As2O3 into multidrug postremission treatment in
future clinical trials.
The reason that the effect of ATRA is less durable than
As2O3 in treating APL patients could be
multiple. As a vitamin-like hormone, ATRA may induce more easily a
metabolic resistance than As2O3, an inorganic
small compound. Secondly, ATRA mainly induces differentiation of APL
cells, whereas As2O3 could induce both apoptosis and partial differentiation of these leukemia cells. Thirdly,
although the two drugs share a common target, PML-RAR, As2O3 may exert an effect on a wider spectrum
of proteins, whereas the action of ATRA is limited to RA receptors
only. For example, As2O3 could modify the
phosphorylation of transcription factors such as AP1 or DNA
hypomethylation27 or cause downregulation of bcl-2
protein.15
One of the major purposes of this study was to evaluate the outcome of
relapsed APL patients after CR achieved with
As2O3, to find out possible prognostic factors
and what could be the best postremission treatment. Among the 33 relapsed APL patients available for follow-up, the median DFS time was
17 months, whereas relapse occurred in 17 patients. As expected,
patients at the second or third relapse before
As2O3-induced CR seemed to relapse more
frequently (3 of 4) than those at the first relapse before As2O3-induced CR (14 of 29), although more
cases should be studied. Next, patients with lower tumor burden as
reflected by low WBC counts (below 10 × 109/L) showed
statistically better DFS than those with higher tumor burden (WBC > 10 × 109/L) (P = .038). When different
treatment protocols were compared, clinical outcome seems to be
associated with postremission therapy, since there was only 2 relapses
of 11 cases in the combination therapy group, compared with the 12 of
18 with As2O3 treatment (P = .01).
These results indicate that combination therapy may be the choice of
treatment to achieve a longer survival. Furthermore, ATRA could still
have a role to play even in patients relapsed after
As2O3, because among 17 cases who relapsed
again after As2O3 induced-CR, only one achieved
a DFS for 20 months after reinduction with ATRA and with combined
chemotherapy/ATRA as maintenance treatment. It is possible that APL
cells in this case restored sensitivity to ATRA, as suggested by a
recent study that As2O3 could affect the RA
response through changes in proteins involved in the RA pathway.28 If this is true, then a new strategy to prolong
the survival could be designed in which ATRA is incorporated into an
As2O3 + chemotherapy + ATRA triple combination
after As2O3-induction in relapsed APL patients.
An important issue is how to use the three medications. Adverse
reactions were similar among relapsed patients who received the
combination of As2O3 with ATRA or chemotherapy or As2O3 alone. However, in vitro studies
showed that the use of ATRA and As2O3 might
interfere with the full effect of each drug. Shao et al29
found that the ATRA-induced cell differentiation was interfered by
As2O3 addition, and the addition of ATRA
reduced As2O3-induced cell apoptosis. Hence,
sequential use of the two drugs may be better than simultaneous use, as
also suggested in SCID mouse-APL model (Chen et al, in preparation).
In conclusion, As2O3 treatment can lead to a
high CR rate in both newly diagnosed and relapsed APL patients.
However, the severe hepatic adverse effect in some patients does not
support As2O3 to be used as a first-line
remission-inducing drug. Finally, the
As2O3/chemotherapy combination after
As2O3-induced CR in relapsed patients yielded
better DFS than As2O3 or chemotherapy alone. Long-term use of As2O3 alone is able, at least
in part of the patients, to induce a molecular remission, justifying
further investigation of its use in a multidrug maintenance therapy
after ATRA induced CR and chemotherapy consolidation, in a hope that the DFS in APL could be further improved.
| |
NOTE ADDED IN PROOF |
The data listed as unpublished in Discussion were published in J
Natl Cancer Inst 91:772, 1999.
| |
ACKNOWLEDGMENT |
The authors thank all members of Shanghai Institute of Hematology for
their support and encouragement and thank Prof Anne Hagemeijer for
kindly giving us the P1 164 and Co664 clones that were used as probes
for FISH in the detection of AML1-ETO. We also thank Prof Dao-Pei Lu
from Beijing Medical University, Beijing, China, Prof. Laurent Degos
from Saint-Louis Hospital, Paris, France, and Prof Ryuzo Ohno from
Hamamatsu University School of Medicine, Japan, for constructive discussion.
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FOOTNOTES |
Submitted January 5, 1999; accepted July 7, 1999.
C.N. and H.Y. contributed equally to this work.
Supported in part by the Chinese Climbing Project, National Natural
Sciences Foundation of China, Shanghai Municipal Commission for
Sciences and Technologies, Samuel Waxman Cancer Research Foundation, and the Clyde Wu Foundation of Shanghai Institute of Hematology.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sai-Juan Chen, MD, PhD, or Jiong Hu,
MD, Shanghai Institute of Hematology, Department of
Hematology/Oncology, Rui Jin Hospital, Shanghai Second Medical
University, 197 Rui Jin Road II, 200025, Shanghai, China; email:
zchen{at}ms.stn.sh.cn.
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ABSTRACT
INTRODUCTION