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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Hematopathology, Biostatistics,
and Leukemia, The University of Texas MD Anderson Cancer Center,
Houston.
Myelodysplastic syndrome (MDS) is a disease characterized by
ineffective hematopoiesis. There are significant biologic and clinical
differences between MDS and acute myeloid leukemia (AML). We studied a
cohort of 802 patients, 279 (35%) with newly diagnosed MDS and 523 (65%) with newly diagnosed AML, and compared clinical and biologic
characteristics of the 2 groups. Complete clinical and cytogenetic data
were available on all patients, and a subgroup of patients was studied
for apoptosis, angiogenesis, proliferation, and growth factors. Our
results demonstrate that MDS is a discrete entity that is different
from AML and is characterized primarily by increased apoptosis in early
and mature hematopoietic cells. Using cell sorting and loss of
heterozygosity, we demonstrate that the leukemic cells from MDS
patients are capable of differentiation into mature myeloid cells and
monocytes. We also demonstrate that there is a significant overlap
between AML and MDS when MDS is defined on the basis of an arbitrary
percentage of blasts of 20% or 30%. These data suggest that despite
similarities between AML and MDS in their responses to treatment and
outcomes, MDS is biologically and clinically different from AML and
should not be considered an early phase of AML. The data indicate that
MDS must be better defined on the basis of its biology rather than the
percentage of blasts; further, the data suggest that there is a need to
develop therapeutic approaches that specifically address the biologic abnormalities of MDS.
(Blood. 2002;100:791-798) Myelodysplastic syndromes (MDS) are a heterogeneous
group of diseases characterized by active but ineffective hematopoiesis leading to pancytopenia.1-5 MDS has been recognized for
more than 50 years and has been called preleukemia, smoldering
leukemia, oligoblastic leukemia, and refractory anemia. Most patients
with this syndrome die without progressing to overt acute
leukemia.1-5 The term MDS reflects the presence of
dysplasia in bone marrow and peripheral blood. Dysplasia may reflect
disordered maturation and fragmentation of the nuclear structures, both
of which are signs of increased apoptosis.6
There is significant clinical variability in MDS.7-10
Patients with severe cytopenia, increased percentage of blasts, or
cytogenetic abnormalities have clinical outcomes that are not
significantly different from those seen in acute myeloid leukemia (AML)
(P = .1, log-rank test).10 On the other hand,
patients with none of these features are likely to live several years.
According to the French-American-British (FAB) classification, MDS is
said to be present in patients who have less than 30% blasts in bone
marrow and peripheral blood and have evidence of ineffective
hematopoiesis.11,12 If 30% blasts are present, AML is
diagnosed. The 30% cut-off rate is arbitrary. A new classification proposed by the World Health Organization (WHO) reduces the maximum percentage of blasts from 30% to 20%, taking into consideration the
fact that patients with 20% to 30% blasts (previously called refractory anemia with excess blasts in transformation [RAEB-T]) might have AML.13-15 The proposed new classification was
based on several reports suggesting that in addition to similarities in
the natural history of RAEB-T and AML, RAEB-T responds to combination chemotherapy in a fashion similar to that of AML. However, it is
important to note that this similarity in outcome does not necessarily
imply that AML and MDS are biologically similar. Here we compare the
biologic characteristics of AML and MDS. We hypothesized that the
clinical differences between MDS and AML reflect biologic differences.
We investigated the basis for the peripheral pancytopenia and confirmed
that apoptosis in bone marrow prevents cells from reaching peripheral
blood. We also hypothesized that the leukemic cells are capable of
differentiation. Using loss of heterozygosity (LOH) and
X-chromosome activation, we demonstrated that malignant cells in MDS
patients could differentiate to mature hematopoietic cells. We
hypothesized that the biologic differences between MDS and AML are
clinically relevant, and we studied the clinical impact of these
biologic markers when MDS is treated as AML. We also found that there
is significant overlap between MDS and AML when the 2 diseases are
separated based on the percentage of blasts. Although our data suggest
that the separation of the 2 diseases as recommended by the FAB
classification is helpful, classification based on the biology of MDS
is needed.
Eight hundred two patients with newly diagnosed AML or MDS who
were treated at The University of Texas MD Anderson Cancer Center
between 1994 and 1998 were reviewed. Included were 133 patients with
RAEB-T, 85 with RAEB; 38 with chronic myelomonocytic leukemia (CMML),
15 with refractory anemia (RA), 6 with refractory anemia with ring
sideroblasts (RARS), and 523 with AML. The diagnosis of RAEB-T was
based on the presence of Auer rods in 13.3% of the patients, more than
5% blasts in peripheral blood in 26% of the patients, and more than
20% blasts in bone marrow in the rest of the patients. All patients
classified as having RAEB-T based on the presence of Auer rods had
increased blasts (more than 5%). Patients with the t(15;17)
translocation were excluded from the analysis because of the specific
molecular abnormality and clinical course. Patients with inversion 16 and t(8;22) are automatically classified as AML in our institution
regardless of their percentage of blasts. Patients who had a diagnosis
of MDS that did not require immediate therapy were not included in this
study. MDS patients were treated if they required transfusion, had
platelet counts less than 50 000/µL, had infection or bleeding, or
had blast counts in bone marrow greater than 10%. This group of
patients is heterogeneous, and diagnoses could not always be
established with certainty. Therefore, these patients were excluded
from our analysis. When the disease progressed, they were re-evaluated
and treated with chemotherapy. Based on recent evaluation of the
International Prognostic Scoring System (IPSS), the population of MDS
patients seen at MD Anderson may be different than what is diagnosed in general hospital populations or even in other referral centers because
the IPSS system does not confirm the clear separation of IPSS groupings
reported by many other studies in the literature.16 CMML
patients are overall different from MDS patients, and the new WHO
classification suggests separating CMML from MDS. However, CMML
patients show high levels of apoptosis, and it remains controversial whether CMML can be divided into dysplastic disease and proliferative disease. All data and studies were analyzed after excluding CMML, and
we found no change in our conclusion. For these reasons, we did not
separate patients with CMML from the rest of the MDS patients. All MDS
patients underwent AML therapy, which was based on ara-C. Therapy in
these patients can be divided into 3 arms: idarubicin + ara-C
(IA), topotecan + ara-C (TA), and fludarabine + ara-C + idarubicin (FAI).
Clinical and laboratory data were collected from the Leukemia
Department database. Plasma, serum, and bone marrow samples were
collected on subgroups of patients in random fashion without specific
selection. To eliminate any possible confounding in study results by
freezing and thawing of samples, additional patient samples were
analyzed prospectively for apoptosis and proliferation without freezing
and thawing. These patients were diagnosed and treated fairly recently
and had only short follow-up; thus, they were not included in most of
the clinical analysis. The distribution of overall values was similar
using all methods.
Antecedent hematologic disease (AHD) is defined as a history of
abnormal blood count (hemoglobin less than 12 g/dL, or neutrophils less
than 1500/µL, or WBC greater than 10 000/µL or less than 4000/µL, or platelet count less than 150 000/µL) documented to be
present for at least 1 month before patient evaluation at our center.
AHD is considered 0 when there is no history of AHD. Clinical remission
(CR) is defined as a marrow sample showing less than 5% blasts,
peripheral platelet count more than 100 000/µL, and peripheral
neutrophil count more than 1000/µL.
Enzyme-linked immunoadsorbent assays
Protein extraction
Measurement of caspase-3 activity Caspase-3 was measured using a tetrapeptide Ac-DEVD-pNA (prepared by Calbiochem, San Diego, CA). As recommended by the manufacturer,21 100 µL reaction mixture consisted of 50 µg cellular protein extracts and 200 µM Ac-DEVD-pNA in 1× assay buffer (100 mM NaCl, 50 mM HEPES,10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1% CHAPS at pH 7.4). A positive control consisted of the same components plus 30 U human recombinant caspase-3 (1 U enzyme is the amount required to release 1 pmol pNA from 200 µM DEVD-pNA per minute at 25°C). Two negative controls were also used, in which either the cell extract or the substrate was not added to the reaction mixture. An additional negative control was included in which cell extracts were treated with caspase inhibitor before the reaction. All reactions were allowed to proceed for 3 hours at 25°C, and optic density at 405 nm was measured every 30 minutes using a spectrometer (Elx808; BioTek Instruments, Winooski, VT). Optical densities were plotted as a function of time, and the slope of the initial linear portion of the curve was used as a measurement of the amount of caspase-3 activity. Mean caspase-3 activity of peripheral blood mononuclear cells from 22 healthy controls was assigned a value of 1. Activity in the leukemic and MDS samples was normalized to the mean of controls. Spontaneous hydrolysis of substrate in negative controls was negligible (less than 0.01).Caspase-3 activity testing was repeated on 60 samples on 2 different days. No significant differences in results were found for the repeated tests. Mitochondrial potential measurement (DePsipher assay) Bone marrow samples were collected in EDTA tubes (minimum of 106 cells), and the red cells were lysed and washed twice. An aliquot of 0.5 µL DePsipher assay (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide) (Trevigen, Gaithersburg, MD) was added, and the mixture was incubated at 37°C in 5% CO2 for 20 to 30 minutes. Cells were washed with phosphate-buffered saline (PBS) then analyzed on FACScalibur (Becton Dickinson, Mansfield, MA) immediately.22-25Measurement of annexin V Cells were isolated using double-density Histopaque 1119 and 1077 to capture mononuclear and polymorphonuclear cells. Both cell populations were mixed, washed, and stained with annexin V and propidium iodide as recommended by the manufacturer (Becton Dickinson, Mansfield, MA).26,27 Cells were also costained with CD14 and CD34. Briefly, phosphate-buffered saline (PBS)-washed cells were incubated with propidium iodine and fluorescein isothiocyanate-conjugated annexin V antibodies for 15 minutes, washed, processed, and acquired by FACScalibur within 5 minutes of staining.Measurement of bromodeoxyuridine incorporation The commercial kit provided by PharMingen/Becton Dickinson (San Diego, CA) was used. Briefly, cells were washed twice, and 0.5 mL 1× PBS w/NaAz was added with 4 mL RPMI. Cells in similar number were prepared similarly in a different well. Bromodeoxyuridine (BrdU) was then added (1 µL/mL) to one of each pair of wells and was incubated for 45 minutes. Cells were then washed and costained with CD34 according to a standard procedure.28,29Loss of heterozygosity and X-chromosome activation studies Various cell subpopulations (CD34+, CD14+, CD19+, CD3+) were sorted using magnetic beads and AutoMACS columns as recommended by the manufacturer (Miltenyi Biotec, Auburn, CA).30,31 Sorted cells fractions were more than 50% pure when analyzed using CD34, CD64, CD20, and CD7. Maturing myeloid cells and polymorphonuclear cells were separated by negative selection.DNA was isolated using standard techniques as previously described.32 Microsatellite markers were purchased from Applied Biosystems (Foster City, CA). In our study of X-chromosome activation, we amplified the human androgen receptor locus (HUMARA) using primers and a method described by Busque et al.33 All primers were labeled with FAM, HEX, or TAMRA fluorescent dye (Perkin-Elmer, Norwalk, CT). In the HUMARA assay, the DNA was digested with HpaII and RsaI using a standard procedure. DNA was amplified using standard techniques.33-38 Briefly, samples were activated at 95°C for 12 minutes, then amplified at 94°C for 30 seconds and at 60°C for 30 seconds for 30 cycles on a 9700 Perkin-Elmer thermal cycler in a total volume of 25 µL. Polymerase chain reaction (PCR) was performed using AmpliTaq Gold DNA polymerase. Aliquots (0.6 µL) of the PCR reaction were mixed with 0.1 µL of size standard (GENESCAN 2500-ROX) and analyzed using the ABI 310 machine. Automatically collected data were analyzed by using GENESCAN software (version 1.2) as described in the manufacturer's manual. Statistical analysis Wilcoxon rank-sum tests were used to compare baseline clinical and biologic characteristics of the MDS and AML groups for continuous risk factors, whereas 2 analysis (or Fisher exact test)
was used for categorical variables. Survival distribution curves were
estimated by the method of Kaplan and Meier. The univariate Cox
proportional hazard model was used to evaluate a possible association
between survival duration and each risk factor.
Univariate analysis was used to identify adverse risk factors
for achieving complete remission (CR) by using
Clinical features Of the 802 patients for whom clinical data were available, 279 (35%) had MDS and 523 (65%) had AML. Approximately 80% of patients with advanced MDS were dead within 2 years of induction therapy, a mortality rate not significantly different from that seen in patients with AML. Despite this similarity in overall survival rates, MDS in these patients represented a distinct disease that is clinically different from AML. In fact, most of our MDS patients lived with and died of MDS without it transforming to AML. MDS evolved to acute leukemia (30% or more blasts) in only 36 (13%) of the patients. However, these patients were on therapy, and most died of infection or bleeding. In contrast, significant numbers of AML patients had high percentages of blasts despite the fact that they died of infection or bleeding. The possibility remains that the low percentage of transformation was caused by the death of cells through chemotherapy. Regardless of transformation, MDS is an aggressive and deadly disease. Table 1 compares the clinical and laboratory characteristics of the MDS and AML groups. The MDS patients were older, more frequently had poor prognosis cytogenetics ( 5, 7, 11q23, +8), and had lower platelet, bone marrow
blast, and WBC counts. The initial distinction between AML and MDS for this study was based on the presence of less than 30% blasts in the
bone marrow and peripheral blood. However, evaluation of the percentage
of blasts in the bone marrows of these patients clearly shows gradual
changes in number without clustering (Figure
1).
The lack of bimodal distribution suggests that the percentage of blasts is a continuous variable and may not be the best way to distinguish MDS from AML, thus raising questions regarding the validity of using a cut-off point, whether 20%, 30%, or another rate. Better criteria based on the biology of the disease should be used to distinguish MDS from AML. Increased apoptosis in MDS MDS is characterized by the presence of dysplasia in myeloid, erythroid, and megakaryocyte cells. Close examination suggests that the dysplastic changes may represent increased apoptosis. Condensation and fragmentation of the nucleus and clumping of the chromatin seen in MDS are all known characteristics of apoptosis. Raza et al39-42 reported increased apoptosis in MDS using a methodology based on in situ end-labeling. We used annexin V analysis, mitochondrial membrane potential, and caspase 3 activity to compare apoptotic activity in AML and MDS samples. Annexin V and mitochondrial potential analyses were performed in prospective fashion on patients with newly diagnosed disease seen at our institution. As shown in Figure 2, the expression of annexin V was significantly higher in patients with MDS than in those with AML (Wilcoxon rank-sum test, P < .0001) (Figure 2). By costaining with CD34, we demonstrated that the increase in apoptosis was not restricted to mature cells but was also seen in CD34+ immature blasts (Figure 2) (Wilcoxon rank-sum test, P < .0001). Further analysis showed that increased apoptosis in CD34+ cells can be demonstrated in RA, RARS, RAEB-1 (5%-9% blasts), RAEB-2 (10%-19% blasts), and RAEB-T (20%-29% blasts) compared with AML.
We also evaluated mitochondrial membrane potential as a means of
measuring apoptosis. We used a dye (DePsipher) that aggregates and
turns orange-red when mitochondrial membrane is polarized (Figure
3) but remains monomeric green when the
membrane potential is disturbed. Disturbance of the mitochondrial
membrane has been demonstrated to be a sign of apoptosis. Upon
analyzing bone marrow samples from patients with MDS and AML, Wilcoxon
rank-sum analysis showed a significantly greater loss of mitochondrial
potential in MDS than in AML (P = .007) (Figure 3).
Further analysis showed increased apoptosis in RA, RARS, RAEB-1,
RAEB-2, and RAEB-T as compared with AML. Similar results were also
obtained when we gated only the polymorphonuclear cells, suggesting
that the difference in apoptosis between AML and various subgroups of
MDS are not simply caused by higher percentages of blasts in AML
(Figure 3). As shown in Figures 2 and 3, there is some difference
between annexin V and mitochondrial potential in the RAEB-T group. This may reflect the fact that mitochondrial potential measures early apoptosis, whereas annexin V measures late apoptosis. Overall there was
excellent correlation between annexin V and mitochondrial potential
(P < .0001) when all samples were considered and when only RAEB-T patients were considered.
Apoptosis was also measured using caspase 3 activity in cell lysates
from 36 patients with MDS and 54 with AML; this assay showed
significantly greater caspase 3 activity in MDS than in AML
(P = .04, Wilcoxon test) (Figure
4). When high or low caspase 3 activity
levels (using the median as a cut-off point) was used in predicting
diagnosis, the predictive association was significant (P = .01). These data confirm that increased apoptosis is
one of the characteristics distinguishing MDS from AML.
Increased proliferation in MDS patients Using BrdU incorporation to measure DNA synthesis, we demonstrated that cell proliferation was greater in MDS than in AML (Figure 5) (P = .03). Analysis of CD34+ cells also showed increased proliferation in CD34+ cells in RA, RARS, RAEB-1, RAEB-2, and RAEB-T compared with AML (P = .01) (Figure 5).
MDS cells are capable of differentiation Several fluorescence in situ hybridization studies in patients with MDS with cytogenetic abnormalities have demonstrated the capability of MDS cells to differentiate to mature myeloid and erythroid cells.1 Clonality assays using the X chromosome also showed the ability of MDS cells to differentiate. We used magnetic beads to sort blasts (CD34+), monocytes (CD14+), T cells (CD3+), and polymorphonuclear cells from 10 female patients who had MDS and cytogenetic abnormalities involving chromosome 5 or 7, or both, and studied clonality using X chromosome activation and LOH (Figure 6). As shown in Table 2, mature polymorphonuclear cells always showed clonality, confirming the ability of leukemic cells to differentiate. Interestingly, monocytes in some patients with MDS without monocytosis can also be clonal. The possibility of residual normal mature polymorphonuclear cells cannot be ruled out using this methodology. All studied patients had either5 (or 5q) or 7 (7q) to demonstrate LOH. Interestingly, in 2 patients we found clonality in T cells using X-chromosome activation,
but we did not find clonality using LOH, which may represent
X-chromosome usage bias inactivation (the tendency to inactivate one
and not the other X chromosome) rather than actual clonality. In that regard, most of the patients with AML had too few mature cells for
isolation and clonality study. We were able to study mature polymorphonuclear cells in 2 AML patients and found no clonality by LOH
in mature cells in one patient. The second patient demonstrated clonality in mature cells, and the possibility of contamination by
immature cells cannot be ruled out.
Inverse correlation between apoptosis and percentage of blasts To investigate the relationship between percentage of blasts and apoptosis, we grouped the AML and MDS patients and correlated apoptosis in the CD34+ cells (blasts) as measured by annexin V with the number of CD34+ cells counted. As shown in Figure 7, the extent of apoptosis decreased significantly with an increased percentage of CD34+ cells (R =0.2; P = .00001). However, a
significant number of patients were identified who had low numbers of
blasts and low apoptotic activity (Figure 7). At the same time, some
patients had apoptotic activity and high percentages of blasts.
Furthermore, the changes in apoptotic activity appear to be gradual and
show no sharp change at the blast counts of 10%, 20%, or 30%. An
inverse correlation between percentage of blasts in the bone marrow and caspase 3 activity was also identified by the Spearman correlation test
(P = .002; R = 0.21). A low percentage of
blasts was associated with low caspase 3 activity, and, in rare
patients, a high percentage of blasts was associated with high caspase
3 activity. These data suggest that there is some overlap between AML
and MDS when the division is based on blast count only. Clearly,
apoptotic activity is a dominant feature that distinguishes MDS from
AML, and the exceptions (MDS patients without increased apoptosis) may
represent patients with early AML discovered while the percentage of
blasts is still low.
Clinical relevance of the biologic differences between AML and MDS The data described above demonstrate that significant differences exist between AML and MDS. Major differences between the group of patients with MDS and those with AML are listed in Table 1. We evaluated whether these variables have a different prognostic value in AML than in MDS. Table 3 shows the results of the univariate survival analysis of these factors in AML and MDS. Overall, the prognostic values of most of these factors are similar in AML and MDS, which reflects the lack of significant difference in survival between AML and MDS using the current therapeutic approaches. These patients were treated using 1 of 3 arms: idarubicin + ara-C (IA), topotecan + ara-C (TA), and fludarabine + ara-C + idarubicin (FAI). There were significant differences in survival between the 3 arms when univariate analysis was used. However, this difference was not significant when adjusted for age. Multivariate analysis showed no difference between AML and MDS in survival regardless of the treatment arm or age. Multivariate analysis using the logistic regression model was performed and established that only age, TNF- , and cytogenetics were
significant (P = .0002, .003, and .03, respectively).
When we compared these variables in patients who achieved CR with those
who did not achieve CR, some differences in the levels of these
variables were seen (Table 4). Patients
who did not achieve CR had a greater tendency toward high levels of
caspase 3 activity. In addition, when we analyzed annexin V in
CD34+ cells, patients with higher percentages of annexin
V+/CD34+ cells had lower chances for achieving
CR. This suggests a distinct clinical behavior for patients with
increased apoptosis. Nonresponders also had higher levels of
These data suggest that a biologic difference exists between patients with high apoptotic activity and those with low apoptotic activity. Because outcomes in AML and advanced MDS using the current therapy were no different, delineating significant clinical differences between the 2 diseases is difficult.
The concept of MDS as a preleukemic or early leukemic process may not be accurate. Despite the similarities between MDS and AML, most patients with MDS die without their disease evolving to leukemia. Currently, treatment outcomes for AML and advanced stages of MDS remain poor, without significant differences in survival rates between the 2 diseases. Although further studies using large numbers of patients are needed, our data suggest that patients with high apoptosis are more likely not to respond to current therapy. Similarity between AML and MDS in survival using the current therapeutic approaches does not imply that the 2 diseases are the same. For example, survival in small cell lung cancer is similar to that in AML, but we do not consider the 2 diseases the same because there are biologic differences. Diagnosis of MDS based on the percentage of blasts allows for
significant overlap between AML and MDS, making assessment of differences in clinical characteristics and responses to therapy between the 2 diseases more difficult. When we grouped AML and MDS
patients together and investigated whether any of the biologic markers
make a difference in achieving CR, caspase 3 activity and annexin V
positivity in CD34+ cells
Submitted May 23, 2001; accepted April 2, 2002.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maher Albitar, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Box 72, 1515 Holcombe Blvd, Houston, TX 77030-4095; e-mail: malbitar{at}mdanderson.org.
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Top
Abstract
Introduction
2-microglobulin, IL-6, TNF-
the major biologic markers that
distinguish AML from MDS
cells to undergo apoptosis in myelodysplastic marrows.
Int J Hematol.
1999;69:152-159