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Keywords:

  • myelodysplastic syndromes;
  • flow cytometry;
  • response prediction;
  • azacitidine

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

Background

In intermediate-2 (Int-2) and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological improvement and prolonged overall survival (OS) in patients who respond to therapy. However, only half of the patients who are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients.

Methods

Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia, or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS).

Results

The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p = 0.02). A low pretreatment FCSS was associated with significantly better OS compared with a high pretreatment FCSS (p = 0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p = 0.006).

Conclusions

Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine. © 2014 International Clinical Cytometry Society


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of bone marrow (BM) disorders characterized by ineffective hematopoiesis resulting in peripheral cytopenias and increased risk of acute myeloid leukemia (AML). The WHO classification, International Prognostic Scoring System (IPSS) and the revised IPSS provide prognostic information in MDS patients [1]. By using IPSS, MDS patients can be divided into four risk groups with respect to survival and progression to AML: low, intermediate-1 (Int-1), intermediate-2 (Int-2), and high risk [2]. Approximately 29% of newly diagnosed MDS cases are Int-2 and high risk MDS [3]. Intermediate-2 and high risk patients may benefit from intensive treatment approaches. At present, the only curative treatment is allogeneic stem cell transplantation. However, the majority of MDS patients are not eligible for intensive treatment schedules due to increased age in combination with comorbidities [4]. Low-intensity treatment regimens are currently available for these patients. Azacitidine has been approved for treatment of Int-2 and high risk MDS, chronic myelomonocytic leukemia (CMML), and AML patients with 20–30% blasts. Recently, prognostic scores were designed for MDS patients treated with azacitidine, using platelet doubling, defined as an at least two-fold increase in platelet count after the first cycle of azacitidine or performance status, cytogenetics, the presence of circulating blasts, and red blood cell transfusion dependency as components of the score [5, 6]. Lower response rates were found in patients with abnormal karyotype and >15% BM blasts. Previously, it was shown that a flow cytometric scoring system (FCSS) was able to estimate survival and relapse after allogeneic hematopoietic stem cell transplantation in patients with MDS [7, 8]. The FCSS is a scoring system that allows for a numerical display of immunophenotypic aberrancies in the (im)mature myelomonocytic lineage in the BM [7]. Scores are generated by weighed scoring for the number of abnormalities in the myelomonocytic compartment and for the percentage of progenitor cells; that is, high scores reflect high number of aberrancies and/or high percentages of progenitor cells. Furthermore, flow cytometric analysis of BM cells in low and Int-1 risk MDS is instrumental to identify subgroups with distinct clinical behavior regarding transfusion dependency and progression [9]. A clinical decision model was designed using presence of aberrant myeloid progenitors determined by flow cytometry (FC) in BM in combination with endogenous erythropoietin (EPO) levels to predict response to growth factor treatment [10]. Low and Int-1 risk MDS patients with normal myeloid progenitors and low endogenous EPO have the highest probability of responding to growth factor treatment.

In this study, we aimed to investigate the role of FC in prediction of response and treatment monitoring of Int-2 and high risk MDS patients treated with azacitidine.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

Patient Selection

Patients were eligible for treatment with azacitidine if diagnosed with MDS, MDS/myeloproliferative neoplasm (MPN), CMML, or AML with 20–30% blasts and not eligible for standard induction chemotherapy and/or allogeneic stem cell transplantation regimens. As azacitidine is also approved for patients with CMML and AML with 20–30% blasts, these patients were also included in the study. However, the study describes a series of patients who are enriched for Int-2 and high risk MDS. From 2009 to 2012, 42 patients [28 male/14 female; median age 71 years (range 56–82)] were enrolled in this retrospective cohort study. Diagnosis were made according to WHO2008 classification [11]. MDS patients were assigned into prognostic groups by applying the IPSS and an adjusted IPSS with extra weight to poor risk cytogenetics [2, 12]. BM samples were evaluated for chromosomal anomalies according to International System for Human Cytogenetic Nomenclature guidelines [13]. In those cases where no metaphases could be analyzed, additional fluorescence in situ hybridization was performed as recommended [14].

All samples were drawn after informed consent and in conformance with the Declaration of Helsinki. The study was approved by the Institutional Medical Ethics Committee of VU University Medical Center, Amsterdam, Netherlands.

Treatment

All patients were treated according to standard protocols with azacitidine (75 mg/m2/day), administered subcutaneously during the first 7 days of a 28 day cycle. Dose modifications were applied according to standard guidelines.

Response Criteria

Response was evaluated according to IWG2006 criteria [15]. Responses were categorized according to hematologic improvement and disease status. Hematologic improvement was further categorized into erythroid (HI-E), platelet (HI-P), and neutrophil response (HI-N), according to IWG2000 criteria to investigate whether the degree of hematologic improvement was of relevance [16]. Disease status comprised patients with complete remission (CR), stable disease (SD), and progressive disease (PD).

Flow Cytometric Analysis of BM Samples

Immunophenotypic analysis was performed using four-color FC. BM samples for FC were drawn at baseline, after every third cycle or in case of suspicion of disease progression. Analysis was performed on total nucleated BM cells after ammonium chloride lysis of erythrocytes as proposed by the European LeukemiaNet Working party [17, 18]. All samples were processed and analyzed within 24 h. Monoclonal antibodies that were used in this study included fluorescein isothiocyanate conjugated: CD5 (clone DK23), CD13 (WM-47), CD16 (DJ130c) from DakoCytomation, Glostrup, Denmark; CD15 (MMA), CD34 (8G12) from BD Biosciences (San Jose, CA); CD36 (CLB-IVC7) from Sanquin, Amsterdam, Netherlands; phycoerythrin conjugated: CD7 (M-T701), CD11b (D12), CD13 (L138), CD19 (SJ25C1), CD33 (P67.6), CD56 (My31), CD117 (104D2), and CD123 (9F5) from BD Biosciences; CD10 (SS2/36), CD25 (ACT-1), CD64 (10.1) from DakoCytomation; peridinin-chlorophyll protein (PerCP) conjugated: CD45 (2D1) from BD Biosciences; allophycocyanin conjugated: CD11b (D12), CD13 (WM15), CD14 (MoP9), CD33 (P67.6), CD34 (8G12), HLA-DR (L243) from BD Biosciences, and CD117 (104D2) from DakoCytomation. Measurements were performed on a FACS Calibur and data were analyzed by Cell Quest Pro Software (BD Biosciences).

Cell populations of interest were selected by sideward light scatter (SSC) and CD45 properties. Mature myeloid cells were defined as CD45dim(inished) and SSChigh. Monocytes were identified by CD45bright and SSCintermediate in combination with CD14 or CD33bright expression. Myeloid progenitor cells were defined as CD45dim, SSClow in combination with CD34 and/or expression of a myeloid marker such as CD13 and/or CD117 [17, 19]. B cell progenitors were discriminated from myeloid progenitors by lower CD45, lower SSC properties, and back gating with CD19 and were excluded from myeloid progenitor analysis [17, 18]. A minimum number of 250 events within the myeloid progenitor compartment was measured. The myeloid progenitors were considered as positive for asynchronous or lineage infidelity marker expression (LIM) if there was a cluster of ≥20% of cells with expression of CD11b, CD5, CD19, CD56, and/or CD25 based on cut-off values in routine immunophenotyping diagnostics of leukemia [20]. Aberrant expression of CD7 was assessed in the context of CD13 expression. In normal differentiating hematopoietic cells, CD7 can be expressed on CD34posCD13dim cells. Abnormal expression of CD7 on myeloid progenitors can be distinguished from normal expression by quantifying CD7 on CD13bright cells. If CD7 was present on 10% or more of CD13 pos-bright myeloid progenitors, the myeloid progenitors were regarded positive for this aberrant marker.

BM aspirates of 16 age-matched healthy volunteers and patients undergoing cardiothoracic surgery (median age 65, range 45–79) were used as a reference (after informed consent). Aberrant expression of a marker was defined as ≥ two standard deviations increase or decrease compared with the mean expression level in the age-matched control group.

The observed number of aberrancies in the maturing myelomonocytic compartment and the percentage of myeloid progenitors were transformed into a weighed flow score using the FCSS as described previously [7]. In short, for aberrancies in the differentiation of maturing myelomonocytic cells a maximum of five points can be scored. Additionally, the percentage of myeloid progenitors is scored for in a weighted manner, to a maximum of four points. In 14 patients, fresh BM samples were not available for flow cytometric analysis at baseline and after three cycles of azacitidine. In these cases, BM mononuclear cells that were frozen after density gradient centrifugation were analyzed. The FCSS could not be calculated in these cases because mature myeloid cells are removed by the latter procedure. Therefore, solely analysis of aberrant marker expression on myeloid progenitors was performed. Myeloid progenitors after density gradient centrifugation were not used for quantification of myeloid progenitors because the proportions are not comparable with whole BM samples. Previously, it was shown that aberrant marker expression on myeloid progenitor cells from fresh samples is comparable with that of cells that have been frozen and thawed [21]. In two patients, the baseline FCSS could not be calculated because of unsuccessful BM aspiration. The circulating myeloid progenitors in the peripheral blood were analyzed for aberrancies in these cases.

The flow cytometric approach for prognostication and prediction of response to treatment was compared with a clinical prognostic scoring system designed for patients treated with azacitidine [5]. The scoring includes four clinical factors: the Eastern Cooperative Oncology Group performance status, presence of circulating blasts, red blood cell transfusion dependency, and cytogenetics.

Statistical Analysis

The relationship between response and flow cytometric aberrancies was tested using the Mann-Whitney U test for continuous data and Fisher's exact test for categorical data. Differences in overall survival (OS) were assessed by Kaplan-Meier analysis and significance by using log-rank testing. The OS was defined as time from start of treatment with azacitidine until death or for patients who were alive at time of data analysis, until the date of last visit. Statistical calculations were performed by SPSS 15.0 (IBM Corp., Armonk, NY). A P-value lower than 0.05 was regarded significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

Patient Characteristics

The diagnoses according to WHO 2008 classification were RCMD (n = 4), RAEB (n = 21), AML with ≤30% blasts (n = 12), MDS/MPN (n = 1), and CMML (n = 4; Table 1). The risk categories according to the IPSS were Int-1 (n = 4), Int-2 (n = 17), and high (n = 16). In total, 45% (17/38) of patients with known cytogenetics had normal karyotype. Median follow up after initiation of the first cycle of azacitidine was 10.1 months (range 1.0–28.5). Median number of cycles received was six (range 1–28). At time of data analysis, 30/42 (71.4%) patients had discontinued treatment. Four patients discontinued treatment due to hematological toxicity, one patient decided to stop treatment after one cycle; in three patients, treatment was stopped at physician's discretion and one patient received allogeneic stem cell transplantation after three cycles. Overall, 19 patients stopped because of PD after median follow up of 8.9 months (range 1.0–26.8). At time of data analysis, 18 patients were deceased. One patient died of progression of a nonhematological malignancy after 9.6 months and was excluded for OS analyses.

Table 1. Patient Characteristics
WHO2008 classification diagnosisn =%
  1. WHO World Health Organization; IPSS international prognostic scoring system; FCSS flow cytometric scoring system; MPs myeloid progenitors; RCMD refractory cytopenia with multilineage dysplasia; RAEB refractory anemia with excess blasts; AML acute myeloid leukemia; MDS/MPN myelodysplastic/myeloproliferative overlap syndrome; CMML chronic myelomonocytic leukemia.

  2. a

    In cases where cytogenetic analysis was not available, the minimum IPSS category is given.

  3. b

    Cytogenetic category according to the IPSS classification. The karyotype of the patient with MDS/MPN patient and one patient with CMML-1 was normal. There were two patients with CMML-1 and one with CMML-2 with a noncomplex karyotype.

  4. c

    Response could not be evaluated in these patients because of follow up time less than 2 months.

RCMD410
RAEB-1614
RAEB-21536
AML (≤ 30% blasts)1229
MDS/MPN12
CMML-137
CMML-212
IPSSa  
Intermediate-1411
Intermediate-21746
High1643
Cytogenetic categoryb  
Good1746
Intermediate719
Poor924
Not available411
Responsec  
Complete response615
Stable disease1744
Progressive disease1641

Response to Treatment with Azacitidine

According to IWG2006 response criteria for MDS, six patients showed CR, 17 patients had SD, and 16 patients had PD; three patients were excluded because response could not be evaluated because these patients received less than three cycles.

OS in patients with CR or SD was significantly better compared with patients with PD [median OS not reached vs. median 9.8 months (range 1.0–26.8)), p < 0.001, respectively].

No significant difference was observed in HI between the group with known karyotype (n = 31), with a normal karyotype 10/15 (67%), and patients with karyotypic abnormalities 7/16 patients (44%), including complex karyotypes (≥3 aberrations, p = 0.2).

Erythroid response was achieved in 13/34 patients, of which nine patients achieved major HI-E after median of two cycles (range 2–4) and minor HI-E was observed in four patients after median number of 3.5 cycles azacitidine (range 3–4).

Platelet response was achieved in 12/32 patients, of which 11 had major response and one patient had minor response. Median number of cycles to achieve HI-P was 2.5 (range 1–5). Major HI-N was achieved in 5/21 patients and minor HI-N was present in one patient after a median number of three cycles (range 2–4).

HI-E, HI-P and/or HI-N could not be evaluated in cases where pretreatment peripheral blood values were either above reference values or patients were transfusion independent [14].

Any HI was seen in 54% of patients (19/35) [after median of two cycles (range 1–4)]. Median response duration of patients who showed any HI was 8 months (range 3–28.5). The majority of patients (11/18, 61%) who showed HI, had combined HI-E, HI-N, and/or HI-P. Nine patients with initial HI lost response or progressed after median time of 6.9 months (range 4–14) and median number of nine cycles (range 5–15).

The clinical prediction model for response to treatment with azacitidine, as proposed by Itzykson et al., which could be calculated for a subgroup of patients (n = 23) due to availability of the parameters, was not able to identify patients who responded to treatment in our cohort [5].

Absence of Aberrant Myeloid Progenitors is Associated with Hematologic Improvement in Patients Treated with Azacitidine

Median percentage of myeloid progenitors by FC, defined as CD45dim, SSClow in combination with CD34 and/or expression of a myeloid marker such as CD13 and/or CD117, was 8.9% (range 1.1–54%) [17, 19]. Of note, progenitor counts as detected by FC correlate with morphologic blast count, but do not necessarily generate the same percentage. Median percentage of myeloid progenitors as detected by FC did not differ between patients with PD, SD, and CR. At baseline, 64% (27/42) of patients had LIM expression on myeloid progenitors such as CD5 or CD7; in 77% (26/34) of patients aberrant over or under expression of myeloid markers such as CD13, CD34, CD45, CD117, and/or HLA-DR was observed. Overall, 79% (33/42) of the patients had aberrant myeloid progenitors defined as either LIM or aberrant expression of lineage-associated markers. As noted above, at baseline the percentage of myeloid progenitors by FC did not differ between response groups. In contrast, aberrant myeloid progenitors were significantly more frequent in those patients with SD and PD. In the CR group, only 33% (2/6) had aberrant myeloid progenitors, compared with 88% (15/17) in SD group and 88% (14/16) in the PD group (Fisher's exact test, p = 0.01). Furthermore, absence of aberrant myeloid progenitors was significantly associated with achievement of any major HI (Fisher's exact test, p = 0.02). Interestingly, although 10 patients with aberrant myeloid progenitors had major HI, there was a trend toward shorter response duration compared with patients without aberrant myeloid progenitors [median response duration 6.5 months (range 3–12.4) vs. 13.2 months (range 4–28.5), respectively, p = 0.08]. These data indicate that immunophenotype of myeloid progenitors is more informative in response prediction than percentage of myeloid progenitors.

Patients with aberrant myeloid progenitors received a median of six cycles azacitidine (range 1–16) and had median survival of 14.2 months after initiation of treatment. In contrast, patients without aberrant marker expression received a median of 12 cycles (range 2–28) and had median survival of 26.8 months (Fig. 1A). Patients with aberrant myeloid progenitors received less cycles because of disease progression and/or death. In other words, patients who received less cycles because of disease progression or death were mainly found in the group with aberrant myeloid progenitors. Thus, OS of patients without aberrant myeloid progenitors was significantly better compared with patients with aberrant myeloid progenitors.

image

Figure 1. Absence of immunophenotypically aberrant myeloid progenitors and a pretreatment low FCSS is associated with better OS in patients treated with azacitidine. Kaplan-Meier plot (A) of patients treated with azacitidine with (n = 33) or without (n = 9) aberrant myeloid progenitors at baseline. Patients without aberrant myeloid progenitors at baseline had a significantly better OS (full line, median survival = 26.8 months) compared with patients with aberrant myeloid progenitors (dotted line, median survival = 14.2 months), Log-rank test p = 0.03. Kaplan-Meier plot (B) of patients treated with azacitidine with a pretreatment FCSS of 3–5 (n = 11) and patients with a FCSS of 6–8 (n = 15). Patients with a FCSS of 3–5 at baseline had a significantly better survival, (full line, median survival = not reached) compared with patients with a FCSS of 6–8 (dotted line, median survival= 9.8 months), Log-rank test p = 0.03.

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Interestingly, loss of aberrant myeloid progenitors during treatment with azacitidine was associated with response to treatment in contrast to persisting LIM expression on myeloid progenitors in patients that did not respond. Figure 2 shows an example of this finding in one patient with loss of aberrant myeloid progenitors and concomitant response to azacitidine, in contrast to a patient with persisting aberrant myeloid progenitors and lack of response.

image

Figure 2. Disappearance or persistence of myeloid progenitors with aberrant immunophenotype corresponds with the nature of response to azacitidine. In the CD45 versus SSC graphs, the myeloid progenitors can be identified by CD45dim, SSClow characteristics (encircled). To assess aberrancies, the myeloid progenitors in these examples are gated by using CD34 (CD34 vs. SSC graphs). In the upper row, a patient (A) with complete response during treatment had an aberrant expression of CD56 (X-axis) on the myeloid progenitors at baseline (indicated by squares, defined as CD45dim, SSClow and CD34pos). After six cycles of azacitidine, these aberrant myeloid progenitors could not be detected (second row). In the third row, at baseline, a patient (B) with SD during treatment had aberrant expression of CD5 (X-axis) on the myeloid progenitors at baseline (indicated by squares, defined as CD45dim, SSClow, and CD34pos). After six cycles of azacitidine, the aberrant myeloid progenitors were still present (fourth row).

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Decrease in FCSS During Treatment with Azacitidine is Associated with Response

Cumulative aberrancies in the myelomonocytic compartment as assessed by FC are reflected in the FCSS. The baseline FCSS was not significantly different between patients achieving CR, SD, and PD (median FCSS = 5.5, range 3–8, median FCSS = 4, range 2–8, median FCSS = 7, range 5–8, respectively).

Patients who achieved CR showed a significant decrease in the FCSS after three cycles of azacitidine compared with patients with PD (median FCSS = 1.5, range 1–3 and median FCSS = 6.5, range 3–8, respectively, p = 0.004) (Fig. 3). Decrease in FCSS was sustained after six cycles azacitidine in patients with CR.

image

Figure 3. Decrease of the FCSS correlates with response to azacitidine treatment. The FCSS represents a weighed score for aberrancies of myelomonocytic cells and the percentage of myeloid progenitors: higher scores indicate a higher degree of disruption of hematopoiesis [6]. The FCSS was measured at baseline and after every third cycle. The FCSS at baseline was comparable in the response categories progressive disease (PD, light gray boxes, n = 9), stable disease (SD, gray boxes, n = 10), and complete response (CR, dark gray boxes, n = 4). The response criteria were based on IWG2006 criteria [12]. The FCSS is significantly decreased in patients with CR compared with patients with PD after three cycles of azacitidine (p = 0.004).

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Median FCSS for patients with SD was decreased after three cycles (FCSS = 4, range 1–6, p = 0.03, compared with patients with PD and not significantly different from patients with CR) (Fig. 3). Decrease in FCSS could not only be assigned to a decrease in the percentage of myeloid progenitors; a decrease in the number of aberrancies in the differentiation of myeloid and monocytic cells was also observed.

There was an association between occurrence of HI-E, HI-P, and decrease of FCSS upon azacitidine treatment (Fig. 4). This is remarkable, as the FCSS only takes dyspoiesis of myeloid cells into account. The majority of patients with HI-E (7/9, p = 0.01) and HI-P (6/8, p = 0.02) had improvement of FCSS upon treatment. Furthermore, patients with HI-N, all showed decrease in the number of aberrancies in the mature myeloid compartment (data not shown). Overall, this indicates that HI-N is reflected by improvement of neutrophil dyspoiesis as detected by FC.

image

Figure 4. Changes in the FCSS correlate with erythroid response in patients with MDS treated with azacitidine. The FCSS was measured at baseline and after every third cycle [6]. The FCSS at baseline was comparable for patients without (no HI-E, light gray boxes, n = 10), with minor (minor HI-E, not depicted in figure, n = 1) and major (major HI-E, dark gray boxes, n = 8) erythroid response according to IWG2006 response criteria [12]. The FCSS is significantly decreased after three cycles of azacitidine in patients with major erythroid response compared with patients without erythroid response, p = 0.002. One patient with minor HI-E is not depicted in the figure. The FCSS was three at baseline and four after three cycles of azacitidine. n.s. not significant.

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High Pretreatment FCSS is Associated with Worse OS in Patients Treated with Azacitidine

All BM samples were analyzed by FC at baseline and after every third cycle. Median FCSS at baseline was six (range 2–8). No difference in FCSS was observed between IPSS Int-2 and high risk groups [median FCSS = 5 (range 3–8) and 7 (range 4–8), respectively] and between IPSS cytogenetic risk categories good, intermediate, and poor. The FCSS did not differ significantly between adjusted IPSS cytogenetic risk groups [12]. Among patients with baseline FCSS of 2–5, OS was significantly better compared with patients with pretreatment FCSS of 6–8, p = 0.03 (Fig. 1B).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

MDS are heterogeneous with respect to clinical course and response to treatment. It was shown previously that aberrancies as detected by FC can be instrumental in prognostication [9, 22] Furthermore, FC can be applied for clinical decisions in low and Int-1 risk MDS patients by identification of patients that are most likely to respond to growth factor treatment [10]. Therefore, we studied the clinical relevance of FC for prediction of response to azacitidine in Int-2 and high risk MDS. Absence of aberrant myeloid progenitors was significantly associated with favorable response to treatment with azacitidine. In our patient group, 79% had myeloid progenitors with aberrant immunophenotype. Moreover, there was a significant association between absence of aberrant immunophenotype and achievement of CR. Furthermore, presence of aberrant myeloid progenitors was significantly associated with lower probability of achieving long-term major HI-E. These findings are in line with data reported on low and Int-1 risk MDS patients [10].

In this study, the absence of aberrant myeloid progenitors was predictive for HI-E to growth factor treatment. Remarkably, although a proportion of patients with aberrant myeloid progenitors had major HI-E, response duration of these patients tended to be shorter than that of patients without aberrant myeloid progenitors. An association between abnormal karyotype and lower response rates and complex karyotypic abnormalities with shorter response rates was described by Itzykson et al. [5]. However, in our group, abnormal cytogenetics show a trend but did not significantly correlate with absence of response to azacitidine.

The rate of any HI was 54% in our patient group and majority of patients had combined HI-E, HI-P, and/or HI-N (61%). These response rates are in line with published data [23-25]. As described earlier, the number of cycles to obtain response varies from five to nine [26, 27]. The FCSS was significantly decreased after three cycles azacitidine in patients with CR compared with PD, whereas patients with SD or PD did not show significant change in FCSS after three cycles. Decrease of FCSS in responsive patients was not only caused by decrease in percentage of myeloid progenitors but also by a qualitative improvement of dyspoiesis in maturing myelomonocytic cells. Majority of patients with HI-E and HI-P had significant decrease of dysmyelopoiesis as reflected by an improvement of the FCSS. This is of interest, as erythroid and megakaryocytic cell analysis is not included in FCSS. This might indicate that in MDS, in which a common hematopoietic progenitor cell is affected, ineffective hematopoiesis is restored upon treatment with azacitidine in responsive patients.

Significantly worse OS was found in patients with high pretreatment FCSS [6-8] and/or myeloid progenitors with aberrant immunophenotype. This might be a reflection of the degree of disruption of normal hematopoiesis. These findings are in line with previously published studies; which showed that flow cytometric scores, including the presence of aberrant myeloid progenitors is associated with disease progression, shorter transfusion free survival and/or OS [28-30]. Notably, FCSS was similar between subgroups within IPSS and IPSS cytogenetic risk groups. Therefore, FC may provide valuable prognostic information in addition to currently validated prognostication systems.

In conclusion, the presence of myeloid progenitors with aberrant immunophenotype identified patients with higher risk MDS that are unlikely to achieve CR and long-term major HI upon treatment with azacitidine. Hence, presence of myeloid progenitors with aberrant immunophenotype was associated with worse OS compared with patients without aberrant myeloid progenitors. Decline in FCSS during treatment was correlated with clinical response to azacitidine which indicates that FC might be applied to monitor MDS during treatment and might identify patients who would benefit from prolonged treatment with azacitidine.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

The authors would like to thank Claudia Cali, Kelly Schouten, and Kristin Vandenberghe (Department of Hematology, VU Institute of Cancer and Immunology (V-ICI), Cancer Center Amsterdam (CCA), VU University Medical Center, Amsterdam, Netherlands) for technical assistance. Tanja M van Maanen-Lamme, Linda Luppens-de Graaf, (Department of Internal Medicine, Westfries Gasthuis, Hoorn), Aart Beeker, and Bart de Valk, (Department of Internal Medicine, Spaarne Ziekenhuis, Hoofddorp) for providing BM samples.

CONFLICT OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED

C.E. is a member of the nurse advisory board of Celgene Corporation. G.H. received an honorarium from Celgene Corporation. V.S. received honoraria from Celgene, Novartis, and Janssen corporation. G.J.O. and A.A.L. both did consultancy and received honoraria from Celgene Corporation.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST
  9. LITERATURE CITED
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