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Significant Correlation Between the Degree of WT1 Expression and the International Prognostic Scoring System Score in Patients With Myelodysplastic Syndromes

Daniela Cilloni, Enrico Gottardi, Francesca Messa, Milena Fava, Patrizia Scaravaglio, Marilena Bertini, Mauro Girotto, Carlo Marinone, Dario Ferrero, Andrea Gallamini, Alessandro Levis, Giuseppe Saglio for the Piedmont Study Group on Myelodysplastic Syndromes

From the Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences of the University of Turin; Divisions of Hematology and Internal Medicine, S. Giovanni Battista Hospital, Turin; Immunohematology Service, Ivrea; Division of Hematology, S. Croce Hospital, Cuneo; and Division of Hematology, SS. Antonio e Biagio Hospital, Alessandria, Italy.

Address reprint requests to Giuseppe Saglio, MD, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Hospital, Gonzole 10, 10043 Orbassano-Torino, Italy; email: saglio{at}csi.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: To determine whether pattern of WT1 gene expression is a useful marker for establishing prognosis and tracking disease progression in patients with myelodysplastic syndromes (MDS).

Patients and Methods: We performed a quantitative assessment of the WT1 transcript amount by real-time quantitative polymerase chain reaction (RQ-PCR) in 173 samples (131 bone marrow samples and 42 peripheral-blood samples) from 131 patients with MDS (79 patients with refractory anemia [RA], 31 with RA with excess blasts [RAEB], 18 with secondary acute myeloid leukemia [s-AML] evolved from MDS, and three with deletion of 5q as the sole cytogenetic abnormality). Values obtained were correlated with the blast percentage and International Prognostic Scoring System (IPSS) score.

Results: Sixty-five percent of BM and 78% of PB samples for RA and 100% of BM and PB samples of RAEB and s-AML expressed WT1 transcript amounts greater than the level observed in healthy volunteers. The degree of WT1 expression was highly correlated with the type of MDS, was much higher in RAEB and s-AML compared with RA, and increased during disease progression. Moreover, a significant correlation was found between WT1 expression levels, blast cell percentage, and the presence of cytogenetic abnormalities. Therefore, we found a significant correlation between the amount of WT1 transcripts and the IPSS score, which currently represents the most reliable risk index of disease progression available for MDS patients.

Conclusion: WT1 is a useful molecular marker for risk assessment in MDS patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
MYELODYSPLASTIC SYNDROMES (MDS) are clonal hematopoietic stem-cell disorders characterized by ineffective dysplastic hematopoiesis involving one or more cell lineages and characterized by peripheral-blood cytopenias and a high risk of progression to acute myeloid leukemia (AML).^1,2

MDS are highly heterogeneous with respect to numerous parameters (ie, clinical and hematologic characteristics, cytologic and pathologic features, and cytogenetic and molecular lesions),^3–5 and several attempts to classify them in more homogeneous groups of diseases have been made in the past and more recently.^6–10

To facilitate clinical decision making, risk-based classification systems have been developed for MDS. In particular, the International Prognostic Scoring System^11,12 (IPSS) has achieved international acceptance and assigns scores according to marrow blast cell percentage, karyotype, and degree of cytopenia, providing a useful method for evaluating prognosis in MDS patients and for designing clinical trials. However, because of frequent difficulties in obtaining all parameters needed to establish the IPSS score, probability of disease progression may not always be easy to assess for individual patients. For example, cytogenetic analysis provides essential information to formulate the IPSS score, but a sufficient number of metaphases is detectable at diagnosis in only 70% to 80% of patients with MDS.^13–15

The Wilms’ tumor gene (WT1) is a tumor suppressor gene coding for a zinc-finger transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor.^16–19 In normal peripheral blood (PB) and bone marrow (BM), WT1 expression is reported to be low and sometimes undetectable even by qualitative reverse transcriptase polymerase chain reaction (RT-PCR).²⁰ By contrast, WT1 is highly expressed in most acute leukemias, and we and others have recently shown that its level of expression is associated with the presence, persistence, or reappearance of leukemic hematopoiesis.^21–24 Although at present the factors responsible for increased expression of WT1 in acute leukemias as well as its functional significance within the leukemogenesis process remain elusive, this phenomenon could potentially be used as a marker of leukemic hematopoiesis. The full spectrum of MDS ranges from relatively benign clonal BM disorders to forms with rapid evolution to AML, and recent clinical and biologic studies indicate that in the latter cases, MDS and AML can be considered as part of the same continuous disease spectrum rather than distinct disorders. Therefore, we tested whether pattern of WT1 gene expression evaluated by methods of real-time quantitative polymerase chain reaction (RQ-PCR) could represent a useful marker in MDS and help to establish prognosis and track progression of disease in individual patients.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and Controls
After informed consent, BM samples from 131 MDS patients (79 patients with refractory anemia [RA], 31 patients with RA with excess blasts [RAEB], 18 patients with secondary AML [s-AML] evolved from MDS, and three patients with deletion of 5q as the only cytogenetic anomaly) were tested for WT1 expression. In 42 patients, paired PB samples were also collected and tested. For the purpose of this study and to establish a correct IPSS score, we considered only the MDS cases in which at least 10 analyzable metaphases were present and an immunophenotypic analysis of BM aimed at counting the blast cells according to pre-established criteria (see below) was available. The patients were derived from the cohort of patients enrolled by the Piedmont Study Group on MDS (http://www.mds.piemonte.it/) with the only bias of having all the parameters needed to establish the IPSS score.

Diagnosis was made by cytomorphologic and histologic methods according to the proposals made by the French-American-British group in 1982.⁶ Dysplasia of the three cell compartments in the BM was assessed according to published criteria: dyserythropoiesis, the presence of multinuclearity, nuclear fragments, cytoplasmic abnormalities, or megaloblastoid changes; dysgranulopoiesis, the hypolobulation (including pellagroid forms) and hypogranulation of granulocytes; dysmegakaryopoiesis, the presence of micromegakaryocytes or (large) mononuclear forms. Dysplasias in one cell lineage was diagnosed if at least 50% of the examined cells of this lineage had dysplastic features. The IPSS was established for all patients included in the study.

In addition, samples taken during follow-up of three patients with RA and seven patients with RAEB who were treated only with supportive therapy and whose disease progressed to acute leukemia and of two patients with RAEB who underwent intensive eradicating chemotherapy were analyzed.

Finally, we tested 31 normal BM and 46 normal PB samples from healthy donors and from two patients with megaloblastic anemia and five patients with erythroid hyperplasia resulting from hemolytic anemia as controls.

Cytogenetic Analysis
Cytogenetic analysis was performed in all patients included in the study. Only patients in whom at least 10 analyzable metaphases were present have been considered for this study.

The cytogenetic analysis was carried out after 1-day culture of unstimulated BM cells, and G-banding karyotypes were analyzed and classified according to the International System for Human Cytogenetic Nomenclature (1985).²⁵

Immunophenotypic Characterization
Immunolabelings and flow cytometry analysis of the different BM cell subsets were performed on the whole BM after RBC lysis according to standard procedures.²⁶ The blast cell percentage was calculated on the basis of the CD34+/CD117+, CD33brightCD14- elements present in the CD45low/SS-low gate, after a primary gating of BM cells based on CD45 antigen expression and side scatter laser light diffraction. Monoclonal antibodies originated from Becton Dickinson (Milan, Italy), Beckman Coulter (Milan, Italy), and Dako (Milan, Italy).

RQ-PCR Analysis for WT1
The RT (reverse transcription) step was performed as previously described.²⁷ RQ-PCR reactions and fluorescence measurements were made on the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) as described.²¹

Briefly, the RQ-PCR primers and probe for WT1 were as follows: 5'- CAGGCTGCAATAAGAGATATTTTAAGCT-3', 5'-GAAGTCACACTGGTATGGTTTCTCA-3', and 5'-CTTACAGATGCACAGCAGGAAGCACACTG-3'.

The RQ-PCR primers and probe for ABL were as follows: 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3', 5'-GATGTAGTTGCTTGGGACCCA-3', and 5'-CCATTTTTGGTTTGGGCTTCACACCATT-3'.

All sample analysis was performed in triplicate, and results showing a discrepancy greater than one cycle threshold in one of the wells were excluded and repeated.

For quantitative assessment of WT1, a calibration curve with a plasmid containing the WT1 target sequence was used (Amplimedical s.p.a. Bioline Division, Turin, Italy). The sensitivity of the RQ-PCR assay for WT1 was established as described.²¹ The WT1 transcripts obtained by RQ-PCR were normalized with respect to the number of ABL transcripts and expressed as WT1 or copy numbers every 10⁴ copies of ABL.

Statistical Analysis
The comparison between the WT1 values obtained in the different types of myelodysplastic syndromes was performed using the Student’s t test and Mann-Whitney test.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 131 BM and 42 paired PB samples obtained from 131 patients with MDS (79 patients with RA, 31 patients with RAEB, 18 patients with s-AML, and three patients in whom deletion of 5q was the only detectable cytogenetic abnormality) were analyzed by RQ-PCR for the expression level of WT1. In addition, we tested a set of normal BM (n = 31) and PB (n = 46) samples from healthy donors. The results obtained are reported and graphically represented in Fig 1.

View larger version (23K): [in this window] [in a new window]   Fig 1. WT1 copy number evaluated by real-time quantitative polymerase chain reaction in bone marrow (BM; black dots) and peripheral blood (gray dots) from healthy volunteers and patients with refractory anemia (RA), RA with excess blasts (RAEB), and secondary acute myeloid leukemia (s-AML). The differences in WT1 expression levels in the different subgroups are highly significant from a statistical point of view.

WT1 Expression in RA
As shown in Fig 1, the WT1 values found in both BM and PB samples of RA were significantly increased with respect to the normal values (P = 1.07^-6 in BM and P = 2.3^-5 in PB). The WT1 transcript was increased above the normal range in 65% of the BM samples in RA. In the whole RA series, the median value of WT1 transcript copies (expressed as WT1 copies/10⁴ ABL copies) was 314 (range, 13 to 1,289 copies), whereas the median number found in the RA cases with a WT1 value above the normal upper limit (180 copies) was 366. Of the 23 RA patients in whom we were able to test a paired PB sample, WT1 levels above the upper limit found in healthy volunteers were present in 18 (78%), with a median value of 42 WT1 copies/10⁴ ABL copies (range, 30 to 65 copies). Fifty-nine (83%) of 71 RA patients were characterized by multilineage dysplasia (RC+Dys according to the new World Health Organization classification⁸), whereas 12 patients showed dysplasia only in the erythroid lineage. The RC+Dys patients show WT1 transcript levels significantly higher than RA with unilineage dysplasia (P = .002). However, because of the small number of patients with RA with unilineage dysplasia present in our series, further data are needed to confirm this finding.

All cases with an abnormal WT1 value in PB showed an increased WT1 value in BM, and all the cases with a normal PB WT1 value showed normal values in BM. However, in cases showing abnormal WT1 values in both BM and PB, no exact relationship between the WT1 values observed in BM and PB was present, indicating that the PB values were probably influenced by the percentage of abnormal precursors with respect to the normal cells (mainly lymphocytes) present.

As shown in Fig 2, in BM samples, a strong correlation was present between the number of WT1 transcripts and the blast percentage (r = 0.86).

View larger version (18K): [in this window] [in a new window]   Fig 2. Regression analysis between WT1 transcript amount and the blast cell percentage in refractory anemia patients. A strict correlation is present between the two parameters, resulting in an r value of 0.86.

WT1 Expression in RAEB and in s-AML
WT1 expression was increased in all BM and PB samples obtained from RAEB or s-AML cases. In 31 RAEB patients examined, the median value of WT1 transcripts was 2,262 copies (range, 227 to 11,006 copies) in BM and 816 in PB (n = 10). These values are significantly higher not only with respect to those found in normals (P = 3.27^-8 in BM and P = .000004 in PB), but also with respect to those found in RA (P = 6.7 ^-6 for BM and P = .00025 for PB). In 18 s-AML patients tested, the median number of WT1 transcripts was 14,033 (range, 3,757 to 51,700 transcripts) in BM and 5,509 (range, 579 to 13,034 transcripts) in PB samples (n = 9). Again, these values are significantly increased with respect to those found in RAEB (P = .00027 and P = .002, respectively). This latter result was confirmed by Mann-Whitney U test (P = .003). Abnormal karyotypes were found by cytogenetic analysis in 54% of the RAEB cases and 43% of these abnormalities were complex karyotypes. The percentage of the cases with cytogenetic abnormalities increased to 77% in s-AML, and the majority of them (86%) were represented by complex karyotypes. In the RAEB cases, the WT1 values found in the patients with cytogenetic abnormalities were significantly increased with respect to those associated with normal karyotypes (P = .0037 by Mann-Whitney U test). A similar analysis was not performed in s-AML because of the few cases with a normal karyotype.

WT1 Expression in Cases With Deletion of 5q as the Sole Cytogenetic Abnormality
In three cases, cytogenetic analysis detected a deletion of the 5q chromosome as the only abnormality. From the clinical and hematologic point of view, only one of these cases was fulfilling the criteria of 5q- syndrome, whereas the remaining two, based on the blast percentage, were RAEB.

The 5q- syndrome case, with a 3% blast in BM, showed a WT1 value of 568, whereas in the other two cases, in which 6% and 15% of blasts were shown in BM, WT1 levels were 607 and 12,904, respectively.

Correlations Between the Levels of WT1 Expression, Blast Percentage, and the IPSS Score
As shown in Fig 3, considering all MDS cases, a strong correlation was found between WT1 copy number and the percentage of BM blasts (r = 0.83). An even stronger correlation (r =. 92) was present between the WT1 expression levels and the risk categories of the patients defined according to the IPSS (Fig 4). As shown in Fig 5, the WT1 expression levels found in different risk categories defined according to IPSS were highly different from a statistical point of view.

View larger version (17K): [in this window] [in a new window]   Fig 3. Regression analysis performed considering the WT1 copy number and the percentage of blast cells in all myelodysplastic patients. The r value obtained (0.83) indicates a good correlation between the two parameters considered.

View larger version (10K): [in this window] [in a new window]   Fig 4. Regression analysis performed considering the WT1 copy number and the International Prognostic Scoring System (IPSS) score in all myelodysplastic patients. The r value obtained (0.92) indicates a good correlation between the two parameters considered.

View larger version (17K): [in this window] [in a new window]   Fig 5. WT1 expression level in different risk categories defined according to the International Prognostic Scoring System (IPSS). The differences in WT1 transcript amount evaluated between the IPSS risk categories are highly significant from a statistical point of view.

Follow-Up of WT1 Expression During MDS Progression
WT1 expression level was evaluated in 12 MDS patients at different time points during follow-up. Three patients with RA and six patients with RAEB who showed increased BM WT1 levels at the time of the first observation and were treated only with supportive therapies converted subsequently to acute leukemia. In two of these patients, at the time of leukemic progression, additional cytogenetic abnormalities were detected, whereas in another, an Internal Tandem Duplication of the FLT3 gene appeared. As represented in Fig 6, all of them showed a marked increase in the WT1 transcript amount at the time of disease progression. A similar increase in WT1 expression levels was present in a patient with RA who at the time of first observation presented with BM WT1 levels within the normal range but who subsequently progressed to RAEB and s-AML.

View larger version (20K): [in this window] [in a new window]   Fig 6. WT1 expression levels during follow-up of 10 myelodysplastic patients (four with refractory anemia and six with refractory anemia with excess blasts) treated only with supportive therapy who subsequently converted to acute leukemia. In all cases, a marked increase of WT1 transcript amount was noted in parallel with disease progression.

View larger version (27K): [in this window] [in a new window]   Fig 7. WT1 expression level in patient with refractory anemia with excess blasts characterized by the trisomy of chromosome 11 evaluated during follow-up by fluorescent in situ hybridization analysis. After chemotherapy, the patient reached a short hematologic but not cytogenetic remission (3% of trisomic cells). WT1 transcript never returned to within normal values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

WT1 expression has been previously reported to be increased also in myelodysplastic syndromes.^30–32 So far, however, the limited number of MDS cases studied for WT1 expression and the semiquantitative RT-PCR methods used did not allow to fully perceive the important clinical and prognostic significance that the quantitative assessment of the level of expression of this marker can endow.

In this study, using a sensitive and precise RQ-PCR approach, we studied WT1 expression in a large series of MDS patients (including also a number of secondary leukemias) from whom all essential data to establish a correct IPSS score at presentation were available. The WT1 levels have been expressed in terms of number of copies per every 10⁴ ABL copies, reference gene whose expression has been established to be particularly stable and constant in all cell types in both BM and PB by a large cooperative international study.³³ The data obtained show that in most MDS, including approximately two thirds of RA, WT1 is expressed above the range observed in normal controls in both BM and PB and that its expression is directly correlated with the type of MDS, being higher in a statistically significant manner in RA with respect to RAEB and to s-AML. In addition, even within each subgroup, a strong association is present between the level of WT1 expression and the blast percentage and the presence of cytogenetic alterations. This finally results in a very strict correlation, highly significant from the statistical point of view, between the BM amount of WT1 transcripts and IPSS, the most widely accepted risk scoring system for MDS. The capacity of WT1 levels to distinguish between different prognostic IPSS subgroups in MDS seems particularly high for patients in the intermediate-low risk group as compared with patients in the intermediate-high risk group, where a level of 10³ copies of WT1 per every 10⁴ ABL copies seems to represent a discriminating threshold.

Moreover, as already suggested,³⁰ our study confirms that a longitudinal monitoring of the WT1 levels may represent a good marker to establish disease progression in MDS and, although this needs to be proved in a prospective study, it may also help to distinguish patients who are at high risk for progression from those who are not at such high risk. Finally, as already demonstrated for AML,^21,23,24 WT1 levels seem to represent a good marker for minimal residual disease detection.

The important implications of these findings in clinical practice are obvious, as the identification of a molecular marker so strictly corresponding to a prognostic score and able to establish the tendency of myelodysplastic hematopoiesis to progression can be of great help in decision making for MDS patients. Moreover, the fact that WT1 may be abnormally expressed even in the PB of MDS patients can also facilitate a correct diagnosis in cases in which the bone marrow aspirate is inadequate because of hypocellularity or fibrosis or if the diagnosis is in doubt. In conclusion, we believe that the level of WT1 potentially fulfills all the requirements to be introduced as a additional marker to the standard parameters already considered in risk assessment for MDS, which is particularly useful in cases in which BM aspiration and/or cytogenetic analysis fail or are not informative. For this purpose, although new quantitative real-time procedures promise to simplify the protocols for WT1 transcript quantitative assessment that are currently in use, standardization and the introduction of rigorous, internationally accepted controls are required to enable RQ-PCR to become a robust and routine basis for therapeutic decisions.

At the moment, however, besides the potential clinical exploitation as a marker, the biologic significance of WT1 overexpression in myelodysplastic hematopoiesis remains elusive. Several sets of data support the notion that increased levels of WT1 expression are indeed specific of leukemic or in some way abnormal progenitors, as in paroxysmal nocturnal hemoglobinuria with respect to their normal counterparts and not a simple consequence of the degree of differentiation of the hematopoietic cells.²² Consistent with this suggestion is the fact that we found that WT1 is overexpressed in leukemic but not in sorted normal primitive progenitor cells.^21,22 Based on these considerations, several approaches of immunotherapy directed against cells overexpressing WT1 have been recently proposed as a potential treatment for a variety of hematologic malignancies.^32,34 The present study provides further details on the significance of WT1 expression in malignant hematopoiesis. We found abnormal values of WT1 transcript even in cases of RA in which, as established by the immunophenotypic analysis, the percentage of blast cells was not increased. This suggests that WT1 expression reflects not only the presence of leukemic blasts but also the presence of genetic lesions in progenitor cells apparently normal from the morphologic and immunophenotypic point of view. In agreement with this finding and in addition to the strict correlation found between the blast cell count and WT1 expression levels, an even stronger correlation was found between WT1 expression levels and IPSS. This further suggests that, in addition to blast cell count, even the number and the type of the cytogenetic abnormalities present and the number of cytopenias can influence WT1 expression. Therefore, WT1 transcript levels mirror quite well the risk of progression of these diseases as assessed by the IPSS.

Taken together, all these data suggest that WT1 overexpression represents the result of several factors, including the occurrence of genetic damages on the progenitor cell compartment, which are clinically evident in advanced MDS or may still remain silent for some time in the early phases of the disease.

	NOTES

 
Supported by grants from Associazione Italiana per la Ricerca sul Cancro, Ministero Universita Ricerca Scientifica Tecnologica Cofinanzianento 2002, and Associazione Italiana contro le Leucemie.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted October 21, 2002; accepted March 5, 2003.

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