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(1999) 13, 891–900  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu WT1 and GATA1 expression in and acute leukemia P Patmasiriwat1,2, G Fraizer1,3, H Kantarjian4 and GF Saunders1

Departments of 1Biochemistry and Molecular Biology, and 4Hematology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

The Wilms’ tumor , WT1, represses transcription from stem cells but it is only weakly expressed in normal mature several growth factor genes. WT1 transcription is regulated in blood cells.15 Our analysis of murine hematopoietic WT1 erythroid and myeloid lineages by the GATA-1. Using a sensitive, isotopic duplex RT-PCR procedure expression in embryonic tissues has shown that WT1 tran- amplifying WT1 or GATA-1 together with ␤-actin as the internal scripts are highest in the liver at 12.5 dpc, suggesting that WT1 control in a single reaction mix, we quantitated the expression is expressed in the primary hematopoietic organ during of WT1 and GATA-1 mRNA of 16 patients with myelodysplastic definitive hematopoiesis. WT1 expression in hematopoietic syndrome (MDS), 56 with (AML) and 22 malignancies has been reported,16,17 and the high WT1 with acute lymphoblastic leukemia (ALL). K562 was used as expression levels correlate well with a poor prognosis of reference positive control for this cell line expresses both WT1 18 and GATA-1. Among MDS patients, increased WT1 expression patients with acute leukemia. 19 Ј was found in refractory with excess blast (RAEB) and Wu et al identified a hematopoietic enhancer located 3 RAEB in transformation (RAEB-T) subtypes compared to the of the WT1 gene, and showed that GATA-1 is able to trans- normal controls, whereas WT1 expression in refractory anemia activate this enhancer. The GATA-1 gene is located on the X (RA) was not different from the normal control level. All of AML and encodes a hematopoietic transcription fac- cases of subtypes M0, M1, M2 and M3 expressed WT1 more tor.20,21 Recently, Zhang et al22 demonstrated that the 3Ј WT1 than three times the normal WT1 level. Subtypes M4 to M7 showed significantly lower WT1 levels than M1 to M3 and AML enhancer is responsible for the expression of WT1 in the cases with CD14+ expressed less WT1 than CD14−. Higher than erythroid lineage and confirmed that this enhancer is trans- normal WT1 levels were also expressed in cases of ALL. activated by GATA-1. In addition, another enhancer located Keywords: WT1; GATA; MDS; AML; ALL at the third intron of WT1 (intronic enhancer) is responsible for the expresssion of WT1 in the myeloid granulocytic lin- eage and is also GATA-1-responsive.22 Coexpression of WT1 Introduction and GATA-1 mRNA was observed in murine spleen, K562, HEL cells and human acute leukemia.19,23 Wilms’ tumor (WT), or nephroblastoma, is a childhood malig- GATA-1 protein recognizes the TGATAA/G DNA consensus nancy of renal blastemal cells. These mesenchymal cells nor- sequence, the site initially identified in the promoters of mally differentiate to form nephrons when induced by the erythroid-␣ and -␤ globin genes and their control regions, as ureteric bud.1,2 Cytogenetic studies have provided evidence well as in the promoter of erythropoietin .21 Gene tar- of chromosomal localization when deletions in a region of the geting in mouse embryonic stem cells at the GATA-1 short arm of chromosome 11 at 11p13 were commonly found has shown that GATA-1 is essential for erythroid develop- in patients with the WAGR syndrome of Wilms’ tumor, aniri- ment.21,24–26 The GATA-1 binding sites are also located within dia, genito-urinary abnormalities and mental retardation.3,4 the promoter and enhancer regions of WT18,22 as well as in The human Wilms’ tumor gene (WT1) is a potential tumor globin gene.27 From this accumulating evidence, GATA-1 is suppressor gene that encodes a transcription factor containing thought to play a major role in the regulation of WT1 a proline-rich amino terminus and four contiguous zinc fing- expression in the hematopoietic system. ers of the cys2-his2 type at its carboxy end. The proline-rich In this report, we examined WT1 and GATA-1 expression region functions as a transcriptional repressor,5 and the zinc and the factors influencing such expression in myelodysplastic finger region binds DNA in vitro.6,7 This WT1 gene product syndrome (MDS), a condition previously designated as preleu- represses transcription from promoters of many growth factors kemia, and in acute leukemia (acute myeloid leukemia (AML) and receptor genes.8,9 WT1 has also been shown to repress and acute lymphoblastic leukemia (ALL)). Our results support the promotors of several genes expressed in hematopoietic a role for WT1 and GATA-1 in abnormal hematopoiesis and cells (ie CSF-1, TGF␤-1, RAR␣, c-, and bcl-2).10–12 This acute leukemia. suggests that WT1 may function as a transcriptional repressor of genes that control hematopoietic proliferation, differen- tiation or both. Materials and methods The expression pattern of WT1 during development is lim- ited to cell types of mesothelial origin.13,14 A homozygous Patients point within the WT1 gene in human mesothelioma has been reported.13 We have previously shown that WT1 is This study group included 16 patients with MDS, 56 with strongly expressed in immature CD34+ (BM) AML, 22 with ALL and nine normal controls. MDS and human leukemia were classified according to the French–American– British (FAB)28–31 and morphologic, immunologic and cyto- Correspondence: GF Saunders, Department of Biochemistry and Mol- genetic (MIC)32,33 criteria. Among 16 cases of MDS there were ecular Biology, Box 117, The University of Texas MD Anderson Can- seven cases of refractory anemia (RA), four RA with excess cer Center, Houston, Texas 77030, USA blasts (RAEB), two RA with excess blasts in transformation Present addresses: 2Faculty of Medical Technology, Mahidol Univer- sity, Bangkok, Thailand, and 3Department of Urology, The University (RAEB-T) and three with chronic myelomonocytic leukemia of Texas MD, Anderson Cancer Center, Houston, TX, USA (CMML). Of the 56 cases of AML, 55 were de novo AML Received 20 March 1998; accepted 4 February 1999 patients and one had CML in blastic transformation to AML. WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 892 Forty-six of the 55 de novo AML cases were FAB subclassified in a 1.5% low melting point agarose gel containing ethidium as M0 to M7. The number of cases in each FAB subtypes were bromide (0.1 ␮g/ml) and photographed under ultraviolet M0, 1; M1, 8; M2, 12; M3, 4; M4, 12; M5, 5; M6, 2 and M7, illumination. Figure 1 shows examples of RT-PCR-amplified 2 cases. AML subclassification were not available for the other WT1 expression. One-half of the GATA-1 PCR products were nine cases. Eight of AML cases and three of ALL cases were separated on 4% polyacrylamide gel electrophoresis. Sizes of Thai patients treated at Siriraj Hospital Mahidol University, the PCR products were 480 bp of WT1 zinc finger region Bangkok; the other patients were treated at the Department of ( 7 to exon 10), 226 bp of GATA-1 coding sequence, and Hematology, The University of Texas MD Anderson Cancer 541 bp of ␤-actin coding sequence. Both agarose and polyac- Center, Houston, Texas, USA. rylamide gels were dried and the radioactivity was quantitated Bone marrow (BM) and peripheral blood (PB) were by phosphoimaging (Molecular Dynamics, Sunnyvale, CA, obtained from the patients before they received chemothera- USA). The ratio between the ␣32P-dCTP-labeled WT1 and ␤- peutic treatment. PB specimens were obtained from all 94 actin products as well as the ratio between GATA-1 and ␤- patients, whereas both BM and PB specimens were obtained actin products were calculated for each sample. The ratios for from 44 of the 94 patients. Leukopheresed leukemic blood each PB and BM sample were normalized by dividing each samples were obtained from 37 of the remaining 50 patients, ratio by the ratio for the positive control K-562 for each so were WBC-enriched. Normal PB were used as controls for experimental assay. WT1 and GATA-1 expression. Mononuclear cells were separated from leukemic and normal heparinized PB and BM aspirates by Ficoll–Hypaque Chromosome analysis centrifugation. Total RNA was isolated by the method of Chomczynski and Sacchi34 by using STAT-60 (Tel Test, Standard cytogenetic techniques were used35 and Friendswood, TX, USA) according to the manufacturer’s were described according to International System for Human recommendations. Quality of the RNA was analyzed by Cytogenetic Nomenclature (ISCN) recommendation.36 electrophoresis in a 1% agarose-formaldehyde minigel.

Statistical analysis Reverse-transcriptase polymerase chain reaction (RT- PCR) Statistical evaluation of WT1 and GATA-1 expression levels were accomplished by Mann–Whitney U test, using NPAR Reverse transcription was carried out as previously module of the SYSTAT computer software. Results were com- described.18 In brief, 1 ␮g of total RNA was diluted in diethyl- puted as mean values ± standard error of the mean. Corre- pyrocarbonate (DEPC)-treated water, denatured at 70°C for lation between bone marrow and peripheral blood levels of 10 min and then quick-chilled on ice. Reverse transcription WT1 and GATA-1 expression were tested by regression analy- (RT) was performed by adding RT reaction cocktail containing sis – MGLH module of the SYSTAT programme. Relationship

50 mm Tris-HC1 (pH 8.3), 70 mm KCl, 3 mm MgCl2,10mm between WT1 levels and cytogenetic findings dithiothreitol, 200 U of Moloney murine leukemia virus were analyzed by Yates corrected Chi-square test. reverse transcriptase (Superscript II; Life Technology, Gaithers- burg, MD, USA), 500 mm each dNTP (Pharmacia, Piscataway, NJ, USA), 50 ␮m random hexamer primer, and 33 U of RNase Results inhibitor (Boehringer, Mannheim, Germany). The reaction was incubated at 37°C for 90 min and then heated at 95°C We report here the WT1 and GATA-1 gene expression in 103 for 7 min. After quick-chilling, the cDNA products were stored PB samples from 16 patients with myelodysplastic syndromes, at −20°C before use. PCR amplification of one-half of the 55 with de novo AML, one with AML following CML blastic reverse transcription reaction was performed as duplex PCR transformation, 22 with ALL and nine normal individuals. for amplifying either WT1 and ␤-actin (35 cycles) or GATA-1 Bone marrow (BM) WT1 and GATA-1 expression levels were and ␤-actin (30 cycles). ␤-Actin was used as the RNA internal also examined in 44 of the 94 cases of MDS and acute leuke- control of the reaction. The optimal conditions for amplifi- cation of duplex PCR (␤-actin-WT1 and ␤-actin-GATA-1) were described previously23 with a thermal cycler (Perkin- Elmer, Norwalk, CT, USA). One microcurie of ␣32P-dCTP (spec act = 3000 Ci/mmol) was added to each 50 ␮l PCR reac- tion for quantitation purposes. Primer concentrations were 200 nm for WT1 and GATA-1 and to compensate for the over abundance of ␤-actin mRNA we used 1/10th concentration of ␤-actin primer (20 nm). PCR amplification was carried out using the following primer pairs: 5Ј ␤-actin (5Ј- GTGGGGCGCCCCAGGCACCA-3Ј), 3Ј ␤-actin (5Ј- GTCCTTAATGTCACGCACGATTTC-3Ј). 5Ј WT1 (exon 7) (5Ј- GGCATCTGAGACCAGTGAAA-3Ј), 3Ј WT1 (exon 10) (5Ј- GAGAGTCAGACTTGAAAGCAGT-3Ј); 5Ј GATA-1 (exon 5) (5Ј-ATTGTCAGTAAACGGGCAGG-3Ј)3Ј-GATA-1 (exon 6) (5Ј-CACCACCATAAAGCCACCAGC-3Ј). Distilled DEPC water Figure 1 WT1 transcript levels determined by reverse-transcription PCR. 540 bp ␤-actin cDNA was amplified as the internal control. WT1 was used as negative control and RNA isolated from K-562 cDNA was amplified transcript from normal controls (lanes 1–7), cells was used as a positive control for both WT1 and GATA- acute myelogenous leukemia (AML; lane 8), MDS (lane 9, 10, 13), 1 expression. One-fifth of WT1 PCR products were separated ALL (lane 11, 12), and K562 (lane 14). Lane 15 is the 100 bp marker. WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 893 Among the 16 MDS patients, three of seven RA, four of six RAEB/RAEB-T and two of three CMML cases exhibited elev- ated PB WT1 levels. The WT1 expression levels in each of MDS subtypes, as well as WBC counts and the percentage of PB and BM blasts, are given in Table 2. Elevated PB GATA-1 levels were found in two of seven RA, three of six RAEB/RAEB- T, and none of CMML cases. RAEB and RAEB-T expressed relatively higher level of WT1 than RA (P = 0.086). However, the overall PB GATA-1 gene expression levels in MDS were not different from normal controls, with the average value of 0.19 ± 0.03. Since the majority of PB samples were obtained by leukapheresis of leukemic and MDS patients, a comparison was made between the paired BM and PB samples for both WT1 and GATA-1 expression in 12 MDS and 32 acute leuke- ␤ Figure 2 Normalized ratios of WT1 to -actin levels were used as mia cases summarized in Table 3, showed a good correlation internal controls for each sample. The sample ratios were normalized between the BM and PB levels of both WT1 and GATA-1. to K562 cell ratios as described in the text. Each bar represents the mean; the standard error of the mean is depicted above the bar. (1) One MDS case of RAEB subtype (patient No. 50) exhibited Normal PB (n = 9); (2) MDS RAEB/RAEB-T (n = 6); (3) MDS RA (n = elevation of both WT1 (0.72) and GATA-1 (0.44) transcript 7); (4) AML FAB M1, M2, and M3 (n = 21); (5) AML FAB M4, M5, levels in peripheral blood MNC. Complex chromosome M6, and M7 (n = 20); (6) ALL (n = 22); (7) B-ALL (n = 9); and (8) B- abnormalities were also detected in this patient. = and T-ALL with myeloid markers (B,T/My ALL) (n 7). MDS patients whose PB strongly expressed WT1 also showed the most complicated chromosomal abnormalities. mias. The relationship between WT1 expression level and Comparison of the cytogenetic findings in each MDS patient cytogenetic/immunophenotypic findings are described. with different WT1 levels are in Table 4. Although MDS is a rare hematological condition and the number of MDS patients in this study is rather small (n = 16), we found a relationship WT1 and GATA-1 expression in normal individuals between MDS subtypes, WT1 gene expression levels and cytogenetic findings among these patients. When 15 cases Average PB MNC levels of WT1 from nine normal individuals with MDS were analyzed, the MDS cases whose WT1 levels was 0.12 ± 0.02 (range 0.02 to 0.20) (Figure 2). The same were lower than 0.35 showed normal diploid karyotypes or a average level of 0.12 ± 0.02 was found for PB MNC GATA-1 single chromosome abnormality while MDS cases with WT1 gene expression (range 0.09 to 0.17). The individual normal expression higher than 0.35 exhibited complex chromosome ␹2 = = control level of WT1 and GATA-1 are shown in Table 1. aberrations ( 7.85, P 0.007). Six of the seven RA cases had either normal diploid karyotypes or a single chromosome defects (loss of Y or gain 8). Chromosome analysis was not WT1 and GATA-1 expression in myelodysplastic possible in one patient with RA (No. 39). Four patients with syndromes RAEB/RAEB-T whose PB WT1 expression levels were above 0.35 exhibited complex chromosomal abnormalities (patients Among MDS patients, higher WT1 levels in PB were found in 28, 30, 36 and 50). Two of them had very high PB WT1 the subtypes RAEB and RAEB-T (Figure 2, Table 2), with the expression levels (0.88, patient 36; and 0.72, patient 50) and mean values of 0.46 ± 0.12 (n = 6) or 3.8 times higher than showed several types of both numerical and structural chro- the average normal control level (P = 0.029). Four of the six mosome aberrations. Addition of the short arms of chromo- RAEB and RAEB-T cases strongly expressed WT1 and their some 11 (add(11)(p15)) and 17 (add(17)(p13)), as well as WT1 gene expression levels were more than 0.35 (Table 2). deletions of the long arms of chromosome 5 (del(5)(q32)) and In contrast, WT1 expression levels in RA was not different 20(del(20)(q11.2) were detected in patient No. 36. In patient from normal control and the average WT1 levels in RA (n = No. 50, the clonal structural chromosome defect includes 7) and CMML (n = 3) were 0.19 and 0.26, respectively. t(7;17)(q21;q11.1) and t(1;16)(p36.3;p13.3) in combination with many numerical defects. In addition, del(5)(q13) and i(17)(q10) were observed in patient 28 and del(12)(p12) in Table 1 Peripheral blood MNC WT1 and GATA-1 levels in nor- patient No. 30. The other two RAEB/RAEB-T cases (patients mal individuals 29 and 33) whose PB had normal or slightly elevated WT1 levels, showed normal diploid karyotypes. A normal diploid − − Control ID WT1 × 10 2 GATA-1 × 10 2 , however, was observed in a CMML patient who had an elevated WT1 level of 0.53 (patient 62). 114 12 17 115 12 10 116 11 14 117 14 9 WT1 and GATA-1 expression in acute myeloid 118 13 14 leukemia 119 4 14 120 19 12 Peripheral blood WT1 and GATA-1 expression levels in MNC 121 2 11 of 56 AML patients are summarized in Table 5. Increased PB 122 20 11 WT1 gene expression levels were found in 50 of 56 cases Mean 12 ± 212± 2 (89%) as compared with levels in the normal controls (Figure Range 2–20 9–17 2). The average WT1 level in patients with de novo AML was 0.7 (n = 54) or 5.8 times the mean normal control value (P Ͻ WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 894 Table 2 WT1 expression levels, WBC counts and percentage of blasts in MDS patients

ID No. Sex/Age FAB types WT1 levels × 10−2 WBC × 109/l % PB blast % BM % BM normoblast

31 F/45 RA 34 3.2 0 4 NA 32 M/72 RA 19 5.1 0 0.7 7 34 M/72 RA 9 1.7 0 5 17.2 35 M/65 RA 23 4.8 0 NA 48.4 37 F/49 RA 31 1.3 0 0 26 38 M/80 RA 16 3 0 0.2 30.2 39 M/49 RA 2 1.8 0 3.8 29.3 28 F/84 RAEB 42 0.8 0 14 3 33 M/72 RAEB 19 2.2 0 9 30.2 36 F/76 RAEB 88 1.4 0 10 NA 50 M/76 RAEB 72 1.8 2 12 55.6 29 M/59 RAEB-T 9 NA NA 30 NA 30 M/66 RAEB-T 50 6.5 22 22.8 0 62 M/62 CMML 53 7.5 2 2 44 63 F/72 CMML 6 17.3 1 5 6.8 64 M/73 CMML 21 20.0 NA 7.2 26.2

Table 3 Correlation between peripheral blood and bone marrow expression of WT1 and GATA-1 in AML, ALL and MDS

Type of diseases (n) WT1 (PB and BM) GATA-1 (PB and BM)

rr2 P value of regression r r2 P value of regression

AML (19) 0.977 0.951 Ͻ0.001 0.864 0.732 Ͻ0.001 ALL (13) 0.989 0.975 Ͻ0.001 0.953 0.899 Ͻ0.001 MDS (12) 0.996 0.992 Ͻ0.001 0.789 0.585 0.002

r, standard correlation coefficient.

Table 4 Cytogenetic findings in 16 MDS patients

ID No. FAB types WT1 × 10−2 Cytogenetic findings

31 RA 34 46,XX 32 RA 19 46,XY 34 RA 9 46,XY/45,X, −Y 35 RA 23 46,XY/47,XY, +8 37 RA 31 46,XX 38 RA 16 45,X, −Y 39 RA 2 NA 28 RAEB 42 46,XX/Hypotetraploid: 91,XXXX, del(5)(q13),del(5)(q13),− 17,i(17)(q10)/46,XX,del(5)(q13) 33 RAEB 19 46,XY 36 RAEB 88 46,XX/46,XX,del(5)(q32), add(11)(p15), add(17)(p13),del(20)(q11.2)/85–93,XXXX, exhibiting del(5)(q32) x2, add(11)(p15) x2, −14, −14, −16, −16, add(17)(p13) x2, del(20)(q11.2) x2, +2 to 6 mars 50 RAEB 72 46,XY,der(7)t(7;17)(q21;q11.1), −13,−17,−18,−19,−20,+21,+4mars/ 43,XY, −1, −5, der(7)t(7;17)(q21;q11.1), −13, −15, der(16)t(1;16)(p36.3;p13.3), −17, −20, +3mars/42–44,XY, exhibiting: −5, der(16)t(1;16)(p36.3;p13.3), −13, −17, +mar 29 RAEB-T 9 46,XY 30 RAEB-T 50 46,XY/47,XY, idem, +8/46,XY, del(12)(p12) 62 CMML 53 46,XY 63 CMML 6 46,XX 64 CMML 21 46,XY/45,XY, −7 WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 895 Table 5 Summary of PB WT1 levels, PB GATA-1 levels, percent- of the relationship between WT1 expression levels and cytog- age of BM and PB blast and promyelocyte in each AML subtype enetic findings revealed that AML cases with high WT1 expression (WT1 у1.0) associated with complicated chromo- × −2 + FAB (n) WT1 10 GATA-1 % blast pro some abnormalities of two or more types, whereas AML cases × 10−2 with lower WT1 expression (WT1 Ͻ1.0) tend to have normal BM PB diploid karyotype or a single type of chromosome aberration (␹2 = 9.35, P = 0.002). Cytogenetic findings of AML cases M1 (8) 105 (17) 28 (16) 83 (4.4) 69 (6.9) whose PB cells expressed moderately high WT1 levels M2 (12) 85 (12) 23 (6) 83 (6.1) 57 (5.0) M3 (3) 54 (16) 33 (15) 62 (17) 54 (20) (between 0.50 to 0.99) are shown in Table 6b. M4 (12) 48 (10) 15 (3) 66 (6.4) 43 (7.7) The types of chromosome aberration observed in M5 (5) 37 (8) 57 (41) 56 (11.9) 25 (9.4) overexpressing WT1 are noteworthy. Both of the AML cases M6 (2) 43 42 15 0 with an abnormality of chromosome 19p13.3 (patients 58 and 19 19 16 2 59) (Table 6a) had very high WT1 expression levels of 1.12 M7 (2) 39 9 32 26 and 1.10, respectively. In patient 58, the abnormality involv- 81432NA AML (9) 72 (25) 26 (10) 69 (7.7) 51.4 (11.2) ing the short arm of chromosome 11 (add(11)(p15)) was also found. Abnormal 11p15 was also observed in another patient AML, AML cases for which FAB subtypes were unavailable. with AML who had t(11;17)(p15;q23) and in one patient with Values are mean (s.e.m). MDS (patient 36) who also had add (11)(p15), and both of these cases also showed high WT1 expression levels of 0.69 and 0.67, respectively. In AML case No. 65 arising from blas- 0.001). Patient No. 58 with AML subtype M0 and all cases of tic transformation of CML, significant levels of both WT1 the granulocytic subtypes (M1, M2, M3, n = 23) showed WT1 (1.75) and GATA-1 (0.41) were expressed. Again, cytogenetic expression levels of more than three times the mean normal analysis of this patient revealed complex abnormalities control level (Ͼ0.35). Subtypes M4, M5, M6, M7 had lower involving t(9;22)(q34;q11), del(11)(q13q21), del(12)(q14), WT1 expression than the granulocytic (M1, M2, M3) subtypes add(16)(q24), del(3)(p11p21) and t(2;17)(p21; p13). (P Ͻ 0.001) but higher levels than the normal controls (P = 0.001). Additionally, two of the nine FAB unclassified cases expressed CD33+CD14− (non-monocytic) and three were WT1 and GATA-1 expression in acute lymphoblastic CD33+CD14+. WT1 expression in the two non-monocytic leukemia (ALL) AML were 0.97 and 1.57 while the WT1 levels observed in the three cases with CD14+ were 0.11, 0.32 and 0.27. Data Sixteen of 22 cases of ALL (72.7%) had higher WT1 levels on CD33 and CD14 from the other four FAB unsubclassified than the normal individuals (P = 0.002) (Figure 2). The average cases were not available. When the WT1 gene expression lev- WT1 level among patients with ALL was 0.43 ± 0.06. Details els of patients with AML and MDS were compared, the levels of FAB classes, immunophenotypes, percentage of PB and BM in AML subtypes M1, M2 and M3 were significantly higher blasts, WT1 expression levels and cytogenetic findings in each than those in MDS in both subtypes RAEB and RAEB-T (P = ALL patient are listed in Table 7. The FAB type L1 and type 0.044) and subtype RA (P Ͻ 0.001). L2 did not differ in terms of WT1 expression levels, with the Average peripheral blood GATA-1 expression in all 56 AML means of 0.36 (n = 7) and 0.43 (n = 10), respectively. Of all cases is 0.29 ± 0.06. A wide variation of GATA-1 levels was patients with ALL, B-precursor ALL was diagnosed in 16 of 22 observed among AML cases and there is no statistical differ- cases, T precursor ALL in two cases and unclassified pheno- ence among AML subtypes. Also, no consistent difference in type in four cases. A patient of B-precursor ALL was diagnosed GATA-1 levels was found between the granulocytic subclasses as pro-B-ALL (CD34+, CD10−, CD19+, CD20−) and 15 cases and M4 to M7 subclasses. However, 24 of 56 AML cases co- as pre-pre-B (CALLA)/or pre-B-ALL (CD10+, CD19+, CD20+/or−, expressed both WT1 and GATA-1 in higher than normal lev- C␮ data were not available). Of these 15 B-precursor inter- els. In our previous report of AML cases whose leukemic cells mediate ALL cases, six also expressed myeloid markers (CD33 exhibited chromosome abnormalities involving 16q22,23 or CD13 or both) and so-called B-precursor/My ALL. Both expression of high WT1 and very low GATA-1 levels was cases of T-precursor ALL expressed CD7+, and had high WT1 found. However, here we report that only 10 of the 56 AML levels of 0.9 and 1.0. One of these two T-precursor ALL cases cases expressed higher than normal WT1, but lower than also expressed TdT+, CD34+, CD33+, CD13+ and so-called T- normal GATA-1. precursor/My ALL. These mixed B (n = 6) or mixed T (n = 1), Cytogenetic analyses were done for 52 of 56 AML cases. In precursor/My ALL cases showed relatively higher WT1 14 of these cases, WT1 expression levels were very high (the expression levels than the cases of pure B-precursor ALL, with levels of у1.0) and details of their cytogenetic findings are average WT1 levels of 0.56 ± 0.13 s.e.m. (n = 7) and shown in Table 6a. Among this group, abnormal karyotypes 0.36 ± 0.08 s.e.m. (n = 9), respectively. Five of the seven were found in 11 of 14 cases (77%). In 10 of these 11 cases, mixed ALL/My had WT1 levels above 0.35 two or more types of chromosomal abnormalities were whereas this level was observed in only three of the nine B- observed. Three or more types of chromosomal abnormalities precursor-ALL. The immunologic phenotypes were not avail- were found in seven of the 14 high WT1 expressors. Of the able in four ALL cases; but three of four showed elevated 38 patients with AML whose cells expressed WT1 at lower levels of WT1 expression. than 1.0, 25 cases (66%) exhibited abnormal karyotypes. Only Among six cases with CD10+ and CD19+, the CD20 pheno- eight of these 25 cases showed two or more types of chromo- type was examined: four were CD20+ without any myeloid somal defects (compared with 10 of 11 in the former group), markers and the other two cases were CD20−, both expressed and only five of the 38 cases whose WT1 expression ranging CD13+. The average WT1 levels were 0.17 in the former four from normal limit to 0.99 showed three or more types of cases and 0.79 in the latter two. chromosomal abnormalities. Interestingly, statistical analysis Cytogenetic analysis was done in 17 of 22 ALL cases and WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 896 Table 6 Cytogenetic findings in AML patients whose WT1 expression is increased

ID No. FAB types WT1 × 10−2 Cytogenetic findings

(a) AML cases with greatly increased WT1 levels (у1.00) 40 M4 128 46,XX/45,X,−X/46,X,del(X)(p11.3) 41 M2 138 46,XY 56 NA 110 46,XY/41–44,XY, exhibiting common abnormalities of: t(3;5)(q10;p10), add(12)(p10), −19 57 M1 118 47,XY,t(6;9)(p23;q34), +13/46,XY,t(6;9)(p23;q34) 58 M0 112 46,XY/46,X, −Y,der(1)t(1;9)(q12;q12),add(2)(p21), −8, −10, add(11)(p15), add(16)(q23), I(17)(q10),der(19)t(1;19)(q11;p13.3)×2, +20, +21, +mar, +1 to 2R/42–44,X, −Y, exhibiting common of: der(1)t(1;19)(q12;q12),add(2)(p21), −8, −10, add(11)(p15), −13, −14, add(16)(q23),I(17)(q10),der(19)t(1;19)(q11;p13.3)x2, +20, +21, + mar, +R and random numerical changes 59 M1 110 46,XX, −5, +8, +11, −17/45,XX,add(1)(p36.3), −5, +11, −17,add(19)(13.3)/46,XX, −5, +8, +11, −17 74 M2 165 46,XX/46,XY,3p+,7p−, inv(16) 85 NA 157 46–55,XY, exhibiting: −5, 6p+,9p+, −11, 13q−, 15p+, 17p−, 17p−, −18, −21, −22, +mar 86 M2 128 46,XX 87 M4 100 47,XX +22, inv(16)/46,XX,inv(16),t(22;22)(q+;q−) 91 M2 120 47,XX, +11/46,XX 93 NA 210 46,XX 94 M1 220 46,XX/46,XX,1p−, t(11;16)(q−;p+) 65a AML 175 46,XY,t(9;22)(q34;q11)/46,XY,t(9;22)(q34;q11), del(11)(q13q21), del(12)(q14), add(16)(q24)/46,XY,del(3)(p11p21),t(9;22)(q34;q11)/ additional chrom. Abnormality: 46,XY,idem,t(2;17)(p21;p13)

(b) AML cases with moderately increased WT1 levels (у0.50 to 0.99) 51 M2 95 46,XY/48,XY, +4, +8 55 M4 57 42–46,XY,exhibiting:del(3)(q23),del(4)(q12), −5, −6, del(6)(q15),add(7)(q22), −11,+add(12)(p11), add(15)(q26), −17, −18, −21, −22, +1 to 5 mars 71 NA 97 46,XY 73 M1 78 46,XX 75 M4 72 46,XX,inv(16)(p13q22) 78 M2 62 46,XY 80 M2 68 46,XY 83 M4 53 46,XX 84 M2 79 45,X,−Y/46,XY 88 M1 81 46,XY 89 M2 91 46,XX,4q+ 95 M1 96 46,XY,20q− 98 M3 87 45,XX,−9 100 M5 69 46,XX/46,XX,t(11;17)(p15;q23)

aAML case arising from CML in blastic transformation.

chromosome abnormalities were seen in 14 of the 17 cases del(13)(q12q22) and another with del(4)(p11). Five B-precur- (82%). Thirteen of these 14 cases with abnormal karyotypes sor ALL cases exhibited t(9;22)(q34;q11) and three of five showed elevated WT1 transcript levels, whereas elevation of showed high WT1 transcript levels. Since the majority of ALL GATA1 transcript was found in only three cases. Two ALL cases in our study were diagnosed as B-precursor ALL, we did cases, one with translocation t(9;22)(q34;q11) and another not find t(9;22) in the two cases of T-precursor ALL. WT1 lev- with del (2)(p11.2), showed elevated levels of both WT1 and els within normal limits were observed in four cases with GATA1 gene transcripts. Chromosome 19p abnormalities abnormal karyotypes. Conversely, increased WT1 levels were were seen in three of the 14 ALL cases with abnormal kary- detected in all three cases with normal karyotype. Increased otypes: patient 6 with del(19)(p13.3), patient 10 with sample size of ALL cases will be necessary to verify the statisti- t(1;19)(q23;p13) and patient 15 with add (19)(p13), along with cal significance of ALL phenotypes in relation to WT1 hyperdiploid and other complex structural abnormalities, expression/or cytogenetic findings. including t(14;18)(q32;q21), add(1)(p36) and add(14)(q32). All The overall GATA-1 expression in ALL was not different three cases exhibited high levels of WT1 transcripts. from the normal control, with the average value of 0.19 ± Seven ALL cases showed chromosomal deletions and high 0.05. Only two out of 20 ALL cases showed increased levels levels of WT1 were expressed in six of these seven cases. The of both WT1 and GATA-1 coexpression (patient 4, WT1 0.65 highest WT1 level in the ALL group was observed in a case and GATA-1 1.07; patient 17, WT1 1.03, GATA-1 0.22). Inter- of T-precursor/My ALL who had del(2)(p11.2). As mentioned estingly, the immunophenotype and cytogenetic study of these earlier, moderately elevated GATA1 was also detected in this two patients revealed that patient 4 had mixed B-precursor/My patient. High elevation of WT1 levels were also found in ALL and t(9;22)(q34;q11) while patient 17 had mixed T- the other two deletion cases, one with del(6)(q23), precursor/My ALL and cytogenetic findings showed complex WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 897 Table 7 Details of clinical and hematological features, immunophenotypes, peripheral blood MNC WT1 expression levels and cytogenetic findings in 22 ALL patients

ID S/A FAB Imm % Blast WT1 Cytogenetic findings Phea PB BM × 10−2

2 F/74 L2 1 92 76 14 46,XX/46,XX,t(4;11)(q21;q23),I(17)(q10)/46,XX,t(4;11) (q21;q23) 1 M/35 L2 2 66 79 5 46,XY/46,XY,del(7)(p15),t(9;22)(q34;q11),del(12)(p11) 3 M/41 L1 2 66 87 9 NA 6 F/90 NA 2 91 92 37 del(19)(p13.3) with random structural and numerical abnormalities 9 M/43 L2 2 7 94 87 46,XY 11 F/35 L2 2 76 89 22 46,XX,t(9;22)(q34;q11) 12 M/38 L1 2 56 90 32 46,XY,del(9)(p11p21) 13 F/60 L1 2 41 94 34 46,XX,/52,XX, +2, +6, del(9)(p22),t(9;22)(q34;q11), +13, +14, +21,+der(22)t(9;22)(q34;q11)/53–54,XX exhibiting common of +2, +4, del(9)(p22),t(9;22)(q34;q11),+13, +14, +21, +der(22)t(9;22)(q34;q11),+2mars 15 M/45 L1 2 26 95 29 Hyperdiploid: 54,XY, +X,+Y, add(1)(p36), add(4)(q32), +7, b +11, t(14;18)(q32;q21), add(14)(q32), +der(18)t(14;18) (q32;q21), add(19)(P13)×2, +20, +2 mars 18 F/7 L1 2 21 30 67 NA 4 F/30 L1 3 43 46 65 46,XX/46,XX,t(9;22)(q34;q11) 5 F/65 L2 3 76 91 38 46,XX 7 M/20 L2 3 8 93 14 46,XY/47,XY,t(9;22)(q34;q11),+der(22)t(9;22) (q34;q11)/47,XY,dup(1)(q31q44),t(9;22) (q34;q11),+der(22)t(9;22)(q34;q11) 8 M/36 L1 3 22 97 14 46,XY/45,XY,−14 10 M/33 L2 3 74 94 81 46,XY/46,XY,der(19)t(1;19)(q23;p13) 16 M/18 L2 3 72 80 77 46,XY/46,XY,del(6)(q23),del(13)(q12q22)/ 46,XY,del(13)(q12q22) 19 M/54 L2 4 96 96 91 46,XY,del(4)(p11) 17 M/60 NA 5 99 96 103 46,XY/45,XY,del(2)(p11.2), −5, −8, −16, −18, −22, +4 mars/ 45,XY, del(2)(p11.2), −5, −8, −18, −22, −22, +4 mars 14 M/57 NA NA NA NA 44 46,XY 20 F/? NA NA NA NA 35 NA 105 M/55 NA NA NA NA 44 NA 123 NA L2 NA 42 45 3 NA aImmunophenotypes of ALL cases: ‘I’ early B precursor ALL (pro B ALL); ‘2’ pre-pre B (CALLA)/or pre-B ALL; ‘3’ pre-pre B/or pre-B ALL with myeloid markers; ‘4’ T-precursor ALL; ‘5’ T-precursor ALL with myeloid markers; NA, samples were not available for analysis, or inadequate samples. numerical and structural abnormalities with deletion of chro- BM. However, the percentage of blasts and promyelocytes mosome 2 at 2p11.2. There were inadequate samples for which could affect WT1 levels were not described in their GATA-1 determination in two of 22 ALL cases. study. By using the sensitive, isotopic-quantitative duplex primed RT-PCR approach, with ␤-actin as an internal control in the same PCR reaction used for amplifying WT1 or GATA- Discussion 1, we demonstrated a detectable amount of WT1 transcripts in PB mononuclear cells of normal individuals. Bone marrow WT1 transcript levels in patients with MDS and acute CD34+ cells also expressed a substantial level of WT115 which leukemia may be affected by stages of cellular has been confirmed by other investigators.39,44 Maurer et al40 differentiation/maturation, proliferation capacities, cellular recently reported that WT1 mRNA is equally expressed in lineages, and genetic changes. We and others have previously blast cells from AML and normal CD34+ progenitors. On the reported the differences in WT1 transcript levels in various other hand, Inoue et al41 found that leukemic cells expressed subtypes of AML.16–18,23,37,38 Less differentiated subtypes of WT1 in the range between 2.4 to 9.3 × 10−1 and the levels of AML, such as M0 and M1, expressed higher WT1 levels than WT1 expression in normal CD34+ subsets of bone marrow the more differentiated subtypes. We have presented evidence and cord blood were very low compared to the leukemic that WT1 mRNA was produced at higher levels in patients cells, although significantly higher than the CD34− subsets. with undifferentiated (M0) and granulocytic AML (M1 to M3) The observation that WT1 was transiently expressed in normal than in patients with the other AML subtypes (M4 to M7). In CD34+ cells42 in early hematopoietic differentiation and that accordance with Bergmann et al,39 AML patients with mono- it was downregulated during differentiation of leukemic cell cytic subtypes (M4, M5) expressed either lower WT1 levels lines43,44 suggests that the WT1 gene plays a role in hematopo- (ours) or the frequency of WT1 expression in (WT1+)39 in AML ietic differentiation in normal and leukemic cells. patients with monocytic subtypes was less. We also found that The clinical relevance of WT1 is of current interest. Inoue all cases of M0–M3 (n = 25) and half of M6 and M7 (n = 4) et al18,45 reported the detection of minimal residual disease expressed high WT1 levels, whereas Bergmann et al reported (MRD) by monitoring of WT1 expression levels in BM and PB WT1+ in M0, M1, M3 and M6 cases but only 74% of their and found that peripheral blood samples were superior to M2 cases overexpressed WT1 compared to the normal control bone marrow samples for detection of MRD.45 Quantitation WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 898 of WT1 expression levels has been proposed as a method to while only three of nine cases of B-precursor ALL without assess the effectiveness of individual treatment and the diag- myeloid markers reached this level. Combination of this find- noses of clinical relapse,45 as well as prediction of the overall ing with that of our previous work8,23 suggests that WT1 survival of leukemia patients.39 expression is cell type-specific and expressed more in myeloid The normal PB WT1 levels in this study are five to 10 times than in lymphoid or monocytic cells. Alternatively, elevated higher than those determined by Inoue et al41,45 but variation WT1 levels in ALL expressing myeloid markers may simply in the quantitation procedures used could explain differences reflect the early stage of differentiation of their abnormal in the levels of WT1 expression observed among investigators. multipotent leukemic ancestors that inherited the We used duplex single reactions for amplifying WT1 and ␤- ability to express both lymphoid and myeloid markers. We actin, standard amplification cycles, and lower concentration and others have demonstrated that leukemias with early differ- of ␤-actin relative to WT1 primers. All of these could affect entiation-stage markers express more WT1 than the late stage, the WT1/␤-actin ratio calculated by different investigators. more mature leukemias.18,23,37 Our observation that ALL with Optimization of RT-PCR conditions used in our studies was CD20−, CD10+, and CD19+ expresses more WT1 than the ALL reported previously15,23 and only high quality total RNAs were with CD20+, CD10+, and CD19+ also supports the hypothesis used in order to prevent failure of the RT-PCR reaction from that WT1 plays role in the stages of cellular degraded RNA. The ethidium bromide stained PCR products differentiation/maturation. Thus, function of WT1 gene may (␤-actin and WT1) from normal controls and patients with influence both stage of differentiation of the leukemic clones MDS and leukemia are apparent in Figure 1. and the specific lineage programme for cellular differentiation. To understand a possible interaction between WT1 and Expression pattern of transcription factors may reflect the GATA-1, a regulator of WT1 expression, we examined WT1 programming for specific lineages. Increased GATA-1 and GATA-1 gene expression in MDS and acute leukemia. expression was observed in 27 of our 56 AML cases, the fre- Above normal levels of WT1 gene expression were observed quency comparable to those of Shimamoto et al.46 In their in PB and BM of patients with MDS (especially subtypes RAEB report, the detectable levels of GATA-1 expression (GATA-1+) and RAEB-T), AML and ALL. MDS-RA (the mildest form of were found in 30 of 62 AML patients (48.4%). Cell lines exhi- MDS) samples did not show significantly different WT1 biting erythrocytic and megakaryocytic phenotypes expressed expression levels compared with normal controls. Patients GATA-1, and downregulation of GATA-1 in HL-60 cells was with RA presented with reticulocytopenia, oval macrocytes, found during the process of monocytic differentiation. In morphologically normal and in addition, AML patients with GATA-1− showed significantly BM and rare blast cells (less than 1% in PB and less than 5% higher complete remission rate (CR) than the AML patients in BM). The more aggressive MDS subtypes RAEB and RAEB- with GATA-1+ suggesting a prognostic value for GATA-1 T were examined together in this study because of the limited expression in AML. In this study, we found that 24 of 56 AML number of cases. These two MDS subtypes showed obviously patients showed elevation of both WT1 and GATA-1, whereas higher WT1 expression levels than the normal level and - only one of 12 MDS and two of 20 ALL showed this type of tively higher than RA (P = 0.086). These subtypes presented coexpression. Both cases of ALL which had high WT1–GATA- with trilineage cytopenias with significant dysmyelopoiesis 1 coexpression exhibited myeloid markers. The compilation and dysmegakaryopoiesis. PB and BM blasts are 1–5% and 5– of these data partly supports the previous studies19,22 that WT1 20%, respectively, for RAEB, but Ͼ5% and 20–30%, respect- transcription is transactivated by GATA-1 through 3Ј and 5Ј ively in RAEB-T. Differences in WT1 expression levels WT1 enhancers in a cell type-specific fashion. These between RA (mean 0.19) and RAEB/RAEB-T subtypes (mean enhancers are responsible for expression of WT1 in erythroid 0.46), along with the similarity of WT1 expression levels (WT1 3Ј enhancer) and myeloid (WT1 5Ј enhancer) lineages. between RA and the normal individuals suggests that WT1 Several observations suggest that GATA-1 is expressed in the gene expression probably increases in the abnormal MDS same cell type-specific manner as WT1: high expression of cells of more severe subtypes. When WT1 gene expression GATA-1 in primary AML and erythrocytic and megakaryocytic levels of AML and MDS samples were compared, we found cell lines, downregulation of GATA-1 during monocytic differ- that AML (especially subtypes M1, M2, M3) showed even entiation of HL60 cells,46 downregulation of WT1 during dif- higher WT1 expression levels than MDS in both RAEB/RAEB- ferentiation of K562 cells to erythroid and megakaryocytic T and RA. cells43 as well as downregulation of WT1 in HL-60 cells dur- Leukemic cells in PB are descendants of their BM ancestors. ing myelomonocytic differentiation.44 These findings suggest WT1 gene expression in AML cases with 50% or more BM that GATA-1 and WT1 may have certain regulatory blasts is higher than that in cases with fewer than 50% BM cooperation in terms of the programming for monocytic, blasts (P = 0.002, Table 5). These results imply that WT1 erythrocytic and megakaryocytic differentiation from their expression may be directly related to the number of BM blasts myeloid progenitors. which reflect proliferative capacities of leukemia cells. WT1 Other transcription factors or regulatory mechanisms could gene expression may be useful in determining the prognosis affect WT1 expression. For the 32 AML patients whose GATA- of MDS patients whose disease is tending to progress into the 1 expression levels were not correlated to WT1 expression more aggressive forms, RAEB and RAEB-T and possibly to levels, GATA-1 may not be necessary for transactivation of acute leukemia. However, further studies are needed before WT1 in these patients and WT1 may be regulated by other the conclusion is made concerning the predictive value of factors. WT1 is also regulated by c-Myb,22 PAX8,47 leukemia WT1 expression regarding progression of MDS. inhibitory factor (LIF)48 and WT1 itself (autoregulation).49 All cases expressed WT1 levels that were significantly Zhang et al22 found that c-Myb can transactivate WT1 hema- greater than normal control levels but less than those of AML. topoietic specific enhancer by up to five-fold in a co-transfec- Increased WT1 expression was found in ALL cells expressing tion system, using WT1 enhancer-CAT reporter construct and both lymphoid and myeloid markers. Five of seven ALL cases c-Myb expression vector. We and others50,51 previously dem- (either B- or T-precursor ALL) exhibiting myeloid markers onstrated coexpression of WT1 and PAX8 in both renal and CD33+ and CD13+ expressed WT1 levels of more than 0.35 hematopoietic neoplastic cell lines and we demonstrated that WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia P Patmasiriwat et al 899 PAX8 can transactivate WT1 promoter.47 Upregulation of 5 Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP, WT1 gene by LIF in M1 leukemic cells has been reported,48 Rauscher FJ III. Transcriptional repression mediated by the WT1 and this WT1 activation is associated with monocytic differen- Wilms’ tumor gene product. Science 1991; 253: 1550–1553. 6 Rauscher FJ III, Morris JF, Tournay OE, Cook DM, Curran T. Bind- tiation of the M1 cells. On the other hand, WT1 has also been ing of the Wilms tumor locus zinc finger protein to the EGR-1 known to control expression of hematopoietic growth control consensus sequence. Science 1990; 250: 1259–1262. genes such as CSF-1, TGF␤-1, RAR-␣, c-Myc and bcl-2.12,52 7 Bickmore WA, Oghene K, Little MH, Seawright A, van Heyningen Coexpression of WT1 and bcl-2 in BM cells of 152 AML V, Hastie ND. Modulation of DNA binding specificity by alterna- patients, and association of this coexpression to therapeutic tive splicing of the Wilms’ tumor gene transcript. Science response and long-term outcome of the AML patients have 1992; 257: 235–237. been reported, suggesting the use of WT1–bcl2 coexpression 8 Fraizer GC, Wu YJ, Hewitt SM, Maity T, Ton CC, Huff V, Saunders GF. Transcriptional regulation of the human Wilms’ tumor gene as a new prognostic factor. WT1 may be a key factor that (WT1). Cell type-specific enhancer and promiscuous promoter. J controls not only the hematopoietic system but also genito- Biol Chem 1994; 269: 8892–8900. urinary organ development. Regulation by WT1 of genes 9 Hewitt SM, Fraizer GC, Saunders GF. Transcriptional silencer of involved in sexual development has recently been reported. the Wilms’ tumor gene WT1 contains an alu repeat. J Biol Chem Shimamura et al54 demonstrated that SRY (sex determining 1995; 270: 17908–17912. region of the Y chromosome) promoter was strongly activated 10 Hewitt SM, Hamada S, McDonnell TJ, Rauscher FJ III, Saunders by the WT1 (KTS-) isoform while the AR () GF. Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms’ tumor suppressor gene WT1. Cancer Res 1995; 55: promoter was strongly repressed by this isoform. These find- 5386–5389. ings suggest that WT1 plays an important role in embryonal 11 Dey BR, Sukhatme VP, Robert AB, Sporn MB, Rauscher FJ III, Kim sexual development and it can function as a repressor or an SJ. Repression of the transforming growth factor-beta 1 gene by of its target genes. the Wilms’ tumor suppressor WT1 gene product. Mol Endocrinol In conclusion, we report WT1 and GATA-1 expression, 1994; 8: 595–602. cytogenetic findings and specific cellular markers in acute leu- 12 Goodyer P, Dehdi M, Torban E, Bruening W, Pelletier J. kemia and myelodysplastic syndrome. High WT1 levels were Repression of the -alpha gene by Wilms’ tumor suppressor gene product, wt1. Oncogene 1995; 10: found in RAEB and RAEB-T types of MDS, and in M0 to M3 1125–1129. types of de novo AML. For ALL, cases with B-, or T-precusor 13 Park S, Schalling M, Bernard A, Maheswaran S, Shipley GC, Rob- ALL having myeloid markers showed elevation of WT1 levels ert D, Fletcher J, Shipman R, Rheinwald J, Demetri G, Griffin J, greater than pure B-precursor ALL. Increased WT1 expression Minden M, Hausman DE, Haber DA. The Wilms tumor gene WT1 was associated with AML with increased percentage of blasts is expressed in murine mesoderm-derived tissues and mutated in and leukemic promyelocytes, as well as with MDS and acute a human mesothelioma. 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