Acute leukemic transformation of myelodysplastic syndrome—Immunophenotypic, genotypic, and cytogenetic studies

Leukema

Pergamon

Research Vol. 19, No. 9, pp. 595-603, 1995. Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126195 $9.50 + 0.00

0145-2126(95)00015-l

ACUTE LEUKEMIC TRANSFORMATION OF MYELODYSPLASTIC SYNDROME-IMMUNOPHENOTYPIC, GENOTYPIC, AND CYTOGENETIC STUDIES Hwei-Fang Tien,* Chiu-Hwa Wang,? Sou-Ming ChuangJ Fen-Yu Lee,1 Ming-Chi Liu,$ Yao-Chang Chen,t Ming-Ching Shen,? Kai-Hsin L.in§ and Dong-Tsam Lint *Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; +Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; SDepartment of Pathology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; and SDepartment of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, Republic of China (Received and accepted 29 November 1994) Abstract-The clinical and biological characteristics of myelodysplastic syndrome (MDS) in acute leukemic transformation were studied in 23 patients. All had myeloid transformation according to FAB criteria, but coexpression of lymphoid-associated antigens was detected in five of the 20 patients who underwent an immunophenotypic study. Rearrangement of the immunoglobulin heavy chain gene was also observed in one of the five patients who coexpressed lymphoid markers and that of the T-cell receptor p chain gene in another one. None had pure lymphoid transformation. Clonal chromosomal abnormalities were noted in 12 (63%) of the 19 patients who underwent cytogenetic study, most commonly -7 (six patients or 32%). In the 18 patients who underwent serial analyses both at MDS diagnosis and at acute transformation, seven (39%) underwent karyotypic evolution. The most common new or additional aberrations were +8 and +21. N-ras gene mutation was detected in two of the nine patients at acute leukemic transformation. The median interval from diagnosis of MDS to onset of acute transformation was 10 months (1-36 months). Patients with a normal karyotype at diagnosis had a significantly longer chronic phase duration than those with chromosomal abnormalities (median of 20 months vs. 5 months). However, all had a short survival time after diagnosis of acute leukemia, whether chromosomal anomalies were present or not. Key words: Myelodysplastic syndrome, genotype, karyotypic evolution.

acute leukemic

Introduction

transformation,

immunophenotype,

management of patients. However, only a few immunophenotypic and genotypic studies, the former being very helpful for the diagnosis of lymphoblastic or mixed-lineage leukemia, have been done systematically [13, 141. The mechanism of acute leukemic transformation in MDS may involve successive genetic changes [15] which are shown partly by karyotypic evolution during disease progression [2,3, 5, 8, 161. Ras gene mutations and other undetermined genetic lesions may also play some roles [15, 17, 181. The present study was conducted to clarify the nature of blast cells in patients with acute leukemic transformation oF a previous MDS by combined morphologic, cytochemical, immunophenotypic, and genotypic studies. The clinical features and new or additional chromosomal abnormalities at the time of acute transformation, compared with the change at diagnosis of MDS, were also analyzed.

The myelodysplastic syndromes (MDS) are characterized by proliferation of blood cells with abnormalities of differentiation leading to peripheral blood cytopenia and a preleukemtc state [l]. Evolution to acute leukemia occurs in about 10-3.5s of MDS [2,3,4,5,6,7,8]. The blast cells from leukemic transformation of MDS have been assumed to be of myeloid lineage but transformation to a lymphoblastic or hybrid leukemia has been reported [9, 10, 11, 121. It is important to correctly identify the cell lineage of leukemic blasts in the Abbreviations: MDS, myelodysplastic syndrome; CD, cluster designation; TCR, T-cell receptor; Zg, immunoglobulin; PCR, polymerase chain reaction. Correspondence to: Hwei-Fang Tien, M.D., Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan, Republic of China (Tel: 02 3970800 ext 3955; Fax: 02 3222431). 595

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Materials

and Methods

Patients Twenty-three consecutive patients with MDS in acute leukemic transformation (230% blast cells in the bone marrow) were studied from March 1986 to September 1993 at the National Taiwan University Hospital. Diagnosis and classification of MDS and acute leukemia were made on the basis of the FAB criteria [19, 201. The initial diagnosis of the MDS in these patients was: refractory anemia (RA), two cases; RA with excess of blasts (RAEB), nine cases; RAEB in transformation (RAEBT), eight cases; chronic myelomonocytic leukemia (CMMOL), four cases; and RA with ring sideroblasts (RARS), no cases. One of them had been initally diagnosed as aplastic anemia 9 years before the development of RAEB. There was no underlying hematologic disease or history of chemotherapy or radiotherapy in the other 22 patients. There were 10 males and 13 females. Four were children, and 19 were adults with a median age of 56 years. Cell morphology and cytochemistry Morphologic observation was carried out on air-dried smears of bone marrow (BM) aspirates, stained by the Romanowsky system [21,22]. Cytochemical stains for myeloperoxidase (MPO), periodic acid Schiff reagent, combined chloroacetate esterase and cc-naphthyl butyrate esterase (nonspecific esterase) were performed as described previously [22]. Immunophenotyping Immunocytochemical studies of leukemic cells obtained at the time of acute transformation of MDS were performed on 20 patients (on BM cells from 17 patients and peripheral blood cells from three) using an indirect immunoalkaline phosphatase method, as described previously [22]. A panel of monoclonal antibodies (MoAbs) to lymphoid-associated antigens, including B4 (CD19), J5 (CALLA, CDlO), Bl (CD20), Tll (CD2) (Coulter Immunology, FL, U.S.A.), Leul (CD5), Leu9 (CD7) (Becton Dickinson, CA, U.S.A.), as well as myeloid-associated antigens including My7 (CD13), My9 (CD33), My4 (CD14) (Coulter Immunology), OKMl (CDllb) (Ortho Diagnostic System, NJ, U.S.A.), Leu Ml (CD15) (Becton Dickinson), HPI-1D (CD4Ia, kindly provided by Dr W. L. Nichols, Mayo Clinic), and glycophorin A (Dakopatts, Glostrup, Denmark) were selected for the study. Leukemic cells isolated from BM or peripheral blood (PB) by Ficoll-Hypaque gradient centrifugation were incubated for 10 min sequentially with 5% pooled human AB serum and 5% normal goat serum to block non-specific antibody binding. The cells were then incubated sequentially with appropriately diluted MoAbs and alkaline phosphatase-conjugated goat antimouse Ig (Zymed Laboratories Inc., San Francisco, USA). Non-immune normal mouse ascites was used as a negative control. Antibody binding was shown by enzyme alkaline phosphatase reaction. Samples in which more than 20% of the leukemic cells were positively stained with the antibody were considered positive for that marker. Simultaneous labeling of the cells with MPO and lymphoidassociated marker was performed on two patients whose leukemic blasts expressed the characteristics of both myeloid and lymphoid cells. The cells were first stained with CD19, CDlO, or CD7 using the indirect alkaline phosphatase method mentioned above, and then MPO [22]. Two-color flow cytometry (FACScan, Becton Dickinson) analysis using direct immunofluorescence technique was also carried out on one of them, using CD7/CD13 and CD19ICD13 marker combinations

labeled with fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively. Irrelevant isotype matched monoclonal antibodies were used as negative controls. Cytogenetic study Chromosome analysis was performed as described previously [7]. Briefly, BM or PB cells were harvested directly or after l-3 days of unstimulated culture. Metaphase chromosomes were banded by the conventional trypsin-Giemsa banding technique, then karyotyped according to ISCN [23]. Southern blot analysis Analysis of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements was performed on 16 patients as described previously [24]. High-molecular-weight DNAs were extracted from cryopreserved BM or PB cells by cell lysis, proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation. Five micrograms of each DNA were digested with restriction enzyme EcoRI and BamHI in conditions recommended by the supplier (Boehringer, Mannheim, Germany); they were then size-fractionated by electrophoresis in a 0.7% agarose gel and transferred to Hybond membrane (Amersham International, Amersham, U.K.) with a standard Southern blot technique. DNA probes were labeled with 32P-dCTP (Amersham International) by a random primer DNA labeling system (Bethesda Research Laboratories, MD, U.S.A.) to a specific activity of l-2 x 10’ cpm/ug DNA, followed by overnight hybridization at 42°C. The filters were then washed under stringent conditions and autoradiographed at - 70°C for 5-7 days. The probes used were DNA fragments from a TCR fl chain gene (BglII-digested C p 2 region of TCR /3 chain gene, kindly provided by T.W. Mak); and Ig gene JH fragment (BamHI-Hind111 fragment of JH region, kindly provided by P. Leder). Detection of the N-ras mutation Point mutation of the N-ras oncogene was detected by amplifying ras-specific sequences using the polymerase chain reaction (PCR), followed by hybridization with mutationspecific oligomers as described previously [7]. Direct DNA sequencing was also performed to confirm the point mutation. The PCR-amplified DNA was gel-purified, then sequenced by the dideoxy chain termination method [25] using a dsDNA cycle sequencing system (Life Technologies, Inc., Gaithesburg, MD, U.S.A.). Sequencing primers for sense and antisense orientation were end labeled with y -[32P]-ATP and T4 polynucleotide kinase and then mixed with amplified DNA and Tag DNA polymerase. The tubes were incubated at 95°C for 5 min. Chain extension and termination took place in a thermal cycler (Perkin Elmer Cetus). The reaction was stopped by the addition of formamideidye stop mixture; samples were heated to 90°C for 5 min, chilled, and then electrophoresed on a 6% polyacrylamide gel at 50°C. The gel was dried and autoradiographed at - 70°C overnight. Statistical analysis Curves of survival after diagnosis of acute transformation were plotted using the method of Kaplan and Meier [26]; differences between curves were analyzed by the log-rank test [27]. Duration from diagnosis of MDS to onset of acute leukemia was compared by the Wilcoxon rank sum test [28].

15F 81F 62F 83M 33M 56M 35F 33F 56F 43F 60M 4M 67M 15F 67F 57F 34F 61F 65F SF 25M 64M 67M

RA RA RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEB RAEBT RAEBT RAEBT RAEBT RAEBT RAEBT RAEBT RAEBT CMMOL CMMOL CMMOL CMMOL

MDS FAB*

M2 M2 Ml M2 Ml M2 Ml M2 M4 MO M2 M4 M6 Ml M4 M4 M2 M4 M2 MO M4 M4 M2

AL FABT CD33 ND 24 ND 34 83 0 33 ND 64 60 91 22 70 21 31 60 36 61 41 28 63 44 71

CD13

ND 27 ND ND 38 4 23 ND 25 66 63 53 36 30 30 5 72 18 48 4 56 21 83

ND 2 ND 11 16 ND 0 ND 7 3 6 52 0 3 24 35 15 54 1 21 21 82 1

CD14 ND ND ND ND ND ND ND ND ND 2 98 ND ND ND ND 30 ND 98 ND 4 ND 53 99

CD15 ND 2 ND 80 14 6 12 ND 2 1 94 50 0 18 37 59 37 80 1 4 64 50 80

CDllb

1 0 3 ND 0 0 ND 0 0 9 1 0 0 0 0 0 ND 0 ND

ND 0 ND

CD4la ND ND ND 0 ND ND ND ND ND ND ND ND 50 ND 2 0 0 ND ND ND ND ND ND

GA ND 0 ND 3 0 5 0 ND 0 2 0 15 0 7 0 0 3 0 0 0 ND 0 99

CD19 ND 3 ND 0 0 8 0 ND 0 0 0 0 0 5 0 0 24 0 1 13 ND 0 0

CD10

Antigen expression (%)$

ND ND ND ND 2 ND 0 ND 0 0 0 4 0 1 ND 0 ND 1 ND 0 ND 0 0

CD20 ND 0 ND 5 0 11 0 ND 3 0 4 0 0 7 0 0 0 0 2 0 ND 0 0

CD2 ND 10 ND 0 0 2 0 ND 3 0 0 0 0 0 15 0 0 0 0 0 ND 0 0

CD5 ND 89 ND 0 0 0 5 ND 29 3 0 0 0 82 0 0 1 0 1 0 ND 0 22

CD7 ND ND ND ND ND ND ND ND ND 93 37 ND ND ND ND ND ND 0 ND 0 ND 0 11

14 25 74 46 82 100 93 ND 49 51

ND ND

44 54 ND 75 70 87 40 66

-

+ ND ND ND

ND -

ND -

JH ND 76 ND 70 59

CD34 HLA-DR

ND ND ND +

-

ND

ND ND -

ND -

TB

Gene rearrangement5

*FAB classification of MDS at diagnosis. TFAB classification of acute leukemia at the time of acute transformation. :Results are expressed as the percentage of leukemic cells with positive staining. The criterion of positivity was that > 20% of the leukemic cells reacted with the antibody. aGene rearrangement was studied at the time of acute transformation except case 3 (performed at the stage of RAEB) and cases 9, 13, 15, and 17 (performed at the stage of RAEBT). GA, glycophorin A, JH, immunoglobulin heavy chain gene; Tfl, T-cell receptor /I chain gene; ND, not done; -, negative; +, positive.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Case no. Age/sex

‘Table 1. Phenotype and genotype of 23 patients with MDS in acute transformation

P 2 s K Fc 3. n gct z 2 B E. 5 0,

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598

N 17

N 23

kb 16-

Fig. 1. (A) Southern blot analysis of DNA showing rearrangement of immunoglobulin heavy chain gene in patient 17 (using EcoRI restriction enzyme and JH probe). (B) Rearrangement of T-cell receptor (TCR) fi chain gene in patient 23 (using EcoRI restriction enzyme and TCR /I constant probe). N,

normal control. The rearrangedbands are indicated by arrows. Results

Phenotype and genotype Leukemic cells from all 23 patients with acute transformation of MDS showed the characteristic morphology and cytochemical staining pattern of myeloid cells. The FAB classification of acute leukemia [20] in these patients is shown in Table 1. Two patients transformed to MO subtype of AML as identified by negative MPO, negative B and T lineage markers and expression of myeloid antigens on leukemic blasts [29]. Immunophenotying was performed at the time of acute change on 20 patients. Coexpression of lymphoidassociated antigens on leukemic blasts was detected in five patients (cases 2, 9, 14, 17 and 23). Four patients showed positive staining with T-cell-associated antigen CD7; one of them (case 23) also expressed B-cell antigen CD19. The remaining one had CD10 expression. Repeated study on leukemic cells from case 23, 8 months after the first analysis, revealed persistent expression of lymphoid markers though the CD19 positive leukemic cells had decreased in number (from 99 to 33%), while the CD7 positive cells had increased (from 22 to 89%). Double staining of the cells with MPO and lymphoid markers (CDlO, CD19 or CD7) was performed on case 17 and on the second sample of case 23. It demonstrated that some individual blasts were simultaneously expressing MPO and lymphoid marker (CD10 for case 17, CD19 or CD7 for case 23), while

some blasts were expressing either one of the two characteristics. Two-color flow cytometry analysis on leukemic cells from case 23 showed that almost all CD7 or CD19 positive cells carried myeloid antigen CD13. Rearrangements of Ig heavy chain and TCR b chain genes were analyzed on 16 patients. All but five were studied at the time of acute transformation (Table 1). One patient (case 17) showed Ig heavy chain gene rearrangement, but germline configuration of TCR p chain gene, and another (case 23) showed rearrangement of TCR /I chain but not Ig heavy chain gene (Fig. 1). Both patients were among the five patients with coexpression of lymphoid markers. Other patients including cases 2, 9 and 14 who also had lymphoidassociated antigen expression on leukemic cells, showed germline configuration of both genes.

Chromosomeanalysis Results of cytogenetic studies are summarized in Table 2. Clonal chromosomal abnormalities were detected in 12 (63%) of the 19 patients at acute transformation (17 cases) or shortly before it (two cases). Monosomy 7( - 7) could be found in six (32%) (cases 3, 8, 14, 19, 20 and 21). Of the 18 patients where the studies were carried out both at diagnosis of MDS and at transformation, karyotypic evolution was noted in seven (39%). The new or additional changes which occurred more than once were trisomy 8(+8) and trisomy 21(+21). No specific chromosomal abnormal-

N +8,+9 Same, with additional non-clonal changes +8 N ND

N N 5q - , - 7,der( 17),2Oq - , - 18 add(21),22q - ,+mar N N add(4),5q - , - 7,der( 12), - 13, add(14),17q-,-18,+mar N -7 N N N -6,7qTetraploidy - 7,+21,+mar t(2;11) N cv) N ND - 7,t(4; 12) - 7,22q +8 N

9 4 19

20 21 24

26 35 37 69 72 41 42 45 47 48 52 56 70

65 59 67 71

1 2 3

4 5 6

7 8 9* 10 11 12 13 14 15 16* 17 18 19

20 21 22 23

ND i ND ND ND ND + ND

21 4 5 10

7 5 20 20 20 2 5 1 7 11 IA 36 9 5

ND ND ND ND ND ND -

28 4 4

36 33 9

Duration from dx to transf (months)

ND

ND ND

N-ras mutation

16 2+ 1 8+

1 3+ 1.5 5 14+ 7 2 8 3.5+ 14 7 21+ 1

1.5 8 1.5

2 0.3 1

Survival after acute transf (months)

*Follow-up cytogenetic studies of these two patients were carried out at 4 and 5 months, respectively, before the diagnosis of acute transformation. dx, diagnosis; transf, transformation; N, normal karyotype; ND, not done; -, negative; t, positive. Part of the cytogenetic findings have been reported previously [7].

N -7 +21 N N ND ND - 7,+mar ND N t(3;3),1 lPN der(3), - 4,+5q - ,5q -, - 7, - 8, - 12,+mar - 7J(4; 12) -7 +8,+21 inv(3),+13

At transformation

At diagnosis of MDS

MDS no.

Case no.

Cytogenetic results

Table 2. Cytogenctic results and survival of 23 patients with MDS in acute transformation

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Fig. 2. Dot blot analysis of the N-ras gene mutation in patient 22 showing a codon 12 mutation substituting GGT with AGT (arrow). P, positive control.

ities were found in the five patients with coexpression of

lymphoid-associated antigens. Mutation

of the N-ras gene

Analysis of the N-ras mutation by dot blotting was performed on nine patients at acute transformation (Table 2). A point mutation of N-t-as was detected in two patients: one (patient 16) at codon 61 with a glutamine (CAA) to histidine (CAT) substitution and the other (patient 22) at codon 12 with a glycerine (GGT) to serine (AGT) substitution (Fig. 2). Both mutations were confirmed by repeating the PCR amplication and dot blotting, and the former by direct sequencing, as well. The same mutations had been demonstrated in these patients at the stage of RAEBT 6 and 5 months, respectively, before an acute change (Fig. 3). No point mutation of the N-ras gene was found in the other seven patients studied. Clinical

features

All patients showed anemia at the time of acute transformation. Thrombocytopenia (< 100 x 109/1) was found in all but three patients. About one third of the patients (eight) had leukopenia (< 4.0 x 109/1)while ten patients had leukocytosis (>lO x 109/1) and three had counts more than 100 x lO”/l. Hepatomegaly was detected in ten patients, splenomegaly in seven and lymphadenopathy in four. Nine patients received Adriamycin containing chemotherapeutic regimens after diagnosis of acute leukemia; ten were treated with low

dose cytosine arabinoside or 6-mercaptopurine; the remaining four received conservative treatment only. None of the patients showed a good response to the treatment. The median survival time was 7 months after diagnosis of acute transformation. There was no diiference in survival time among various FAB subtypes of transformed acute leukemia or among subgroups of underlying MDS. Lymphoid marker expression had no prognostic implication in these patients. The median interval from diagnosis of MDS to onset of acute transformation (chronic phase duration) was 10 months (l-36 months). The interval was significantly longer in patients with underlying RA than others, but showed little difference among other subtypes. Patients with a normal karyotype at diagnosis of MDS had a longer chronic phase duration than those with chromosomal abnormalities (median 20 months vs. 5 months, P=O.O38). However, the survival time was short after diagnosis of acute leukemia, irrespective of whether clonal chromosomal abnormalities were present or not (median 7 months vs. 8 months, P>O.O5). Discussion Acute leukemia after MDS has, classically, been assumed to be acute myeloid leukemia (AML), but few studies have been performed to analyze systematically the immunophenotype and genotype of these leukemias [13, 141. In the present study, all 23 patients with acute change of MDS had myeloid transformation according

601

Acute leukemic transformationof MIX

ACG

T

G T T-

Fig. 3. Direct sequencing of PCR-amplified DNA of the antisense strand of the N-ras gene from patient 16 at the onset of acute transformation (B) and 6 months before (A) showing a codon 61 mutation with a glutamine to histidine substitution, TTG+ATG (antisense reading) or CAA-KAT (sense reading), at both stages.

to FAB criteria [20], but five of the 20 patients who had immunocytochemical staining showed coexpression of lymphoid and myeloid markers on leukemic blasts. Ig heavy chain and TCR fl chain gene rearrangements were also detected (cases 17 and 23, respectively) in two of the five patients with lymphoid marker expression. Double staining of the cells with MPO and lymphoid markers in these two cases showed that some blasts were simultaneously expressing MPO activity and lymphoid markers; they were considered to have acute transformation of hybrid leukemia [30]. The remaining three patients who expressed CD7, a pan T-cell marker which can also be detected in some de nova AML [22,31], but showed germline configuration of Ig heavy chain and TCR j3 chain genes, should be considered to have acute myeloid transformation. None of the patients showed pure lymphoid transformation. San Miguel et al. [14] and Masuya et al. [13] have reported similar results. These findings are consistent with the recent observation that myeloid cells from MDS patients are clonally derived, but only a small percentage of these patients have clonal involvement of lymphoid cells [32, 33,341. It is likely that the clonally derived myeloid cells are more easily involved in acute transformation; thus, most MDS transform to AML while very few progress to pure lymphoid leukemia [9, lo]. On the other hand the lymphocytes, at least the B-cells, from patients with CML are considered to be clonally derived [35] and are more frequently involved in acute blast crisis of CML.

Among the 18 patients who had serial cytogenetic analysis both at the diagnosis of MDS and acute transformation, karyotypic evolution was noted in seven (39%). Two patients (11%) acquired new chromosomal anomaly d-8, and another two, +21, at the time of acute transformation; but none had +19 or i(17q) which was also commonly seen in acute change of CML [36]. A review of the literature [2,3, 5, 8, 16,371 indicated that karyotypic evolution could be detected in 38-86% (mean 49%) of MDS patients when the disease had progressed to acute leukemia. The most common new chromosomal abnormalities were +8 and +21, which were found in five (8%) of 65 patients reported; none had karyotypic evolution with +19 or i(17q). Other repeated chromosomal aberrations involving cytogenetic evolution were rearrangements at 17p or lq. The mechanism of acute transformation of MDS is not yet clear. Clonal karyotypic evolution appears to be one of the causes [15]. Patients with multiple cytogenetic changes (two or more aberrations) at initial or subsequent analysis had a significantly higher frequency of acute transformation than others [5,7]. However, in this study 11 patients with acute transformation did not show any chromosomal abnormalities at diagnosis of MDS. Four of them developed clonal anomalies at the time of transformation but the remaining seven still showed normal karyotype. Some submicroscopic genetic changes, such as ras gene mutations, might exist to cause progression of the disease in these patients

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[15, 171. One such patient with normal karyotype was demonstrated to have N-ras mutation in this study. Patients with a normal karyotype at initial diagnosis had a significantly longer chronic phase duration than those with chromosomal abnormalities (20 months vs. 5 months). The former patients might need a longer time than the latter to develop new genetic lesions for leukemic transformation. In conclusion, though most patients with acute leukemic transformation of MDS had a myeloid leukemia, a few had a hybrid leukemia by immunophenotypic and genotypic studies. None transformed to a pure lymphoblastic leukemia. Karyotypic evolution was demonstrated in 39% of patients at acute leukemic transformation and N-ras mutations in 22%. These two factors may play some roles in the progression of MDS to acute leukemia. Patients had a short survival time after development of acute leukemia irrespective of whether clonal chromosomal abnormalities or lymphoid markers were present or not. Acknowledgement-This study was supported in part by grants from the National Science Council of the Republic of China, NSC-83-0412-B002-146 M02.

and NSC-84-2331-B002-220

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