Cytogenetic study of 50 Brazilian patients with primary myelodysplastic syndrome

Cytogenetic study of 50 Brazilian patients with primary myelodysplastic syndrome

Teresa de Souza Fernandez ¹ , Maria Luiza M. Silva ^1,2 , Jamison M. de Souza ^1,3 , Daniel Tabak ² and Eliana Abdelhay ¹

¹Laboratório de Biologia Molecular Maury Miranda, Instituto de Biofísica Carlos Chagas Filho, bloco G,

Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brasil.

Send correspondence to E.A.

²Instituto Nacional de Câncer, Centro de Transplante de Medula Óssea, Praça Cruz Vermelha 23, 7^o andar, Centro, Rio de Janeiro, RJ, Brasil.

³Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rua Frei Caneca 8, Centro, Rio de Janeiro, RJ, Brasil.

ABSTRACT

In this work we analyzed cytogenetically 50 patients with primary myelodysplastic syndrome from several hospitals of Rio de Janeiro, Brazil. The frequency of cytogenetic abnormalities was 32%. Patients with refractory anemia, or refractory anemia with ringed sideroblasts, presented normal karyotypes or single abnormalities such as del(5q) or -Y, while patients with refractory anemia with an excess of blasts, refractory anemia with an excess of blasts in transformation or chronic myelomonocytic leukemia showed complex karyotypes and single abnormalities involving chromosomes 7 or 8, which are related to a bad prognosis and an elevated risk of evolution to acute myeloid leukemia.

INTRODUCTION

Myelodysplastic syndrome (MDS) is a group of bone marrow disorders that originates from a clonal modification affecting a pluripotent stem cell. These disorders are characterized by dysplastic changes in one or more myeloid cell lines, leading to anemia, neutropenia and thrombocytopenia (Taylor et al., 1994). Patients with myelodysplastic syndrome usually present a hypercellular or normocellular bone marrow, increased intramedullary cell death and cytopenia in the peripheral blood (Kouides and Bennett, 1992). In 10% to 40% of cases there is a progression to acute myeloid leukemia (AML), but bone marrow failure is the most common cause of death (Silverman, 1993).

Myelodysplastic syndromes were classified by the French-American-British (FAB) cooperative group in 1982 (Bennett et al., 1982) into five distinct morphological entities, on the basis of morphological characteristics and percentage of blasts in the bone marrow: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t) and chronic myelomonocytic leukemia (CMML).

Chromosomal abnormalities are usually found in 23% to 78% of cases and the most frequently observed abnormalities are: del(5q), +8, -7/del(7q) and del(20q). Less frequently observed are: del(11q), del(12q) or del(13q), inversion of chromosome 3, isochromosome of the long arm of chromosome 17, translocations between chromosomes 1, 3 or 7 and the loss of chromosome Y (Jacobs et al., 1986; Heim and Mitelman, 1987; Noël et al., 1993).

Although the presence of a specific chromosomal abnormality has not been associated with a specific subtype of MDS, some are associated with a bad prognosis and an increased chance of evolution to AML (Jacobs et al., 1986; Noël et al., 1993).

MATERIAL AND METHODS

Patients

Bone marrow cells were obtained from 50 patients with MDS, studied between 1991 and 1994. Chromosome studies were carried out in all cases. The patients were seen at the Hematology/Oncology Units of several hospitals of Rio de Janeiro, Brazil: Instituto Nacional de Câncer (CEMO), Hospital Universitário Pedro Ernesto (UERJ), Hospital Universitário Clementino Fraga Filho (UFRJ), Hospital dos Servidores do Estado, Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Hospital Geral de Bonsucesso and Hospital Universitário Gaffre e Guinle.

There were 28 males and 22 females. The median age was 50 years, with a range of 10 months to 88 years. Twenty-eight patients were classified as RA, four as RARS, 10 as RAEB, four as RAEB-t and four as CMML (Table I).

N FAB Age/sex Number of mitosesanalyzed (normal/ abnormal)Karyotypes Evolution to AML Status 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35a 35b 36 37a 37b 38 39 40 41 42 43 44 45 46 47 48 49 50RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RARS RARS RARS RARS RAEB RAEB RAEB AML RAEB RAEB AML RAEB RAEB RAEB RAEB RAEB RAEB-t RAEB-t RAEB-t RAEB-t CMML CMML CMML CMML40/F 53/M 5/M 8/M 41/F 62/F 64/M 16/M 88/M 73/M 59/F 52/M 57/M 84/F 38/F 56/F 77/F 11/F 18m/F 73/F 7/F 8/F 40/F 57/F 40/F 25/M 30/F 44/M 59/F 83/F 65/M 30/M 35/M 56/M 12/M 12/M 59/M 60/M 60/M 84/F 85/M 61/M 66/M 19/M 64/M 52/F 76/F 21m/M 73/M 10m/M 82/M 71/M31/0 42/0 35/0 33/0 38/0 29/0 32/0 41/0 37/0 0/53 35/0 31/0 37/0 49/4 38/0 46/4 35/0 50/0 40/0 51/10 30/0 32/0 25/0 39/0 40/3 33/0 41/0 47/0 46/0 33/0 52/0 30/0 0/40 22/0 35/0 26/38/4 28/0 23/32 2/15/8/3 19/26 22/15 22/0 15/15 21/30 10/27 4/32 9/18/3 8/14 67/0 31/0 21/0 41/546,XX 46,XY 46,XY 46,XY 46,XX 46,XX 46,XY 46,XY 46,XY 45,X,-Y 46,XX 46,XY 46,XY 46,XX/46,XX,del(5)(q31) 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XX 46,XX 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XX 46,XX 46,XX 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XY 46,XX 46,XY 46,XX 46,XX 46,XY 46,XY 44,XY,del(5)(q15), -7, -13, -18,+mar 46,XY 46,XY 46,XY/90,XXYY/45,XY, -21 46,XY 46,XY/47,XY,+8 46,XY/47,XY,+8/ 47,XY,dup(1)(q21q32), +8/47,XY,+der 8, t(1;8)(q21;p12) 46,XX/46,XX,del(20)(q12) 46,XY/45,XY,-7 46,XY 46,XY/46,XY, dup(1)(q12) 46,XY/47,XY,+8 46,XY/46,XY,del(5)(q15) 46,XX/45,XX,-7 46,XX/47,XX,+8/ 47,XX,del(5)(q31),+8 46,XY/45,XY, -7 46,XY 46,XY 46,XY 46,XY/46,XY,-8,+20No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Yes No Yes AML No Yes AML No Yes No Yes Yes Yes Yes Yes Yes No No No Noalive alive alive alive alive dead dead alive alive alive alive alive alive dead alive alive alive alive alive alive alive alive alive alive alive alive alive alive alive dead alive alive dead alive alive dead alive alive dead dead dead alive alive dead dead dead dead dead dead alive dead

N	FAB	Age/sex	Number of mitosesanalyzed (normal/ abnormal)	Karyotypes	Evolution to AML	Status
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35a 35b 36 37a 37b 38 39 40 41 42 43 44 45 46 47 48 49 50	RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RARS RARS RARS RARS RAEB RAEB RAEB AML RAEB RAEB AML RAEB RAEB RAEB RAEB RAEB RAEB-t RAEB-t RAEB-t RAEB-t CMML CMML CMML CMML	40/F 53/M 5/M 8/M 41/F 62/F 64/M 16/M 88/M 73/M 59/F 52/M 57/M 84/F 38/F 56/F 77/F 11/F 18m/F 73/F 7/F 8/F 40/F 57/F 40/F 25/M 30/F 44/M 59/F 83/F 65/M 30/M 35/M 56/M 12/M 12/M 59/M 60/M 60/M 84/F 85/M 61/M 66/M 19/M 64/M 52/F 76/F 21m/M 73/M 10m/M 82/M 71/M	31/0 42/0 35/0 33/0 38/0 29/0 32/0 41/0 37/0 0/53 35/0 31/0 37/0 49/4 38/0 46/4 35/0 50/0 40/0 51/10 30/0 32/0 25/0 39/0 40/3 33/0 41/0 47/0 46/0 33/0 52/0 30/0 0/40 22/0 35/0 26/38/4 28/0 23/32 2/15/8/3 19/26 22/15 22/0 15/15 21/30 10/27 4/32 9/18/3 8/14 67/0 31/0 21/0 41/5	46,XX 46,XY 46,XY 46,XY 46,XX 46,XX 46,XY 46,XY 46,XY 45,X,-Y 46,XX 46,XY 46,XY 46,XX/46,XX,del(5)(q31) 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XX 46,XX 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XX 46,XX 46,XX 46,XX 46,XX/46,XX,del(5)(q13q33) 46,XY 46,XX 46,XY 46,XX 46,XX 46,XY 46,XY 44,XY,del(5)(q15), -7, -13, -18,+mar 46,XY 46,XY 46,XY/90,XXYY/45,XY, -21 46,XY 46,XY/47,XY,+8 46,XY/47,XY,+8/ 47,XY,dup(1)(q21q32), +8/47,XY,+der 8, t(1;8)(q21;p12) 46,XX/46,XX,del(20)(q12) 46,XY/45,XY,-7 46,XY 46,XY/46,XY, dup(1)(q12) 46,XY/47,XY,+8 46,XY/46,XY,del(5)(q15) 46,XX/45,XX,-7 46,XX/47,XX,+8/ 47,XX,del(5)(q31),+8 46,XY/45,XY, -7 46,XY 46,XY 46,XY 46,XY/46,XY,-8,+20	No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Yes No Yes AML No Yes AML No Yes No Yes Yes Yes Yes Yes Yes No No No No	alive alive alive alive alive dead dead alive alive alive alive alive alive dead alive alive alive alive alive alive alive alive alive alive alive alive alive alive alive dead alive alive dead alive alive dead alive alive dead dead dead alive alive dead dead dead dead dead dead alive dead

Table I - Cytogenetic analysis in 50 patients with primary myelodysplastic syndrome.

RA - Refractory anemia; RARS - refractory anemia with ringed sideroblasts; RAEB - refractory anemia with excess of blasts; AML - acute myeloid anemia; RAEB-t - refractory anemia with excess of blasts in transformation; CMML - chronic myelomonocytic leukemia. m - Months.

None of the patients had been previously treated for malignancy. The diagnosis and subclassification of MDS were made on the basis of morphological and cytochemical studies of the initial peripheral blood films and bone marrow aspirates, according to FAB criteria (Bennett et al., 1982).

Cytogenetic studies

Karyotypes of bone marrow cells were obtained from cultures in RPMI 1640 with 20% fetal calf serum (GIBCO) at 37oC for 24 h. Cell cultures were pulsed with Colcemid to a final concentration of 0.06 mg/ml for the final hour of incubation. Cells were subsequently harvested by standard procedures (hypotonic shock with 0.075 M KCl) and fixed in methanol - acetic acid (3:1). GTG banding was performed as described by Seabright (1971), and chromosomes were identified and arranged according to the International System for Human Cytogenetics Nomenclature (1991).

RESULTS

Among the fifty patients, 16 showed clonal karyotypic abnormalities (Table I). In 34 patients (23 RA, four RARS, four RAEB and three CMML) no clonal chromosome changes were identified, even though an extensive analysis was performed on 20-100 metaphases in each case.

Clonal abnormalities detected in patients with primary MDS

Clonal abnormalities were detected in 16 patients. Gain or loss of whole chromosomes was observed in seven cases. In two others, both numerical and structural abnormalities were present, and only structural rearrangements were detected in seven cases.

The most common numerical abnormalities were monosomy 7 (three cases) and trisomy 8 (two cases), and the most frequent structural rearrangement was partial deletion of the long arm of chromosome 5 (five cases) (Figure 1).

Figure 1 - G-banded partial karyotypes of a bone marrow cell. a) Partial karyotype showing del(5)(q13q33) (patient 16) and b) partial karyotype showing del(5)(q31) (patient 14).

The two patients with complex karyotypes showed association of del(5q) and several monosomies in one case (Figure 2), and del(5q) and trisomy 8 in the other. Pseudodiploidy was detected in one case with CMML (patient 50), with loss of chromosome 8 and gain of chromosome 20.

Figure 2 - G-banded karyotype of a bone marrow cell from patient 33 showing a complex karyotype: 44,XY,del(5)(q15), -7, -13, -18, +mar.

Chromosomal abnormalities frequency and FAB classification

The frequency of each chromosomal abnormality among the 50 patients and their relation with FAB classification are shown in Tables II and ^III.

Diagnosis Total cases analyzed Cases with abnormal karyotypeRA RARS RAEB RAEB-t CMML Total28 4 10 4 4 505 0 6 4 1 16

Diagnosis	Total cases analyzed	Cases with abnormal karyotype
RA RARS RAEB RAEB-t CMML Total	28 4 10 4 4 50	5 0 6 4 1 16

Table II - Frequency of abnormal karyotype in 50 patients with primary myelodysplastic syndrome according to FAB classification.

For abbreviations see legend to Table I.

Table III - Frequency of the different chromosomal abnormalities in primary myelodysplastic syndrome.

Karyotype Cases FAB 5q-
-7
+8
20q-
-Y
dup(1q)
pseudodiploid
complex 5
3
2
1
1
1
1
2 4 RA, 1 RAEB
1 RAEB, 2 RAEB-t
2 RAEB
1 RAEB
1 RA
1 RAEB
1 CMML
1 RAEB, 1 RAEB-t

For abbreviations see legend to Table I.

Follow-up study and prognosis

During the follow-up period some patients progressed from MDS to AML and showed additional chromosomal alterations. In general, RA and RARS patients showed cells with normal karyotypes or cells with karyotypes showing single abnormalities, such as partial deletion of the long arm of chromosome 5 or loss of chromosome Y. These patients did not progress to AML during the follow-up period. The more aggressive subgroups, such as RAEB, RAEB-t and CMML, showed a high percentage of cells with abnormal karyotypes, complex karyotypes or numerical changes, and were associated with a bad prognosis. These patients showed a high frequency of evolution to AML.

Ten of 50 patients showed evolution to AML and two of these patients were cytogenetically studied when diagnosed with AML. One patient (patient 35) initially had a normal karyotype when diagnosis of RAEB was made. After nine months this patient evolved to AML, acquiring two new clones, with the following karyotypes: 90,XXYY (55.8% of cells) and 45,XY,-21 (5.8% of cells).

Another patient initially diagnosed with RAEB (patient 37) presented a normal karyotype in 41.8% of cells and 47,XY,+8 in the remaining cells. After nine months this patient evolved to AML and acquired two new clones, one (47,XY,dup(1)(q21q32),+8) represented 28.6% of cells and the other (47,XY,+der 8,t(1;8)(q21;p12)) was detected in 10.7% of cells.

Two other patients presenting trisomy 8 (patients 42 and 45) evolved to AML, confirming that trisomy 8 is correlated with a bad prognosis and a greater chance of evolution to AML.

DISCUSSION

The frequency of chromosomal abnormalities in our population is similar to other series. The greatest discrepancy appears in the frequency of abnormalities found in RARS patients. While in our study no patients with RARS showed chromosomal abnormalities, in some series, such as the one described by Toyama et al. (1993) for Japanese patients, the frequency of abnormalities reached 75%. The explanation for these dissimilar results can be the different criteria used in the diagnosis of MDS or the small number of patients.

In our case, patients presenting more than 15% of ringed sideroblasts in the bone marrow, but with other features such as megakaryotypic or granulocytic dysplasia and more than 5% of blasts in the bone marrow were reclassified as RAEB. This was the case for two patients (37 and 42) who presented chromosomal abnormalities at diagnosis and evolved rapidly to AML.

The most frequent chromosomal abnormalities found in our patients were del(5q), -7 and +8. It was not possible to establish an association between a specific chromosomal abnormality and FAB subtype, but patients with RA or RARS presented normal karyotypes or cells presenting single abnormalities such as del(5q) or -Y and had a good prognosis, while patients with RAEB, RAEB-t or CMML presented complex karyotypes or single abnormalities such as -7 or +8, and were associated with a bad prognosis.

Evolution to AML was almost constantly associated with karyotypic abnormalities, but patients with chromosomal abnormalities and subtypes RA, RARS and CMML did not evolve to AML during the follow-up study.

Trisomy 8 and monosomy 7 in all cases were associated with evolution to AML, while del(5q) occurred in patients with RA as single abnormalities that did not progress to more aggressive subtypes, and in patients with RAEB or RAEB-t as single or complex abnormalities. However, in these cases progression to AML was constant.

These results are in accordance with data from other populations, although some authors have described evolution in some patients with RA or RARS and del(5q) (Kandioler et al., 1994). However, the time required for this evolution seems to be far greater than the follow-up period of our study.

Myelodysplastic syndrome is a disease affecting elderly persons, but in our study 10/50 patients were children (10 months to 16 years). We cannot say that this frequency reflects some genetic characteristic of our population because our series is still small, but it seems that some genetic or epigenetic factor plays a role.

ACKNOWLEDGMENTS

The authors dedicate this paper in honor of Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary.

RESUMO

Neste trabalho foram estudados citogeneticamente 50 pacientes de diversas unidades hospitalares do Rio de Janeiro, Brasil, diagnosticados com síndrome mielodisplásica primária. Os dados obtidos mostraram uma freqüência de anomalias cromossômicas de 32% nestes pacientes, sendo as anomalias mais encontradas a del(5q) (10%), -7 (6%) e +8 (4%). Pacientes com anemia refratária ou anemia refratária com sideroblastos em anel apresentaram cariótipos normais ou com deleções simples como del(5q) ou -Y, enquanto pacientes com anemia refratária com excesso de blastos, anemia refratária com excesso de blastos em transformação e leucemia mielomonocítica crônica apresentaram cariótipos complexos ou com anomalias simples do tipo monossomia do 7 ou trissomia do 8, tendo um alto índice de evolução para leucemia mielóide aguda.

REFERENCES

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Noël, P., Tefferi, A., Pierre, R.V., Jenkins, R.B. and Dewald, G.W. (1993). Karyotypic analysis in primary myelodysplastic syndrome. Blood Rev. 7: 10-18.

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Silverman, L.R. (1993). Myelodysplastic syndromes. In: Cancer Medicine (Holland, J.S., Frei Terceiro, E., Bast Jr., R.C., Donald, K., Donald, M. and Weichselbaum, R., eds.). Vol. 2. Lea & Fediger, Philadelphia, pp. 1888-1906.

Taylor, K.M., Rodwell, R.L., Taylor, D.L. and Seeley, G.O. (1994). Myelodysplasia. Curr. Opin. in Oncol. 6: 32-40.

Toyama, K., Ohyashiki, K., Yoshida, Y., Abe, T., Asano, S., Hirai, H., Hirashima, K., Hotta, T., Kuramoto, A., Kuriya, S., Miyazaki, T., Kakishita, E., Mizoguchi, H., Okada, M., Shirakawa, S., Takaku, F., Tomonaga, M., Uchino, H., Yasunuga, K. and Nomura, T. (1993). Clinical implications of chromosomal abnormalities in 401 patients with myelodysplastic syndromes: a multicentric study in Japan. Leukemia 7: 499-508.

(Received October 2, 1995)

Publication Dates

• Publication in this collection
  13 Oct 1998
• Date of issue
  Mar 1997

History

• Received
  02 Oct 1995