Cytogenetic Analysis of Bone Marrow from Patients with Primary Myelodysplastic Syndrome 'Laboratory of Haematological Research, Henan Medical University, Zheng-zhou, Peoples' Republic of China; "Central Clinical Laboratory, Faculty of Medicine, University of Tokyo, Tokyo, Japan; "Cytogenic Laboratory, Institute of Family Planning of Henan Province, Zheng-zhou, Peoples' Republic of China

Of 34 patients with primary myelodysplastic syndrome (MDS), 26 (76.5%) were found to have chromosome defects using an improved bone marrow culture method and high-resolution G-banding technique induced by actinomycin D. The frequency of abnormalities varied among the subtypes: 1/2 in refractory anaemia with ringed sideroblasts; 18/24 in refractory anaemia; 516 in refractory anaemia with excess of blasts (RAEB); and 2/2 in refractory anaemia with excess of blasts in transformation. The most frequent abnormalities, either single, double or complex defects, were -5/5q-, -7, +8 and +21; 16q116p-, +19 and -X were also common. The percentage of aneuploid cells, in particular hypodiploid cells, was increased. The abnormalities were detected more frequently in complex aberrations associated with RAEB and RAEB in transformation. The presence of -5/5q- as a sole aberration was associated with longer mean survival time (>18 months) but multiple (more than two) chromosomal abnormalities were associated with a poorer prognosis and a mean survival time of only 7.5 months. Chromosome follow-up studies indicated that patients with -7, +8, +21, -X and complex

254

defects, increased hypodiploid cells and karyotpic evolution were likely to have a high risk oftransformation to leukaemia or to a more severe subtype ofMDS with a short overall mean survival time. These defects, mostly of the deleted type, are assumed to play a specific role in the pathogenesis of myelodysplasia. Repeated chromosomal analyses during the clinical course convey more accurate prognostic criteria for patients. KEY WORDS:

PRIMARY MYELODYSPLASTIC SYNDROME; LEUKAEMIA;

CHROMOSOME DEFECTS; MEAN SURVIVAL TIME

INTRODUCTION Primary myelodysplastic syndrome (MDS), a group of dyspoietic disorders pathogenetically related to acute non-lymphocytic leukaemia (ANLL) that appears in patients with no history of chemotherapy or radiotherapy, 1 is characterized by blood cytopenia and dysplastic changes to haematopoietic cells.' In most patients, the bone marrow is hypercellular, with one or more cell lines having signs of dyspoiesis.v" and approximately half of the patients either develop ANLL or die of marrow failure within 2 years of diagnosis." Clonal chromosome abnormalities can be found in approximately 30 60% of patients with MDS when bone marrow cells are examined by a standard culture technique.t" A high incidence of abnormalities (79%) was found in patients with primary MDS using high-resolution banding." Abrupt transitions from MDS to overt leukaemia were frequently seen in MDS patients with additional cytogenetic abnormalities when cytogenetic investigations were serially conducted.' At present, no generally accepted routine approach exists for the determining the prognosis and therapeutic options in primary MDS, despite the significant ad vances

in h aernato l og y ," cytogen eti cs ' - 7 and therapy. 1.2 The results of cytogenetic analysis of bone marrow cells from 34 primary MDS patients are reported and their clinical prognostic significance is considered. The chromosome high-resolution G-binding preparation technique was thoroughly investigated by an improved method for culturing bone marrow cells.

PATIENTS AND METHODS PATIENTS A cytogenetic study was undertaken in June 1985 of bone marrow cells from unselected patients from the first affiliated hospital of Henan Medical University. All patients were diagnosed according to French - American British (FAB) classification' and had not received previous chemotherapy or radiotherapy. The patients comprised 24 with refractory anaemia (RA), two with RA with ringed sideroblast (RARS), six with RA with excess of blasts (RAEB) and two with RA with excess of blasts in transformation (RAEB-T).

255

The patients (21 male, 13 females) were aged 16 - 62 years (mean 48.9 years); for living patients clinical data given were as of December 1987.

cells with the same missing chromosome. Chromosome defects were subdivided into three types: single, double or complex defects.

CELL CULTURE

Mean survival time was calculated from the time of diagnosis of MDS, when the first successful chromosome analysis was performed. The minimum follow-up period was 6 months; the mean follow-up time for patients alive at the time of the study was 14 months. Mean survival time data were analysed by the generalized Wilcoxon test. Student's t-test was used for the statistical analysis of incidence of leukaemic transformation; P < 0.05 was considered significant.

STATISTICAL ANALYSIS Bone marrow samples (1.0 - 2.0 ml] were collected in preservative-free heparin. Mononuclear cells were prepared and cultured at 1.5 x 10" - 2.0 x 10 o/ml in RPMI 1640 medium (Sigma, USA) containing 10% foetal bovine serum with or without 10 - 15% conditioned medium derived from cultures of human foetal muscle cultured in foetal bovine serum for 7 days. H Cells were incubated for 45 - 69 h at 37°C in 5% carbon dioxide in the dark. Actinomycin D (Sigma, USA) was added (final concentration 4 ug/rnl] for the last 45 - 60 min of culture and colcemid (Gibco, USA) was added at a concentration of 0.05 ug/rnl for 30 min." Hypotonic shock was induced by 0.56% potassium chloride for 30 min and cells were fixed four times with methanol/acetic acid (3:1, by vol.). A single drop of cell suspension was deposited on each slide from a height of 100 - 120 em,

RESULTS High-resolution G-banding induced by actinomycin D and conditioned medium provided greater numbers of metaphases and higher resolution (450 - 800 bands, Table 1). In each patient, the number of metaphases karyotyped was between four and 109. The effect of conditioned medium, and of actinomycinD, on a chromosomally abnormal cell proportion was estimated in four patients with chromosomal abnormalities in 67%, 55%, 83% and 98% of the cells. A total of 1145 metaphases were analysed. The diploid cell count was 781 (68.2%) and the aneuploid cell count was 364 (31.8%). The hypodiploid cell count was 231 (20.2%) compared with a pseudodiploid count of 67 (6.0%) and hyperdiploid count of 64 (5.6%). Clonal chromosome abnormalities occured in 26 (76.5%) cases (Table 2).The distribution, incidence of chromosome abnormalities and mean survival times of FAB subtypes are presented in Table 3 and Fig.l. The frequency of chromosome abnormalities in cases with complex defects (38.2%) was higher than that of cases with a single defect (17.8%) or double defects (23.5%). The most frequent single

CHROMOSOME PREPARATION AND EXAMINATION Chromosome preparations were made by the air-fire drying assay. G-banding of all samples was performed under 0.015% trypsin at 4°C. Giemsa solution was used for staining. To determine the mean number of metaphases per slide, one to four slides were examined for different experimental conditions in four patients. Chromosome identification and karyotype designation techniques were performed as previously described." If possible, at least 30 karyotypes were analysed for each patient to determine changes in chromosome counts. An abnormal clone was defined as the presence of at least two cells with the same structural change or with an extra chromosome, or three

256

N

'"'1

FAB

Incubation

69 69 48 69

2 3 3 4

examined

slides

No.of

Mean no. of

%of

22 15 55 5

per slide

61 54 89 99

cells

metaphases abnormal

3 2 1 3

examined

slides

No. of %of

31 2 12 14

per slide

53 50 77 100

cells

metaphases abnormal

Mean no.of

AMD No. of

1 4 1 2

examined

slides

%of

21 5 11 15

per slide

59 52 88 93

cells

metaphases abnormal

Mean no. of

CM Total no. of

99 76 91 73

analysed

metaphases

aFAB, French - American - British classification; RA, refractory anaemia; RARS; refractory anaemia with ringed sideroblasts; RAEB, refractory anaemia with excess of blasts; RAEB-T, refractory anaemia with excess of blasts in transformation.

RA 6 26 RARS 32 RAEB 33 RAEB-T

No. subtype- time (h)

Case

AMD+CM

Effects of high-resolution G-banding induced by actinomycin D (AMD) and/or conditioned medium (eM) on cultured bone marrow cells from four patients with primary myelodysplastic syndrome

Clonal chromosome abnormalities in cultured bone marrow cells from 34 patients with primary myelodysplastic syndrome

Case No.

Mitoses analysis Sex I Age Sub(years) type" Abnormal Normal

F/35

RA

7

39

2

F/28

RA

11

22

3

F/34

RA

28

10

4

F/33

RA

11

19

5

M/21

RA

5

6

F/24

RA

30

2

7 8 9 10

M/46 M/25 F/19 M/16

RA RA RA RA

26 4 6 12

8 29 31 18

11

F/16

RA

8

27

12 13

F/27 M/41

RA RA

0 16

30 22

14 15 16

M/33 M/50 M/17

RA RA RA

0 0 19

31 30 15

17

F/23

RA

8

36

Chromosome finding

Proqnosis"

46, XX, t(12;13)(q11.1 ;q15) 46, XX, t(12;14)(q22;p11.2), del(12)(p14pter) 45, XX,-22, -19, der(19)(19p13.1 ;22q11.2) 47, XX, +21 46, XX, del(2)(q33qtr), +16 46, XX, t(1 ;6)(p36.3;p21.3) 46, XX, rob(14;15)(p11.1 ;q11.2) 45, XX, -21 46, XX, del(8)(q13qter) 46, XX, del(1 )(p32pter), del(2)(p13pter) 3945, XV, -15 ·45, XV, -16, t(3;14)(q25;p11.1 ) 47, XX, +8 45, XV, -7 46, XV, del(2)(q25qter), del(5)(q15qter) 45, XV, -20 45, XX, -5 45, XV, -7 46, XV, t(5;12)(p51.1 ;q13) 45, XX, -13, -22, der(13)(13p11.1; 22q11.2), t(5;8)(q21 ;q24.3) 45, XXA 45, YO, -X, -8, +21 45, XV, -8 45, XYA 45, XYA 47, XV, +8, 48, XV, +8, +19 45, XX, -8A 45, XX, rob(14;15)(p11.1 ;q11.2)

258

A

P

P

A

P P A A

A

P

P

Case No.

Mitoses analysis Sex I Age Sub(years) type" Abnormal Normal

18 19 20

M/30 M/24 M/22

RA RA RA

0 5 29

48 27 7

21 22

M/18 M/58

RA RA

0 19

37 20

23

M/43

RA

4

29

24 25 26 27

M/36 F/52 M/50 F/25

RA RARS RARS RAEB

0 0 22 21

30 31 11 21

28 29

M/51 F/20

RAEB RAEB

2 21

37 13

30 31 32

M/35 M/23 M/24

RAEB RAEB RAEB

0 6 25

32 34 12

33

F/68

RAEB-T

32

0

34

M/55

RAEB-T

26

17

Chromosome finding 46, XYA 46, XV, t(6;7)(p1.1;q25) 47, XV, +8 45, XY,-5 46,XYA 46, XV, del(5)(q31 qter) 46, XV, t(8;18)(p21;p11) 47, XV, + 21 46, XV, del(5)(q13qter) 46, YO, -X, del(12)(p12pter) 46,XYA 46,XXA 45, XV, -5A 45, XX, +21, del(8)(q13qter) 45, XO, -X, del(5)(p13pter) 45, YO, -X, -8, +19T 46, XX, del(2)(p23pter) 46, XX, del(16)(p11.1 pter) 46, XY 45, XV, -5 47, XV, +19 45, XV, -8, del(16)(q13qter) 46, XO, -X, +21 45, XO, -X 45, XV, -7, del(5)(13qter)

Proqnoslss

A T

P

A

P

P A A T

T T

F, female; M, male. RA, refractory anaemia; RARS; refractory anaemia with ringed sideroblasts; RAEB, refractory anaemia with excess of blasts; RAEB-T, refractory anaemia with excess of blasts in transformation. bA,patients who are alive or in follow-up; P, patients have progressed to a more severe subtype of myelodysplastic a

syndrome; T, patients have transformed to acute non-lymphocytic leukaemia.

defects were monosomy 5 (-5) or del(5) (5q-), monosomy 7 (-7), or del(7) (7q-), trisomy 8 (+8) and trisomy 21 (+21). Less common changes, such as dell lfi) (16q-/16p-), trisomy 19 (+19), trisomy 20 (+20) and X loss (-X),

were also observed. Derived chromosomes der(13) and der(19) were found in two RA patients; their breakdown points were located at the same site on the long arm of chromosome 22 (q11.2). Identical rob abnormalities of

259

(14;15) (p11.1;q11.2) were also found in two RA patients. In one patient, rob of (14;15) was the sole abnormality; in the other, this aberration was accompanied by -8. Karyotypic follow-up studies were

performed in 14 patients after a mean followup time of 10 months. Transformation to overt leukaemia was observed in five (14.7%) patients with -7, +/-8, +19, +21, -X and complex abnormalities occurring following a

260

II K Wu, K Kitamura, Y II Xi et ai.

Primary myelodysplastic syndrome

261

• Refractory anaemia A Refractory anaemia with excess of blasts • Refractory anaemia with ringed sideroblasts

100

o Refractory anaemia with excess of blasts in transformation

--

-

~

.~

50

-

-

AII'

.-

(fJ

o

-

.

--

-

...

...

...

]

:::l

o

- -

12 Mean survival time (months)

T

24

Mean survival times of 34 patients after diagnosis of primary myelodysplastic syndrome according to subtype.

mean time of 12 months after initial chromosome examination. The incidence of leukaemic transformation was significantly higher in RAEB (2/6, P < 0.05) or RAEB-T (2/2, P< 0.05) patients than in RA or RARS (1/ 26) patients; the risk of leukaemic transformation in patients with abnormal karyotypes (5/26, 19.2%) and no abnormal karyotypes (0/8, 0%) was significant (P < 0.05). All patients had some blood cytopenia. In most cases, there was erythroid hyperplasia with dyserythropoiesis or normocellular bone marrow. Slight megakaryocyte increases occurred in seven patients and Auer's bodies were found in three patients who later developed leukaemia. The mean survival times of patients in each MDS subtype are shown in Fig. 2 and Table 3. The mean survival times were significantly (P < 0.01) shorter for RAEB and RAEB-T patients compared with RA and

RARS patients. Among the 26 patients with chromosome abnormalities, the disease progressed to a more severe subtype in 10 (eight with RA, two with RAEB). The cytogenetically delineated subgroup of patients with -5/5q- and another aberration, such as -7, +8, +19, +21 and -X, as well as complex chromosome defects, had very poor prognoses. The 12 cases with multiple chromosome abnormalities had a shorter mean survival time (7.5 months) compared with patients with no abnormalities, or with single or double abnormalities (Fig. 3). Although the overall mean survival time was 12.9 months, a significant (P < 0.05) difference was observed between the cytogenetically normal patients and those with abnormalities. In the 14 patients followed cytogenetically, seven with normal karyotypes were found to have a stable clinical and cytological course. In the other seven, the

262

100

'-'

[P

--

--

-

....

..

..

T ~

.2:

T

50

abnormalities • No 1- 2 abnormalitie s

• 0

..

::J (fJ

't'

o

o

- --

-

1

r-r-r

~

~

-

> 2 abnormalities

.... ....

~

24

12 Mean survival time (months)

Mean survival times of 34 patients after diagnosis of primary myelodysplastic syndrome in relation to the number of chromosome abnormalities in cultured bone marrow cells.

100

-

0



~

Without karyotic evolution With karyotic evolution

'i ~

1

~ Cii >

2:::J

-i

-

50 -

-

1

(fJ

OLL

o

--l.-

---l

12 Mean survival time (months)

---l.-

- -----'

24

Mean survival times after chromosome re-analysis in two groups of patients based on the results of chromosome follow-up studies.

263

chromosome abnormalities (5/6 and 2/2, respectively) was detected. These findings indicate that abnormal neoplastic clones have not yet spread throughout the bone marrow in MDS, as is the case in ANLL. In the present study, aneuploid cells, particularly hypodiploid cells, were increased. Of all the metaphases checked, the hypodiploid cell metaphase rate at 20.2% which was markedly higher than the normal Chinese standard value of 5.4 %," and was close to the 24.2% found in ANLL.'" The two patients with a high percentage of hypodiploid cells (>40%) associated with complex chromosomal abnormalities transformed to ANLL within 4.5 months, indicating that MDS patients have higher chromosome instability more readily transform to ANLL regardless of the associated chromosome defects. It has been suggested that patients with normal metaphases are less likely to develop ANLL. S,7 This was confirmed in this study; none of the patients with normal karyotypes developed ANLL and those with partial/abnormal karyotypes had shorter mean survival times. The F AB classification system has previously been thought to provide better prognostic information in MDS 2 ..1,7,J:I but, in this study, three main chromosome changes (single, double and complex defects) demonstrated differences in mean survival times of prognostic significance and were thus considered to be clinically useful. A clear distinction was found between the mean survival time of patients with normal chromosomes and of those with either -7, +8/-8, +19 or +21, or complex abnormalities. The two RARS patients had a stable clinical and cytogenetic course during the follow-up period; it seems likely, therefore, that such patients with normal or simple chromosome abnormalities will have a longer mean survival time and should be treated symptomatically." - n Although RA has

disease progressed to a more severe subtype or transformed to overt leukaemia with karyotypic evolution after a mean follow-up period of 10 months. The mean survival time after the chromosomes were re-analysed was 3.5 months for patients with karyotypic evolution and more than 20 months for those with no evolution (P < 0.05, Fig. 4).

DISCUSSION In MDS, and ANLL, the reported incidence of chromosome rearrangements has increased with improved cytogenetic techniques.v'" In an attempt to increase the yield of the technique in terms of metaphase number and the quality of chromosome banding resolution, separation of nuclear cells from bone marrow, as well as culture conditions, was tested using synchronization by actinomycin D, with and without conditioned medium. Mononuclear cell separation makes the chromosome preparation easier, since hypotonic shock, fixation and, in particular, confluence are not impaired by the number of cells and debris. The use of conditioned medium improved the yield in terms of the quantity and, consequently, since selection is possible, the quality of metaphases. Despite the small sample size thoroughly analysed, results indicate that conditioned medium does not significantly influence the ratio of abnormal to normal cells in cases with an admixture of normal and abnormal mitoses. With the improved cell culture method and high-resolution G-banding, 76.5% (26/ 34) of the patients had a clonal chromosome abnormality. The incidence of abnormalities was similar to the 79% reported by Yunis et al,' and the types of chromosome defects were basically similar to those found in previous studies. n,,, In the present study, however, there were few RAEB or RAEB-T patients. Such patients should be investigated continually because a relatively high frequency of

264

traditionally been considered to have a good prognosis, recent reports indicate that 15 22% may transform to ANLL! - ILl" In this study, only one of the 24 RA patients developed ANLL; this patient found to have -7 and +8, and died within 5 months of diagnosis. Of the RA patients, eight evolved into a more severe subtype with one or more recurrent chromosome defects, or clinically deteriorated. By contrast, eight RA patients with normal karyotype or only -5/5q - had a stable clinical course for up to 22 months after diagnosis. Although few patients progressed to a more severe subtype, they need to be closely followed for detection of a more aggressive course in the future. Of the six RAEB, patients two transformed to ANLL, two progressed to RAEB- T and two were clinically stable and had survived more than 15.5 months at the time of study. Only 3.5 months after diagnosis, two RAEB-T patients with excess of blasts in transformation and with -X, +21/-7, 5q- had transformed to overt leukaemia. These results indicate that approximately half of the RAEB patients had a poor prognosis, i.e.

Cytogenetic analysis of bone marrow from patients with primary myelodysplastic syndrome.

Of 34 patients with primary myelodysplastic syndrome (MDS), 26 (76.5%) were found to have chromosome defects using an improved bone marrow culture met...
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