Involvement of erythrocytic and granulomonocytic lineages by trisomy 11 in two cases of acute myelomonocytic leukemia with trilineage myelodysplasia

Involvement of Erythrocytic and Granulomonocyfic Lineages by Trisomy 11 in Two Cases of Acute Myelomonocytic Leukemia with Trilineage Myelodysplasia An Interphase Cytogenetic Study Antonio Cuneo, Massimo Balboni, Maria Gretel Carli, Renato Bigoni, Grazia Roberti, Isabella Pazzi, Rita Previati, and Gianluigi Castoldi

ABSTRACT: To study the cytologic profile and lineage involvement in acute myeloid leukemia (AML)

with trisomy 11, cytologic, cytogenetic, and interphase cytogenetic studies were performed at presentation in two cases of acute myelomonocytic leukemia (AML-M4). Patient 1 had + 11 as the sole chromosome aberration in 16/20 karyotypes whereas two related clones with + 11 in all abnormal metaphases (14/18) were detected in patient 2. A proportion of interphase cells with three signals, comparable to the proportion of abnormal metaphases, was detected by fluorescent in situ hybridization (FISH) in these patients. Morphologic aberrations of the nonblast cell population affecting multiple cell lineages, along with a circulating minor megakaryoblastic component, were observed at diagnosis in both patients. By separation of bone marrow cells over a density gradient of Percoll two cell fractions were obtained, the former containing more than 80% erythroid precursors (collected at a density of 1065-1075 mg/ml), the latter containing more than 78% blast cells plus granulomonocytic precursors (collected at a density of 1060-1055 mg/ml). FISH documented the presence of a majority of interphase nuclei with three signals in the erythroblast-enriched cell fraction and in the blast-enriched cell fraction. It is concluded that cytologic features, as well as interphase cytagenetic findings on enriched cell fractions, suggest the occurrence of multipotent stem cell involvement in AML-M4 with + 11.

INTRODUCTION

A number of recurring chromosome aberrations have been described in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [1]. Whereas some of these chromosome changes define specific disease subsets, others are found in a variety of leukemic and preleukemic conditions. Thus, it is well known that t(15;17) is the hallmark of promyelocytic leukemia, that t(8;21) is found in AML with maturation, and that the 5q- chromosome defines a characteristic syndrome of MDS [2, 3]. On the other hand, trisomy 8 and t(6;9) span a spectrum of myeloid neoplasias [4, 5], trisomy 4 can be found in AML with or without myelodysplasfic features of the nonblast cell population, as well

From The Institute of Hematology, University of Ferrara, Ferrara, Italy. Address reprint requests to: Dr. Antonio Cuneo, Institute of Hematology, University of Ferrara, Via Savonarela 9, 44100 Ferrara, Italy. Received January 10, 1994; accepted February 24, 1994.

as in preleukemic conditions [6], and trisomy 14 is usually associated with marked dysplastic features both in MDS and AML [7]. Trisomy 11 is found in approximately 1% of MDS and AML with abnormal karyotypes [8] and attention has been drawn by Takasaki et al. [9] to the fact that this numerical aberration appeared to be preferentially associated with chronic myelomonocytic leukemia {CMMoL) or with AML with myelomonocytic features. Trisomy 11, however, has also been found in refractory anemia [10] and AML with myeloid differentiation [11]. This chromosome anomaly appears therefore to be associated with distinct myeloid neoplasias, the cytologic features of which are poorly defined. In addition, because conventional chromosome analysis does not allow for recognition of the cytologic type of metaphase cells, it is not known which cell lineage is involved in trisomy 11. This issue is of particular interest in view of the heterogeneity of the cytologic features usually observed in this cytogenetic subset of AML and MDS. The aim of the present report is two-fold: a) to describe 33

© 1994 Elsevier Science Inc. 655 A v e n u e of the Americas, N e w York, NY 10010

Cancer Genet Cytogenet 77:33-38 (1994)

0165-4608/94/$07.00

34 the cytologic and interphase cytogenetic findings in two patients presenting with AML and trisomy 11 as the possible primary chromosome change, and b) to assess whether or not involvement of the erythroid and granulomonocytic lineage had occurred in these patients. Results of cytologic and cytogenetic studies are presented here and discussed insofar as they relate to the possible significance of trisomy 11 in leukemia. PATIENTS AND METHODS Two patients with trisomy 11 as the possible primary chromosome change were found among 107 patients (45 with MDS, 62 with AML) in whom cytogenetic studies were successfully performed at our institution over the last 5 years. Diagnoses were made according to the FAB proposals for the recognition of MDS and AML [12, 13]. Trilineage myelodysplasia (TMDS) in patients presenting with AML was defined by the presence of more than 25% morphologically abnormal erythroblasts and more than 50% dysplastic granulocytes and megakaryocytes [14]. Laboratory work-up at presentation included cytogenetic and immunophenotypic studies. FISH was performed in these two patients with trisomy 11 on unseparated BM samples, as well as on an erythroblast-enriched cell fraction and on a blast-enriched cell fraction obtained by separation of BM cells on a density gradient. CASE HISTORIES Patient 1 A 62-year-old previously healthy man was referred to our institution with fatigue and leukocytosis on a routine blood count, which showed anemia and lowered platelet count. The differential count showed 23% neutrophils, 8% granulocyte precursors, 19% lymphocytes, 32% monocytes, and 18% blast cells. Bone marrow (BM) was hypercellular, with 50% blast cells. Nuclear as well as cytoplasmic morphologic abnormalities were detected in some erythroblasts, and in more than 50% of granulocytes and megakaryocytes. Cytochemical stains showed positivity for the peroxidase reaction in 45% of the blasts cells and for the nonspecific esterase with fluoride inhibition in 37% of the blast cells. Serum lysozime was markedly increased. Cytofluorimetric analysis of the cells in the blast gate were performed as previously described [15], yielding the following results: CD34 negativity, CD33 positivity (72%), CD13 positivity (64%), CD14 positivity (24%), and CD15 positivity (34%). The CD41, CD42, and CD61 platelet markers were positive in 6-8 % of blast cells, whereas B- and T-lymphoid markers were negative. A diagnosis of AML-M4 with TMDS was made and treatment with idarubicin, cytarabine, and etoposide was started. Complete remission was not achieved and the patient died 1 year later with disease progression. Patient 2 A 32-year-old man was referred to our institution with a 3-month history of fatigue, pancytopenia, and easy bruising

A. Cuneo et al. with 16% blast cells in a routine blood count. The patient had been exposed to organic solvents in a shoe factory for 4 years. Physical examination was unremarkable. A BM aspirate showed erythroid hyperplasia with marked dysplastic features, hypogranular neutrophils, and several megakaryocytes with multiple separated nuclei. In addition, a 45% blast cell infiltrate was observed. Fifty-five percent of the blast cells were positive for the Sudan black-B stain. The nonspecific esterase reaction was positive in 56% of blast cells. This reaction was inhibited by sodium fluoride. Immunophenotype was consistent with a diagnosis of AML-M4, showing a 20% positivity for the CD34 stem-cell marker, as well as for the CD13, CD33, and CD14 myelomonocytic markers in more than 50% of cells in the blast gate. The presence of a minor megakaryoblastic component was demonstrated by a 5% and 7% reactivity for antiplatelet monoclonal antibodies detecting the CD41 and CD61 antigens. The patient received two courses of multiagent chemotherapy, resulting in a reduction of the percentage of BM blast cells below the 5 % cut-off. A 5-month history of pancytopenia with trilineage myelodysplasia was subsequently recorded until overt leukemic relapse occurred. The patient was unresponsive to chemotherapy and died with disease progression 13 months after diagnosis. Cytogenetic Studies Mononuclear cells obtained by separation of BM samples over a Ficolt gradient were cultured at a concentration of 1.5 x 106 cells/ml in RPMI 1640 additioned with 30% fetal calf serum and incubated for 24-48 hours in a 5% CO 2 humidified atmosphere. Cell synchronization was performed with methotrexate and thymidine. Metaphase spreads were G-banded according to the method described by de le Maza et al. [16]. At least 10 metaphases were karyotyped and chromosome aberrations were described according to the ISCN [17]. Fluorescent In Situ Hybridization (FISH) FISH was performed on these two patients employing a commercially available chromosome 11-specific centromeric probe (Oncor, Gaithersburg, MD) after the presence of trisomy 11 was documented by conventional chromosome analysis. This probe detected less than 1% cells with three signals and less than 10% cells with one signal on control slides from cytogenetically normal BM samples. Interphase cytogenetic techniques adopted at our institution were detailed previously [18]. Slides for conventional cytogenetic analysis, aged at room temperature for 2-30 days, were pretreated by RNAse, dehydrated in methanol, and denatured in 70% formamide in 2 x SSC at 70°C for 2 minutes. The biotinylated probe (100 ng/slide) was dissolved in 45 pl hybridization mixture {total volume) consisting of 60% formamide in 2 x SSC, 10% wt/vol dextran sulfate, and 30 ~g salmon sperm DNA. Following denaturation at 75°C for 5 minutes the hybridization mixture was layered onto each slide. Slides were covered with a coverslip and sealed with nail polish. Hybridization was allowed to proceed overnight at 37°C in a moist chamber. Unspecifically bound probe was removed through repeat

+ 11 in AML with TMDS

35

A

•:iiii!~!!i:~

C

D

Figure 1 Separation of BM cells on a gradient of Percoll and interphase cytogenetic study in patient 2. Erythroid precursors collected at the interfaces between the densities 1075-1070-1065 mg/ml are shown in (A). FISH in this cell fraction shows three fluorescent signals in the majority of cells (B). Blast cells and granulocyte precursors collected at the interface between the density layers 1060-1055 mg/ml are shown in (C). Evidence of trisomy 11 in interphase cells from the blast-enriched cell fraction is provided by FISH (D).

washes in 45% formamide in 2 x SSC with intermittent agitation. Specific signal was visualized by incubation with FITC-avidin (Vector, Burlingame) followed by amplification w i t h biotynated anti-avidin m o n o c l o n a l antibody (Vector) and a second layer of FITC-avidin. P r o p i d i u m i o d i d e was used as counterstain. Slides were examined u n d e r a fluorescence microscope (Diaplan, Leitz). At least 200 ceils w i t h well-delineated fluorescent spots were counted in each patient for signal screening. Separation of BM Cells To obtain an erythmblast-enriched cell fraction and a blastenriched cell fraction, a modification of the method of Oloffsson et al. [19] was employed as previously described [20]. Briefly, 30 × 106 BM cells were layered on top of a discontinuous Percoll gradient in sterile p o l y p m p y l e n e tubes and centrifuged at 600 g for 25 minutes. The densities used from

the bottom to the top were: 1075 mg/ml, 1070 mg/ml, 1065 mg/ml, 1060 mg/ml, and 1055 mg/ml, and the volume of each layer was 1.2 ml. After centrifugation, those cells lying between the 1055-1060 mg/ml and 1065-1070-1075 mg/ml density layers were collected separately a n d cultured in 25-ml culture flasks in RPMI 1640 with 20% fetal calf serum added. Cytospin preparations were made from each culture for cytologic analysis using May-Grunwald Giemsa staining. The cells of each fraction were fixed in methanol acetic acid at a concentration 3:1 and samples were stored at - 2O°C for subsequent FISH study. RESULTS Morphologic, cytochemical, and i m m u n o l o g i c findings in these patients are consistent with a diagnosis of AML-M4 with TMDS (see Case Histories). Complete remission was

36

A. Cuneo et al.

Table 1

Results of cytogenetic and FISH studies on u n s e p a r a t e d BM samples in two patients w i t h AML-M4 a n d trisomy 11 FISH signal screening in interphase cells BM karyotype at diagnosis

Patient 1 47,XY, + 11 Patient 2a 47,XY, + der(8)t(8;13)(p11;q12), + 11, - 13/48,idem, + mar

Abnormal/total

1

2

3

16/20

8%

19%

73%

14/18

6%

31%

63%

° A 47,XY,der(8)t(8;13)(p11;q12)+ 11 karyotype was detected in 2/20 ceils during the MDS phase following inductiontherapy in patient 2.

Table 2

BM cell separation and FISH studies on different cell p o p u l a t i o n s in two patients with AML-M4 and trisomy 11 BM cells after separationa

BM cells before separation (E-bls/GM-cells/blasts°) Patient 1 37%/12%/50% Patient 2 43%/11%/45%

ECF (1065-1075 mg/ml)

BCF (1060-1055 mg/ml)

E-bls, 81% GM-cells 15% Blasts 3% E-bls 83% GM-cells 10% Blasts 4%

FISH: % cells with 3 spots ECF

BCF

Blasts 71% GM-cells 11% E-bls 16%

67%

75%

Blasts 67% GM-cells 11% E-bls 21%

71%

69%

ECF, erythroblast-enriched cell fraction; BCF, blast-enriched cell fraction; E-bls, erythroblasts; GM-cells,cells of the granulomonocytic lineage. a Differentialon 200 cells. Cells below the 1% cut-off not reported.

Abbreviations:

achieved in patient 2, in w h o m a cytologic and clinical picture consistent with MDS was observed following induction therapy.

Cytogenetic and FISH Studies on Unseparated BM Samples Chromosome studies at diagnosis disclosed a clone w i t h trisomy 11 as the sole chromosomal defect in patient I and a complex karyotype with two related clones carrying trisomy 11 in all abnormal metaphases in patient 2. In both cases a minority of normal metaphases was also present. A minority of metaphases with trisomy 11 was present during the MDS phase following i n d u c t i o n therapy in patient 2. The presence of trisomy 11 in interphase cells was d o c u m e n t e d by FISH on unseparated BM samples obtained at diagnosis. The percentage of trisomic interphase cells mirrored the percentage of abnormal metaphases as detected by conventional chromosome study. Results are detailed in Table 1. BM Cell Separation and FISH Studies More than 80% erythroblasts were collected at the interface between the densities 1065, 1070, and 1075 mg/ml in both cases (Fig. 1A). The remaining cells in this fraction were blasts, granulocyte precursors, a n d monocytes.

More than 78% blast cells, granulocyte precursors, and monocytes were collected above the 1060 mg/ml density layer (Fig. 1C). The remaining cells were early erythroblasts. FISH studies demonstrated that 67% and 71% interphase nuclei in the erythmblast-enriched cell fraction h a d three fluorescent spots in patients I and 2, respectively (Fig. 1 B). Evidence of trisomy 11 was detected by FISH in 75% and 69 % cells in the blast-enriched cell fraction in patients I and 2, respectively (Fig. 1 D). Results are summarized in Table 2. DISCUSSION To our knowledge, this is the first report describing cytogenetic and interphase cytogenetic findings in AML with trisomy 11 showing that the percentage of BM interphase cells with three fluorescent signals was similar to that of metaphase cells with trisomy 11. These findings a d d to the growing b o d y of evidence that FISH allows for the reliable detection of some numerical aberrations in interphase cells and for the size estimation of the c h r o m o s o m a l l y abnormal clone in lymphoproliferative disorders and in myeloid neoplasias [18, 21-23]. In line with our data, recent reports documented that comparable results are u s u a l l y obtained w h e n estimating the clone size by conventional chromosome analysis and by in-

+ 11 in AML with TMDS terphase cytogenetics in patients with myeloid neoplasias and aneuploidy [24, 25]. Although exceptions to the rule have been reported [26], there is general agreement that, as FISH is not d e p e n d e n t on in vitro proliferation of neoplastic cells, it gives a better estimation of the real size of the a n e u p l o i d clone than conventional karyotyping. Some cytologic and clinical features in our patients deserve consideration. The presence of morphologic aberrations affecting m u l t i p l e cell lineages identifies a subset of AML u s u a l l y referred to as AML w i t h TMDS [14]. Interestingly, an insidious onset compatible with a preleukemic condition, consisting of a 3-month history of anemia-related symptoms and easy bruisability was anamnestically recorded in patient 2, suggesting that the distinction between MDS evolving into AML and AML w i t h TMDS may be artificial in this case. Myelodysplastic relapse with trisomy 11, occurring in this patient following induction therapy, supports this argument [27]. In addition, morphologic abnormalities of the nonblast cell p o p u l a t i o n that are u s u a l l y regarded as reliable indicators of a preleukemic c o n d i t i o n [12, 28], such as pseudoPelger forms, hypogranular neutrophils, and megakaryocytes with m u l t i p l e separated nuclei, were observed in our two patients, along w i t h the presence of a m i n o r i t y of megakaryocyte precursors in peripheral blood smears. The presence of circulating megakaryoblasts was documented previously in some cases of AML, as well as in myeloproliferative and preleukemic conditions [29, 30] and appear to reflect d i s o r d e r e d maturation of the megakaryocytic lineage. It is noteworthy that AML with myelomonocytic features has been shown frequently to be associated with marked dysmegakaryocytopoiesis [31]. These findings, along with the consideration that trisomy 11 is u s u a l l y found in a spectrum of myeloid stem cell disorders, strongly suggest that multipotent stem cell involvement may occur in myeloid n e o p l a s i a carrying this chromosome abnormality. Indeed, involvement of a multipotent progenitor cell has been documented cytogenetically in AML and MDS patients w i t h trisomy 8 and m o n o s o m y 7 [20, 25, 32], both chromosome abnormalities showing a distribution pattern in myeloid neoplasias similar to that of trisomy 11. For these reasons it was of interest to assess whether or not multipotent stem cell involvement had occurred in our two AML patients. Centrifugation of BM cells over a Percoll gradient has been shown to be a valuable tool for the separation of a lighter cell fraction in w h i c h blast cells represent the prevailing cytologic type from a heavier fraction containing erythroid and granulocyte precursors [11, 33]. The coexistence in diagnostic BM samples of blast cells with a sizeable erythroid population prompted us to perform an interphase cytogenetic study on an erythoblast-enriched cell fraction and on a blast-enriched cell fraction. We excluded from this analysis a cell fraction that was collected at the interface between the densities 1060-1065 mg/ml because it consisted of an a d m i x t u r e of blast cells, granulocyte precursors, and early erythroblasts, which would have p r e c l u d e d a reliable analysis of the data.

37 We were thus able to show that a majority of trisomic cells was present both in the cell fraction containing more than 80% erythroid cells and in the blast-enriched cell fraction, providing evidence that trisomy 11 affected the erythroid lineage, as well as blast cells with myelomonocytic features (Fig. 1). This would suggest that multipotent stem cell involvement is likely to have occurred in these patients. In conclusion we have shown that trisomy 11 may affect different hemopoietic lineages in some patients with AML with myelodysplastic features. Given its association with distinct subtypes of myeloid stem cell disorders, it is likely that trisomy 11 may play an as-yet u n i d e n t i f i e d role in the transformation of a primitive progenitor cell [34] and that additional genetic events may be involved in the generation of such a variable spectrum of diseases. This work was supported 60% by M.U.R.S.To fondi, and supported by fondi regione ER. REFERENCES

1. Mitelman F (1991]: Catalog of Chromosome Aberrations in Cancer, 4th ed. New York, Wiley-Liss. 2. Second MIC Cooperative Group (1988): Morphologic, immunologic and cytogenetic (MIC) working classification of acute myeloid leukemias. Cancer Genet Cytogenet 30:1-15. 3. Van Den Berghe H, Vermaelen K, Mecucci C, Barbieri D, Tricot G (1986): The 5q- anomaly. Cancer Genet Cytogenet 17:189-242. 4. Cuneo A, Kerim S, Vandenberghe E, Van Orshoven A, Rodhain J, Bosly A, Zachee P, Louwagie A, Michaux JL, Dal Cin P, Van Den Berghe H (1989): Translocation t(6;9) occurring in acute myelofibrosis, myelodysplastic syndrome, and acute nonlymphocytic leukemia suggests multipotent stem cell involvement. Cancer Genet Cytogenet 42:209-219. 5. Sandberg AA (1990): Chromosomes in Human Cancer and Leukemia, 2nd edition. Elsevier, New York. 6. Mecucci C, Van Orshoven A, Tricot G, Michaux JL, Delannoy A, Van Den Berghe H (1986): Trisomy 4 identifies a subset of acute nonlymphocytic leukemias. Blood 67:1328-1332. 7. Mancini M, Cedrone M, Nanni M, Rondinelli MB, Petti MC, De Cuia MR, Alimena G (1993): Trisomy 14 in hematologic diseases. Another non-random abnormality within myeloproliferative disorders. Cancer Genet Cytogenet 66:39-42. 8. Helm S, Mitelman F (1992): Cytogenetic analysis in the diagnosis of acute leukemia. Cancer 70:1701-1705. 9. Takasaki N, Kaneko Y, Maseki N, Sakurai M (1988): Trisomy II in chronic myelomonocytic leukemia: report of two cases and review of the literature. Cancer Genet Cytogenet 30:109-117. I0. Morgan R, Green M, Sandberg AA (1988): Trisomy II in myelodysplasia. Cancer Genet Cytogenet 30:159-162. II. Weh HJ, Hoffmann R, Suciu S, Kuse R, Hossfeld DK (1988): Is trisomy II another non-random chromosomal anomaly in acute leukemia and myelodysplasticsyndromes? Cancer Genet Cytogenet 35:205-211. 12. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1982): Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51:189-199. 13. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1985): Proposed revised criteria for the classification of acute myeloid leukemia: A report of the FrenchAmerican-British Cooperative group. Ann Intern Med 103: 626-631. 14. Brito-Babapulle F, Catovsky D, Galton DAG (1987): Clinical and

38

15.

16. 17.

18.

19.

20.

21.

22.

23.

24.

25.

A. C u n e o et al. laboratory features of de novo acute myeloid leukaemia with trilineage myelodysplasia. Br J Haematol 66:445-449. Cuneo A, Michaux JL, Ferrant A, Van Hove L, Bosly A, Stul M, Dal Cin P, Vandenberghe E, Cassiman JJ, Negrini M, Piva N, Castoldi GL, Van Den Berghe H (1992): Correlation of cytogenetic patterns and clinicobiological features in adult acute myeloid leukemia expressing lymphoid markers. Blood 79: 720-727. de la Maza M, Sanchez O (1976): Simultaneous G and C banding of human chromosomes. J Med Genet 13:235-243. ISCN (1991): Guidelines for Cancer Cytogenetics. Supplement to An International System for Human Cytogenetic Nomenclature. Mitelman F (ed). S. Karger, Basel. Cuneo A, Wlodarska I, Sayed Aly M, Piva N, Carli MG, Fagioli F, Tallarico A, Pazzi I, Ferrari L, Cassiman JJ, Van Den Berghe H, Castoldi GL (1992): Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukemia. Br J Haematol 81:192-196. Olofsson T, Gartner I, Olsson I (1980): Separation of human bone marrow cells in density gradients of polyvinylpyrrolidone coated silica gel (Percoll). Scand J Hematol 24:254-262. Cuneo A, Tomasi P, Balboni M, Piva N, Fagioli F, Castoldi GL (1989): Cytogenetic analysis of different cellular populations in chronic myelomonocytic leukemia. Cancer Genet Cytogenet 37:29-37. Le Beau, M [1993): Fluorescence in situ hybridization in cancer diagnosis. In: Important Advances in Oncology, De Vitat V, Hellman S, Rosenberg SA, eds. Philadelphia, Lippincott Company, pp 29-45. Raghoebier S, Kibbelaar RE, Kleiverda JK, Kluin-Nelemans HC, van Krieken JHJM, Kok E Kluin PM (1992): Mosaicism of trisomy 12 in chronic lymphocytic leukemia detected by non-rsdioactive in situ hybridization. Leukemia 6:1220-1226. Jenkins RB, Le Beau M, Kraker WJ, Borell TJ, Stalboerger PG, Davis EM, Penland L, Fernald A, Espinosa HI R, Schald DI, Noel P, Dewald GW (1992): Fluorescence in situ hybridization: a sensitive method for trisomy 8 detection in bone marrow specimens. Blood 79:3307-3315. Kibbelaar RE, Mulder JWR, Dreef EJ, van Kamp H, Fibbe WE, Wessels JW, Beverstock GC, Haak HL, Kluin PM (1993): Detection of monosomy 7 and trisomy 8 in myeloid neoplasia: a comparison of banding and fluorescence in situ hybridization. Blood 82:904-913. Baurmann H, Cherif D, Berger R (1993): Interphase cytogenetics

by fluorescent in situ hybridization (FISH) for characterization of monosomy 7-associated myeloid disorders. Leukemia 7: 384-391. 26. Poddighe PJ, Moesker O, Smeets D, Awwad BH, Ramaekers FCS, Hopman AHN (1991): Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes. Cancer IRes 51:1959-1967. 27. Brito-Babapulle F, Catovsky D, Galton DAG (1988): Myelodysplastic relapse of de novo acute myeloid leukaemia with trilineage myelodysplasia: a previously unrecognized correlation. Br J Haematol 68:411-415. 28. Castoldi GL, Cuneo A, Lanza F, Tomasi P (1990): Problemi di citologia: E' ancom attuale la classificazione FAB? In: Progressi in Ematologia Clinica, Sindromi mielodisplastiche, Vol X, Bernasconi C (ed). Tipografia Viscontea, Pavia, 1990. 29. Breton-Gorins J, Dreyfus B, Sultan C, Basch A, D'Oliveira JG (1972): Identification of circulating micromegakaryocytes in a case of refractory anemia: an electron microscopic-cytochemical study. Blood 40:453-463. 30. Erber WN, Breton-Gorius J, Villeval JL, Oscier DJ, Bay Y, Mason DY (1987): Detection of cells of megakaryoblastic lineage in hematological malignancies by immuno-alkaline phosphatase labelling cell smears with a panel of monoclonal antibodies. Br J Haematol 65:87-94. 31. Jinnai I, Tomonaga M, Kuriyama K, Matano T, Nonaka H, Amenomori T, Yoshida Y, Kusano M, Tagawa M, Ichimaru M (1987): Dysmegakaryocylopoiesis in acute leukaemias: its predominance in myelomonocytic (M4) leukaemia and implication for poor response to chemotherapy. Br J Haematol 66: 467-472. 32. Pasquali F, Bernasconi P, Casalone R, Fraccaro M, Bernasconi C, Lazzarino M, Morra E, Alessandrino EP, Marchi MA, Sanger R [1982): Pathogenetic significance of pure monosomy 7 in myeloproliferative disorders. Analysis of 14 cases. Hum Genet 62:40-51. 33. Piva N, Cuneo A, Carli MG, Cariani D, Fagioli F, Castoldi GL, (1990): Chromosome studies of enriched blast cell fractions in myelodysplastic syndromes terminating in acute myeloid leukemia. Haematologica 75:522-526. 34. Castoldi GL, Cuneo A, Tomasi P (1989): Phenotype-related chromosome aberrations and stem cell involvement in acute myeloid leukemia. Haematologica 74:525-529.