Int J Hematol (2015) 101:58–66 DOI 10.1007/s12185-014-1700-1

ORIGINAL ARTICLE

Clonal origin and evolution of myelodysplastic syndrome analyzed by dysplastic morphology and fluorescence in situ hybridization Chun‑Mei Fu · Zi‑Xing Chen · Dan‑Dan Liu · Jun Zhang · Jin‑Lan Pan · Jian‑Ying Liang 

Received: 31 December 2013 / Revised: 29 October 2014 / Accepted: 11 November 2014 / Published online: 28 November 2014 © The Japanese Society of Hematology 2014

Abstract  Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem/progenitor cells. As bone marrow cells are extremely diverse in these disorders, the origin and evolution of MDS clones are difficult to identify and trace. Cellular dysplasia is a distinct morphologic feature; however, whether the dysplastic cells represent abnormal clones or only nonspecific superficial phenomena remains to be clarified. To address this question, 97 patients were examined for dysplasia features, among them bone marrow slides of 16 patients with chromosomal abnormalities were subjected to fluorescence in situ hybridization (FISH) to determine the karyotype of these dysplastic cells. Furthermore, the emerging frequencies of abnormal karyotypes in various differentiated stages of each lineage were also evaluated by a combination of morphological evaluation and FISH karyotyping. Our results indicate that the overall percentage of dysplastic cells does not differ significantly among the WHO subtypes, while the megakaryoid lineage presents the most frequent dysplasia in all subtypes. A positive correlation between dysplastic cells and FISHdetectable abnormal clones was observed, but the dysplastic morphology was not a specific feature of FISH-detectable abnormal clones. FISH-detectable abnormal clones can

C.‑M. Fu · Z.‑X. Chen (*) · D.‑D. Liu · J. Zhang · J.‑L. Pan · J.‑Y. Liang  Jiangsu Institute of Hematology, The first Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, 188 Shizi Street, Suzhou, 215006 Jiangsu, People’s Republic of China e-mail: [email protected] C.‑M. Fu  Xuzhou Central Hospital, 199 Jiefangnan Street, Xuzhou,  221009 Jiangsu, People’s Republic of China

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differentiate into mature granulocytes and erythrocytes, in coexistence with cells originating from the normal clones. Keywords  Myelodysplastic syndrome · Morphology · FISH · Abnormal clone

Introduction Myelodysplastic syndromes are a group of clonal disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and high risk towards leukemia. In 1982, the FAB group proposed a classification for the diagnosis of myelodysplastic syndromes [1]; refinements of this resulted in the current WHO classification [2, 3]. Both the FAB and WHO group emphasize the morphological dysplasia for the diagnosis of MDS. The minimal morphologic criterion for diagnosis of myelodysplastic syndrome is defined as the dysplasia that occurred in at least 10 % of cells of any one of the myeloid lineages, excluding AML, CMML and other hematological diseases by calculating the percentage of blasts in blood and marrow. However, dysplasia can also be secondary to many conditions including iron deficiency anemia, megaloblastic anemia, nutritional deficiencies, medications, toxins, growth factor therapy, inflammation or infection [3]. Thus, if the dysplastic cells in bone marrow of MDS patients represent the abnormal clonal origin or just are of nonspecific significance needs to be answered. As the cell component of the bone marrow in MDS patients, especially in those of MDS-RA or -RCMD, are extremely diverse, and the cells from the early abnormal clones lacking special biomarkers are rare and varying, the origin of MDS clone is very difficult to identify and trace. Moreover, whether the MDS dysplasia stems from the genetic or epigenetic changes in hematopoietic

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Clonal origin and evolution of MDS

stem/progenitor cells themselves or from the changes and aging-related events in the bone marrow microenvironment remains unclear. To explore these questions, trying to dissect the origin and evolution of the abnormal MDS clones in patients, we firstly combined the observation of cell morphology with fluorescence in situ hybridization (FISH) on the bone marrow smears of 97 MDS patients to determine the dysplastic cells and their clonal origin by using the criteria of dysplastic features previously published [4]. The relationship between the abnormal karyotype and the dysplastic morphological feature of cells was verified by comparing the overall percentage of abnormal karyotype in morphological normal cells and in dysplastic cells. Furthermore, for 16 MDS patients with various chromosomal abnormalities, we differentially counted certain number of bone marrow cells of each lineage at different stages regardless of the dysplastic morphology, analyzed their karyotype by FISH on the bone marrow smears to chase the FISH-detectable abnormal clone evolution as they differentiated into more mature stages and estimate the dynamic situation of coexistence of normal and abnormal clone cell populations in the bone marrow of patients.

Patients and methods Patients A total of 97 MDS patients treated during January 2010 to July 2012 in the First affiliated Hospital of Soochow University were enrolled in this study. All patients were diagnosed based on clinical manifestation, peripheral blood differential count, bone marrow morphology observation

and chromosomal karyotype analysis. According to the WHO 2008 classification, they were classified as 7 cases of MDS-RA, 2 cases of MDS-RN, 5 cases of MDS-RT, 41 cases of MDS-RCMD, 12 cases of MDS-RAEB-1, 23 cases of MDS-RAEB-2, 1 case of 5q-syndrome and 6 MDS-U patients. The clinical and laboratory features of 97 MDS patients are summarized in Table 1. 16 of the above patients who demonstrated abnormal karyotype underwent FISH analysis on their bone marrow slides in addition to regular cytogenetic examination and routine FISH analysis. The median age of this cohort was 45 (ranging 23–77). The patient demography and hematologic and cytogenetic characteristics as well as the probe used in FISH are summarized in Table 2. The clinical study was approved by the Hospital Ethic committee and all patients were informed with documented consent. Analysis of morphology The dysplastic of morphological features were based on previous observations [4–6]. An overall of 14, 14 and 6 types of dysplastic morphological changes could be identified in cells of erythroid, granulocytic and megakaryocytoid lineage [4], respectively. Since the present cohort did not contain a large number of patients, we fractionated these cases into four groups: RCUD, RCMD, RAEB, MDS-U, respectively, based on WHO classification, and also into two groups: the MDS with and without abnormal karyotype based on FISH analysis. For each bone marrow smear (Wright–Giemsa stained) 100 successive erythroblasts and neutrophils, as well as 50 successive megakaryocytoid cells were counted. In case of hypocellularity, the whole bone marrow smear should be surveyed for megakaryocytes. Three parameters were used to justify the

Table 1  Clinical and laboratory features of MDS patients (97 cases) Patients no. Gender male/ female

Median age years (range)

Hemoglobin (g/L)

Neutrophils (×10E9/L)

Platelets (×10E9/L)

Cytogenetics normal/abnormal

5q syndrome MDS-RA MDS-RN MDS-RT MDS-RCMD MDS-RAEB-1 MDS-RAEB-2 MDS-U MDS with abnormal karyotype

1 7 2 5 41 12 23 6 40

1/0 3/4 2/0 3/2 21/18 8/4 18/5 4/2 30/10

25 56 (17–72) 56 (47–65) 60 (39–73) 56 (15–90) 48.5 (25–80) 53 (20–84) 59 (49–80) 59 (21–80)

86 66 (56–81) 59 (53–65) 107 (78–143) 67 (41–130) 72.5 (38–121) 82 (34–119) 57 (28–95) 71 (34–130)

1.98 3.03 (0.39–4.1) 1.9 (1.5–2.3) 1.41 (1.11–3.11) 1.2 (0.2–5.11) 1.19 (0.17–8.24) 1.25 (0.27–9.4) 0.62 (0.44–1.23) 1.17 (0.27–9.4)

221 159 (15–315) 128 (21–135) 57 (22–321) 46 (14–318) 52 (11–199) 38 (10–249) 22 (4–50) 42 (11–274)

0/1 5/2 0/2 1/4 26/15 5/7 11/12 1/5 –

MDS without abnormal karyotype

57

27/30

53 (15–90)

72 (28–143)

1.25 (0.2–8.24)

55 (4–512)

–

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C-M. Fu et al.

Table 2  The demography and characteristics of 16 MDS patients analyzed by FISH on bone marrow slide Patient no.

Sex/age

MDS subtype

Cytogenetics

Probe

1 2 3 4

M/41 F/46 F/44 M/24

MDS-RAEB-1 MDS-RCMD MDS-RAEB-1 MDS-RCMD

47-49,XY,dup(1q),1q-, + 8,?15p + ,? + 22, + mar[CP10] 47,XX, + 8[6] /46,XX[2] 47,XX, + 8[9] /46,XX[1] 47,XY, + 8,der(17)?dup(17)(q11q25)[10]

CEP8 CEP8 CEP8/20q12 CEP8/CEPY

5 6 7 8 9 10 11 12 13 14 15

M/33 F/73 F/61 M/77 M/23 M/35 M/66 M/25 M/56 F/56 M/68

MDS-RCMD MDS-RT MDS-RCMD MDS-RCMD MDS-RAEB-2 MDS-RAEB-1 MDS-RAEB-2 5q- syndrome MDS-RCMD MDS-RAEB-2 MDS-RCMD

47,XY, + 8[10] 45,XX,-7,der(11)t(7;11)(q11;q14),ace[10]/46,XX[3] 45,XX,-7[10] 44-45,XY,5q-,-7,-15,-17,der(17),19p + , + mar1-2[10] 46,XY,der(5),7q-,16q + ,der(17),-17, + mar[CP6] 46,XY,7q-[10] 45,XY,?der(3),5q-,-6[CP2]/46,XY[1] 46,XY,del(5)(q13q33)[9]/46,XY[1] 42-44,XY,5q-,-12,-15,17p-,der(17),-18,20q-,-21[CP8]/46,XY[6] 46,XX,20q-[7]/46,XX[3] 46,XY,20q-[8]/46,XY[2]

CEP8/CEPY CEP7/CEPX CEP7/CEPX CEP7/CEP8 7q31/CEP7 7q31/CEP7 5q31/5P 5q31/5P 20q12/CEP8 20q12/CEP8 20q12/CEP8

16

M/25

MDS-RA

46,XY,20q-[4] 46,XY[14]

20q11/20q12

features and seriousness of cellular morphological abnormality [4]. Firstly, the incidence of specific dysplasia (ISD) was defined as the percentage of cases with a particular morphological feature of dysplasia, and calculated by the (number of) cases with specific dysplasia/total cases. Secondly, the percentage of specific dysplastic cells (PSDC) was defined as the percentage of cells with any particular morphological feature of dysplasia, and was calculated by the number of cells displaying specific dysplastic morphology/total (normal + abnormal) counted cells in a given cell lineage for each patient. PSDC represents the “lineage abnormality (%)”. Finally, the incidence of dysplasia more than 10 % (ID) in a given cell lineage, it represents the seriousness of dysplasia involved in this lineage. One of the major dysplastic features in granulocytic lineage, the pseudo-Pelger–Huët, was further classified as I and II subtypes based on the shape of nuclei (monolobated or bilobated) as previously suggested [7].

for 30 min, followed by 75, 85, 100 % alcohol for gradient dehydration. The hybridization mixture was applied to the slides with corresponding probe chosen based on the patient karyotype changes. The slides were coverslipped, denatured at 75 °C for 7 min and hybridized overnight in a moist chamber at 37 °C. After hybridization, the slides were washed in 0.4× SSC for 2 min at 72 °C and then washed in 2× SSC for 2 min at room temperature. Except case 1 and 2 hybridized with single probe, dual-color probes were used in hybridization for the rest to provide internal control and exclude polyploid. After the processing finished, the dysplasia cells could easily be recognized on the slide under fluorescence microscope according to the marked position and resembled morphological features. The dysplastic cells with fluorescent signal were photographed. Five normal BM smears were used as normal control.

Fluorescence in situ hybridization (FISH) on the bone marrow slides

The statistical analysis was performed by SPASS 17.0 soft ware. The Chi-square test was used to compare the percentage of dysplasia cells between normal and abnormal clones. Difference was considered statistically significant when P