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Quantity of clonal cells detected by conventional cytogenetic analysis correlates with bone marrow blasts and survival in myelodysplastic syndromes
Leukemia Research 36 (2012) 163–168
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Quantity of clonal cells detected by conventional cytogenetic analysis correlates with bone marrow blasts and survival in myelodysplastic syndromes Miyoung Kim a , Soie Chung a , Cha Ja See a , Sung-Soo Yoon b , Byoung Kook Kim b , Hyun Kyung Kim a , Dong Soon Lee a,c,d,∗ a
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, South Korea Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea c Department of Tumor Biology, Seoul National University College of Medicine, Seoul, South Korea d Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea b
a r t i c l e
i n f o
Article history: Received 7 April 2011 Received in revised form 22 August 2011 Accepted 23 August 2011 Available online 13 September 2011 Keywords: Quantity Clonality Conventional cytogenetic analysis FISH MDS
a b s t r a c t We performed quantitative and qualitative analyses of conventional cytogenetic analysis and interphase FISH results in 87 MDS patients. The quantity of clonal cells for each chromosome of CCA did not correlate with the result of iFISH (r, range 0.0761–1.0577). The clonal cell percentage in CCA was higher in patients with >5% bone marrow blasts than those with <5% (44.7% vs. 23.1%, p = 0.017). Multivariate analysis showed that a high quantity of clonal cells in CCA analysis is an independent prognostic factor for overall survival in MDS (p = 0.012). © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In practice, chromosomal abnormalities not only confirm the clonality of a disease but also predict the likelihood of progression into acute leukemia and survival in myelodysplastic syndrome (MDS) [1–6]. The presence of chromosomal abnormalities has turned out to be one of the most important prognostic factors for MDS and has been incorporated into the International Prognostic Scoring System (IPSS), along with the percentage of bone marrow blasts and the number of lineages involved in cytopenia [1–8]. Isolated −Y, −5/5q−, −20q, and normal karyotypes imply a good prognosis, while −7/7q− or complex abnormalities (abnormalities in more than 3 types of chromosomes) suggest a poor prognosis [5,6,8]. Recent studies by the German–Austrian MDS Study Group and the Spanish Group have analyzed the prognostic significance of uncommon abnormalities, including 1q gain, trisomy 8, and 12p− [8,9]. Conventional cytogenetic analysis (CCA) and fluorescence in situ hybridization (FISH) are the most commonly used techniques to detect clonal changes in MDS [1,2,10–12]. FISH, a molecular cyto-
genetic method, has several advantages over CCA. FISH can detect submicroscopic abnormalities owing to its sensitivity [12]. Both CCA and FISH can provide information about clone size since they can quantitate the clonal cells [12]. However, in contrast to CCA, interphase FISH (iFISH) does not require viable cells and can be carried out with non-dividing cells. Nevertheless, CCA is the standard technique recommended by the IPSS algorithm, as it enables the detection of complex abnormalities that could be missed in FISH [13]. Most studies have investigated clonal chromosomal abnormalities in MDS by focusing on the relationship between the presence or absence of an abnormality of a certain chromosome and the prognosis of MDS [1,2,8–12], but not on the quantity of leukemic cells burdened with CCA. Little is known about the utility of the quantitative results of CCA and iFISH in clinical practice. We investigated the roles of CCA and FISH results in predicting the outcome of MDS patients. The results of CCA and FISH were compared both in qualitative and quantitative aspects. We analyzed the prognostic significance of the quantity of clonal cells in CCA or FISH in MDS. 2. Materials and methods
∗ Corresponding author at: Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehang-ro, Jongno-gu, Seoul 110-744, South Korea. Tel.: +82 2 2072 3986; fax: +82 2 747 0359. E-mail address: [email protected] (D.S. Lee). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.08.021
2.1. Patients Data for the study was accumulated from 129 newly diagnosed, de novo MDS patients of Seoul National University Hospital between April 2000 and March 2010. Due to suboptimal number of metaphase cells (less than 20), only 87 patients (54
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Table 1 The incidence of clonal chromosomal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ in conventional cytogenetic analysis (CCA) and interphase fluorescence in situ hybridization (iFISH) in myelodysplastic syndromes.
CCA+ CCA+, iFISH− iFISH+ CCA−, iFISH+ CCA+ and/or iFISH+
−5/5q−
−7/7q−
+8
−20/20q−
+1/1q
9 (10.3%) – 9 (10.3%) – 9 (10.3%)
7 (8.0%) – 9 (10.3%) 2 (2.3%) 9 (10.3%)
10 (11.5%) 1 (1.1%) 14 (16.1%) 5 (5.7%) 15 (17.2%)
5 (5.7%) 1 (1.1%) 6 (6.9%) 2 (2.3%) 7 (8.0%)
9 (10.3%) 1 (1.1%) 9 (10.3%) 1 (1.1%) 10 (11.5%)
men and 33 women) were included from the analysis. The patients’ age was between 19.6 and 83.0 years (mean = 57.6 years). The patient group included 25 cases of refractory anemia (RA), 5 cases of refractory anemia with ringed sideroblasts (RARS), 11 cases of refractory cytopenia with multilineage dysplasia (RCMD), 24 cases of refractory anemia with excess blasts-1 (RAEB-1), 19 cases of refractory anemia with excess blasts-2 (RAEB-2), and 3 cases of unclassifiable MDS (MDS-U), according to the WHO 2001 classification. Data concerning patient deaths and acute myeloid leukemia (AML) progression, obtained in May 2010, showed that 15 had progressed to AML, 21 died, and 66 survived. This study was approved by the Institutional Research Board of the Seoul National University Hospital (IRB No. H-1005-001-316). 2.2. Conventional cytogenetic analysis Cytogenetic studies using standard techniques were performed as a part of the diagnostic work-up. At least 20 metaphases were analyzed, whenever possible. Clonal abnormalities were defined as 2 or more cells with the same additional whole chromosome or chromosome rearrangements, or 3 or more cells with the same chromosome missing. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature 2005 [14].
found in 9 (10.3%), 9 (10.3%), 15 (17.2%), 7 (8.0%), and 10 (11.5%) of the subjects, respectively, by CCA and/or iFISH. The discrepancies between CCA and iFISH were observed in 12 (13.8%) patients, and their clinicopathological characteristics are detailed in Table 2. CCA detected clonal abnormalities of +8, −20/20q−, and +1/1q+ in 1 (1.1%), 1 (1.1%), and 1 (1.1%) patients who did not show those abnormalities in iFISH. The sizes of clones detected in CCA were variable (18.2–83.3%). iFISH detected submicroscopic clonal abnormalities of −7/7q−, +8, −20/20q−, and +1/1q+ in 2 (2.3%), 5 (5.7%), 2 (2.3%), and 1 (11%) patients, and the sizes of clones in iFISH ranged from 5.0 to 84.0%. Among them, 6 patients (010, 016, 019, 028, 046 and 067) showed normal karyotype in CCA (12.8% of 47 patients with normal karyotype). 3.2. Prognostic significance of qualitative results of conventional cytogenetic analysis and interphase fluorescence in situ hybridization
2.3. Interphase fluorescence in situ hybridization We used the iFISH technique to detect the abnormalities of chromosomes 5, 7, 8, 20, and 1 (−5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+). The following probes were used: LSI (locus-specific identifier) EGR1/D5S23, D5S721 Dual Color Probe for chromosome 5q; LSI D7S522/CEP 7 Probe for chromosome 7q; CEP (centromere enumeration probe) 8 DNA Probe for chromosome 8; LSI D20S108 Probe for chromosome 20q; and LSI p58 (1p36)/TelVysion 1p/LSI 1q25 Probe for chromosome 1; all probes were obtained from Vysis Inc. (Downers Grove, IL, USA). The normal cutoff values of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ based on the mean (3 SD) of 40 negative controls, were 2.0%, 2.0%, 2.0%, 2.0%, and 1.5%, respectively. No less than 200 nuclei of a sample were scored for deletion and rearrangement in each sample. Results were described according to the ISCN (2005) criteria [14]. 2.4. Statistical analysis All statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was applied for the comparison of categorical variables. Bivariate correlation analysis was used to evaluate the correlation between two continuous variables, and r (Pearson’s correlation coefficient) and r2 were considered in the evaluation. Mann–Whitney U-test was applied for the comparison of continuous variables. Survival curves were estimated using the Kaplan–Meier method. Log-rank tests were used to conduct univariate comparisons of overall survival and progression-free survival among subgroups. Multivariate analyses adjusted for significant prognostic factors were performed using Cox’s hazard regression model. Prognostic significance was evaluated in terms of 95% confidence intervals.
3. Results 3.1. Clonal abnormalities of chromosomes 5, 7, 8, 20 and 1 in conventional cytogenetic analysis and interphase fluorescence in situ hybridization Conventional cytogenetic analysis (CCA) detected clonal chromosomal abnormalities of any chromosomes in 40 (46.0%), and clonal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ in 31 (35.6%). Twelve (13.8%) had complex chromosomal abnormalities (≥3 abnormalities) in CCA. Interphase fluorescence in situ hybridization (iFISH) for −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ detected clonal chromosomal abnormalities in 36 (41.4%) patients. The incidence of clonal chromosomal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ observed in CCA and/or iFISH is summarized in Table 1. The clonal abnormalities were
Out of 12 with discrepancies between CCA and iFISH, the IPSS risk group changed in 1 case (8.3%: patient 003) from Int-2 to High due to the clonal abnormality detected only by CCA, and in 3 cases (25.1%: patient 016, 028, and 046) from Int-1 to Int-2 due to the submicroscopic clonal abnormalities detected by iFISH. In the total study population, the rate of evolution to AML, leukemia free survival and overall survival of each IPSS risk group are as follows: 0.0%, 654 days and 654 days in Low risk group; 10.2%, 617 days and 627 days in Int-1 risk group; 33.3% 382 days and 515 days in Int-2 risk group; 22.2%, 275 days and 300 days in High risk group. Patient 016 and 028 showed similar survival duration to Int-1 risk group, while patient 003 and 046 showed similar survival duration to High risk group and Int-2 group, respectively. Statistical comparison of survival could not be performed due to too small number of patients (4) who changed IPSS group. 3.3. Qualitative analysis of clonal chromosomal abnormality Clonal cells with chromosomal abnormalities were detected by CCA in 28.0%, 0.0%, 63.6%, 54.2%, 57.9% and 66.7% of the 25 RA, 5 RARS, 11 RCMD, 24 RAEB-1, 19 RAEB-2, and 3 MDS-U patients, respectively. The incidence was similar in iFISH analysis: the presence of any clonal abnormality of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ was detected in 32.0%, 20.0%, 45.5%, 45.8%, 42.1% and 66.7% of RA, RARS, RCMD, RAEB-1, RAEB-2 and MDS-U patients, respectively. There was no association between the incidence of clonal abnormality and the percentage of BM blasts, cytopenia, or MDS subtype (p > 0.05). 3.4. Quantitative analysis of clonal chromosomal abnormality When only the patients with clonal abnormalities detected by CCA and/or FISH are considered, the quantitative results of clonal cells detected in the 2 methods yielded a variable degree of agreement (poor to substantial, depending on the chromosomes involved): −5/5q−: r = 1.0577, r2 = 0.5022; −7/7q−: r = 0.0761, r2 = 0.0181; +8: r = 0.5164, r2 = 0.5179; −20/20q−: r = 0.6805, r2 = 0.8014; and +1/1q+: r = 0.2569, r2 = 0.1547 (Fig. 1). The
429 184 Int-1 (1.0) → Int-1 (1.0) Int-2 (2.0) → Int-2 (2.0)
3.5. Prognostic significance of quantitative results of conventional cytogenetic analysis and interphase fluorescence in situ hybridization In univariate analysis, the quantity of clonal cells in CCA > 50.0%, the presence of clonal cells in iFISH, the quantity of clonal cells in iFISH > 50.0%, isolated abnormalities of −5/5q− (detected by CCA and/or FISH), and the percentage of BM blasts >5% showed prognostic significance for overall survival (p < 0.05). In multivariate analysis, the quantity of clonal cells in CCA > 50.0% (p = 0.012) was the only independent prognostic factor for overall survival in this study population (Fig. 2). The following factors showed prognostic significance for the progression-free survival into AML cases in univariate analysis (p < 0.05): the quantity of clonal cells in CCA > 50.0%, the quantity of clonal cells in iFISH > 50.0%, isolated abnormalities of −5/5q− and +8, and the percentage of BM blasts >5%. In multivariate analysis, the percentage of BM blasts was the only independent prognostic factors for the progression-free survival into AML in this study group (p < 0.001). Individual cytogenetic abnormalities were not considered in the adjustment.
a
Not available due to inadequate quality of BM biopsy section. Leukemia free survival duration in the case evolved to AML.
4. Discussion
b
70–80 80–90 RAEB-1 RAEB-1 7.7–3170–1395–156 8.3–1980–530–19 067 (M/72) 082 (M/75)
165
quantity of clonal cells from total metaphase cells in CCA was higher in the 43 patients with >5% BM blasts than in the 44 patients with <5% BM blasts in CCA (44.7% vs. 23.1%, p = 0.017; Fig. 2). The difference in the quantity of clonal cells in CCA between patients with >10% and those with 5–10% blasts was not statistically significant (p > 0.05). However, the quantity of clonal cells from the total interphase cells in iFISH was independent of the percentage of BM blasts: 44 patients had <5% BM blasts and 43 patients had >5% BM blasts (18.1% vs. 24.0%, p > 0.05). The difference in the quantity of clonal cells in iFISH between patients with >10% and those with 5–10% blasts also showed no statistically significance (p > 0.05).
−20/20q− (15.0%) −5/5q− (40.0%), −7/7q− (20.0%), +8 (16.0%)
452 (434b ) 511 57 Int-1 (1.0) → Int-2 (1.5) Int-1 (1.0) → Int-1 (1.0) High (2.5) → High (3.0) 10–1920–422–82 6.9–7230–4338–224 6.2–1100–231–3 046 (M/70) 048 (M/72) 065 (F/68)
RAEB-1 RAEB-1 RAEB-2
70–80 90–100 80–90
1592 1079 153 Int-1 (0.5) → Int-1 (1.0) Int-1 (0.5) → Int-2 (1.5) Int-2 (2.0) → Int-2 (2.0) 11.1–2390–1279–70 11.1–1640–1032–31 8.9–5730–1547–130 019 (F/44) 028 (M/20) 036 (F/77)
RA RA RAEB-1
20–30 20–30 80–90
6 1016 889 162 Int-2 (2.0) → High (3.0) Int-1 (0.5) → Int-1 (0.5) Int-1 (1.0) → Int-2 (1.5) High (3.0) → High (3.0)
No abnormality detected −20/20q− (27.5%) +8 (36.0%) −5/5q− (81.0%), −7/7q−(84.0%), +8 (5.3%) +8 (9.3%) −7/7q− (31.7%) −5/5q− (65.0%), −7/7q− (59.0%) +8 (11.0%) +8 (79.5%), +1/1q (5.0%) −5/5q (78.5%), +1/1q (35.5%)
47,XY,add(2)(p25),+mar[5]/47,idem,dup(1q)[25] 46,XY[20] 46,XX[20] 44,X,−5,i(8)(q10),der(8;9)(q10;q10),i(13)(q10),−16,+mar[3]/ 45,XY,idem[16]/46,XY[1] 46,XX[20] 46,XY[20] 45,XX,del(5)(q15q33),−7,der(12)t(7;12)(p11.2;p11.2),del(18) (q21)[18]/40–45,sl,del(7)(q22),−16,−17,−20,+1–2 mar[cp4] 46,XY[20] 47,XY,+8[20] 46,XX,del(5)(q13q33),inv(9)(p11q13),−13,der(18)t(13;18) (q14;q23),+ add(?22)(q?13),0–11dmin[5]/45–55,sl,+X,+1,+8,+11[cp7]/ 46,sl,add(16)(q?24)[4]/46,XX,inv(9)(p11q13)[4] 46,XY [20] 39–45,XY,+del(3)(q10),der(?5)t(?5;?12)(q?15;q13)?del(5(p?),− 7,−21[1]/39–42,X,sl,−Y,del(3)(p23),+add(9)(q?10),−12,−15,−17,− 22[13]/40–45,sl,add(19)(q13.1),+2mar[6]/46,XY[3] 50–60 NAa 20–30 NA 6.6–1990–1994–124 4.5–2830–2032–50 10.0–1170–246–85 8.0–9520–3903–57 003 (M/25) 010 (M/60) 016 (F/61) 017 (M/83)
RAEB-2 RARS RAEB-1 RAEB-2
Cellularity (%) Diagnosis Hb (g/dL)–WBC (/uL)–ANC (/uL)–PLT (×103 /L) Case no. (sex/age)
Table 2 Clinicopathological features, conventional cytogenetic analysis (CCA) results and interphase fluorescence in situ hybridization (iFISH) results of 12 patients with discrepancies between CCA and iFISH.
Overall survival (days) IPSS Group (Score) Either CCA or FISH → CCA & FISH iFISH Karyotype by CCA
M. Kim et al. / Leukemia Research 36 (2012) 163–168
Little is known about the clinico-biological significance of the quantity of clonal cells in MDS. We performed both qualitative and quantitative analyses of the results of CCA and iFISH in MDS, and investigated their clinical significance. Qualitative analysis of CCA and iFISH data revealed a 13.8% (12/87) incidence of discrepancies between CCA and iFISH. This incidence could not be compared with others since too few studies have been performed. Among those classified with normal karyotypes by CCA, 12.8% (6/47) showed clonal abnormalities in iFISH, and they showed cytopenia, dysplasia and/or increased BM blasts as typical MDS does. Differences in the sensitivities of the two methods might have caused the discrepancy. Except for 1 case (patient 017, −7/7q−, 84%), the sizes of clones detected only by iFISH were relatively small (5.3–36.0%). It is in the same context with the previous studies with normal karyotype MDS which showed that the proportion of cells with submicroscopic abnormalities detected only in iFISH reached at most 30% [1,2,11]. The low mitotic index of the abnormal clone could be an explanation for the discrepancy. The clone in MDS is not limited to the blast, but is also present in the maturing trilineage cells, therefore the dividing ability of the clone must be variable. Another explanation could be that only a subset of clone is involved in chromosomal abnormalities. The previous studies with normal karyotype MDS suggested the subset of clone with chromosomal abnormalities are related to the disease progression as the size of clone in iFISH correlated with BM blast%, and it increases as the disease progressed to acute leukemia [1,2,11]. Further study is required to clarify this issue since the correlation with BM blast% and the quantity of clonal cells in iFISH were poor in our study with MDS with normal and abnormal karyotype.
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Fig. 1. Correlation of the proportion of clonal cells between conventional cytogenetic analysis (CCA) and interphase fluorescence in situ hybridization (iFISH) in myelodysplastic syndromes. x, the proportion of clonal cells in iFISH; and y, the proportion of clonal cells in CCA. (a) −5/5q−; (b) −7/7q−; (c) +8; (d) −20/20q−; and (e) +1/1q.
The IPSS risk grouping was changed only in a subset of patients with discrepancies (33.3%, 4/12), due to the fact that the remaining patients already presented a complex karyotype in CCA prior to iFISH analysis. Thus, the statistical significance of IPSS risk grouping before and after considering iFISH results could not be evaluated. Nevertheless, the combination of CCA and iFISH is beneficial for predicting the outcome for some MDS patients as shown in case 003 and 046 who have shorter overall survival and leukemia free survival estimated by IPSS determined only either by CCA or by iFISH.
The data from the quantitative analysis of CCA and iFISH in the present study is summarized as follows: (i) the quantity of clonal cells in CCA and iFISH did not correlate well with each other (r, range 0.0761–1.0577); (ii) the percentage of BM blasts correlated with the quantity of clonal cells in CCA (p = 0.017) but not with the quantity of clonal cells in iFISH (p > 0.05); (iii) the high quantity of clonal cells in CCA was a poor prognostic factor in predicting the survival of MDS patients either independently (overall survival, p = 0.012) or in relation to BM blast% (leukemia-free survival). To the best of our knowledge, no study has reported these findings.
M. Kim et al. / Leukemia Research 36 (2012) 163–168
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Fig. 2. Prognostic significance of the proportion of clonal cells in conventional cytogenetic analysis (CCA) in myelodysplastic syndromes. (a) Correlation between the proportion of clonal cells in CCA and the percentage of bone marrow blasts; and (b) correlation between the proportion of clonal cells in CCA overall survival; (c) correlation between the proportion of clonal cells in CCA leukemia-free survival.
The differences we observed could be attributed to the different types of cells used for the 2 techniques, metaphase cells for CCA and interphase cells for iFISH. Cells that are viable and able to proliferate tend to form metaphase cells more easily than quiescent cells in CCA; thus, the high percentage of blasts might have affected the CCA results. In contrast, the results of iFISH might be less affected by the percentage of blasts since the technique is performed on nondividing, interphase cells. It has been suggested that the increase in the size of abnormal clones, which was observed by iFISH, happens in all patients undergoing leukemic transformation [1]. However, Li et al. showed that almost all cases of MDS had higher clonal cells than blast quantity, and that some RA patients showed more than 90% clonal cells [15]. Our unpublished observations are concordant with the latter. It has been demonstrated that the presence of clonal cells is a poor prognostic factor in MDS [1,2]. Bernasconi et al. [2] and
Rigolin et al. [1] showed that MDS patients with a normal karyotype in CCA but with submicroscopic abnormalities in FISH had either a short disease progression-free survival or overall survival. The identity of the chromosomes involved in the clonal change was neither considered by these 2 studies nor in our study. A notable finding of our study is that a high quantity of clonal cells in CCA was an independent prognostic factor in predicting a short overall survival (p = 0.012), and it affects leukemia-free survival as well, in relation to BM blast%. Our study confirmed that CCA and iFISH analysis are complementary to each other by detecting cytogenetic abnormalities overlooked in either method. Quantitative analyses of CCA and iFISH results showed that the quantity of clonal cells in CCA and in iFISH do not correlate with each other, and that the high quantity of clonal cells in CCA could be used as a surrogate marker in predicting survival in MDS patients.
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Acknowledgements This work was supported in part by (i) the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of Education, Science, and Technology (20100020584). Tae Young Kim and Bora Oh are grateful for being awarded a BK21 fellowship, (ii) a grant (10172KFDA993) from Korea Food & Drug Administration in 2010, and (iii) Basic Science Research Program through the National Research Foundation of Korea (NRF) Funded by the Ministry of Education, Science and Technology (2010-0013651). Contributions. M.K. wrote the draft, M.K. and S.C. analyzed data, C.J.S. performed the cytogenetic analysis, S.S.Y. and B.K.K. clinically treated the MDS patients, H.K.K. and D.S.L. diagnosed the MDS patients, and D.S.L. designed the study, supervised the cytogenetic analysis, and reviewed the draft. Conflict of interest statement. Authors do not have any conflicts of interest to disclose. References [1] Rigolin GM, Bigoni R, Milani R, Cavazzini F, Roberti MG, Bardi A, et al. Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype. Leukemia 2001;15(December (12)):1841–7. [2] Bernasconi P, Cavigliano PM, Boni M, Calatroni S, Klersy C, Giardini I, et al. Is FISH a relevant prognostic tool in myelodysplastic syndromes with a normal chromosome pattern on conventional cytogenetics? A study on 57 patients. Leukemia 2003;17(11):2107–12. Nov. [3] Malcovati L, Nimer SD. Myelodysplastic syndromes: diagnosis and staging. Cancer Control 2008;October (15 Suppl.):4–13.
[4] Bennett JM. A comparative review of classification systems in myelodysplastic syndromes (MDS). Semin Oncol 2005;32(August (4 Suppl. 5)):S3–10. [5] Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89(March (6)):2079–88. [6] Morel P, Hebbar M, Lai JL, Duhamel A, Preudhomme C, Wattel E, et al. Cytogenetic analysis has strong independent prognostic value in de novo myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 cases. Leukemia 1993;7(September (9)):1315–23. [7] Pozdnyakova O, Miron PM, Tang G, Walter O, Raza A, Woda B, et al. Cytogenetic abnormalities in a series of 1,029 patients with primary myelodysplastic syndromes: a report from the US with a focus on some undefined single chromosomal abnormalities. Cancer 2008;113(December (12)):3331–40. [8] Detlef, Haase. Cytogenetic features in myelodysplastic syndromes. Ann Hematol 2008;87(July (7)):5157. [9] Haase D, Germing U, Schanz J, Pfeilstöcker M, Nösslinger T, Hildebrandt B, et al. New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 2007;110(December (13)):4385–95. [10] Romeo M, Chauffaille Mde L, Silva MR, Bahia DM, Kerbauy J. Comparison of cytogenetics with FISH in 40 myelodysplastic syndrome patients. Leuk Res 2002;26(November (11)):993–6. [11] Cherry AM, Brockman SR, Paternoster SF, Hicks GA, Neuberg D, Higgins RR, et al. Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study. Leuk Res 2003;27(December (12)):1085–90. [12] Yilmaz Z, Sahin FI, Kizilkilic E, Karakus S, Boga C, Ozdogu H. Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients. Clin Exp Med 2005;5(July (2)):55–9. [13] Slovak ML, Dewald GW. International Working Group on MDS cytogenetics: October 2007 meeting report. Leuk Res 2008;32(September (9)):1329–32. [14] Shaffer LG, Tommerup N, editors. ISCN 2005: an international system for human cytogenetic nomenclature. Basel: S. Karger; 2005, 2005. [15] Li X, Wu L, Ying S, Chang C, He Q, Pu Q. Differentiation and hematopoieticsupport of clonal cells in myelodysplastic syndromes. Leuk Lymphoma 2007;48(7):1353–71. Jul.
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Quantity of clonal cells detected by conventional cytogenetic analysis correlates with bone marrow blasts and survival in myelodysplastic syndromes Miyoung Kim a , Soie Chung a , Cha Ja See a , Sung-Soo Yoon b , Byoung Kook Kim b , Hyun Kyung Kim a , Dong Soon Lee a,c,d,∗ a
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, South Korea Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea c Department of Tumor Biology, Seoul National University College of Medicine, Seoul, South Korea d Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea b
a r t i c l e
i n f o
Article history: Received 7 April 2011 Received in revised form 22 August 2011 Accepted 23 August 2011 Available online 13 September 2011 Keywords: Quantity Clonality Conventional cytogenetic analysis FISH MDS
a b s t r a c t We performed quantitative and qualitative analyses of conventional cytogenetic analysis and interphase FISH results in 87 MDS patients. The quantity of clonal cells for each chromosome of CCA did not correlate with the result of iFISH (r, range 0.0761–1.0577). The clonal cell percentage in CCA was higher in patients with >5% bone marrow blasts than those with <5% (44.7% vs. 23.1%, p = 0.017). Multivariate analysis showed that a high quantity of clonal cells in CCA analysis is an independent prognostic factor for overall survival in MDS (p = 0.012). © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In practice, chromosomal abnormalities not only confirm the clonality of a disease but also predict the likelihood of progression into acute leukemia and survival in myelodysplastic syndrome (MDS) [1–6]. The presence of chromosomal abnormalities has turned out to be one of the most important prognostic factors for MDS and has been incorporated into the International Prognostic Scoring System (IPSS), along with the percentage of bone marrow blasts and the number of lineages involved in cytopenia [1–8]. Isolated −Y, −5/5q−, −20q, and normal karyotypes imply a good prognosis, while −7/7q− or complex abnormalities (abnormalities in more than 3 types of chromosomes) suggest a poor prognosis [5,6,8]. Recent studies by the German–Austrian MDS Study Group and the Spanish Group have analyzed the prognostic significance of uncommon abnormalities, including 1q gain, trisomy 8, and 12p− [8,9]. Conventional cytogenetic analysis (CCA) and fluorescence in situ hybridization (FISH) are the most commonly used techniques to detect clonal changes in MDS [1,2,10–12]. FISH, a molecular cyto-
genetic method, has several advantages over CCA. FISH can detect submicroscopic abnormalities owing to its sensitivity [12]. Both CCA and FISH can provide information about clone size since they can quantitate the clonal cells [12]. However, in contrast to CCA, interphase FISH (iFISH) does not require viable cells and can be carried out with non-dividing cells. Nevertheless, CCA is the standard technique recommended by the IPSS algorithm, as it enables the detection of complex abnormalities that could be missed in FISH [13]. Most studies have investigated clonal chromosomal abnormalities in MDS by focusing on the relationship between the presence or absence of an abnormality of a certain chromosome and the prognosis of MDS [1,2,8–12], but not on the quantity of leukemic cells burdened with CCA. Little is known about the utility of the quantitative results of CCA and iFISH in clinical practice. We investigated the roles of CCA and FISH results in predicting the outcome of MDS patients. The results of CCA and FISH were compared both in qualitative and quantitative aspects. We analyzed the prognostic significance of the quantity of clonal cells in CCA or FISH in MDS. 2. Materials and methods
∗ Corresponding author at: Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehang-ro, Jongno-gu, Seoul 110-744, South Korea. Tel.: +82 2 2072 3986; fax: +82 2 747 0359. E-mail address: [email protected] (D.S. Lee). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.08.021
2.1. Patients Data for the study was accumulated from 129 newly diagnosed, de novo MDS patients of Seoul National University Hospital between April 2000 and March 2010. Due to suboptimal number of metaphase cells (less than 20), only 87 patients (54
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Table 1 The incidence of clonal chromosomal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ in conventional cytogenetic analysis (CCA) and interphase fluorescence in situ hybridization (iFISH) in myelodysplastic syndromes.
CCA+ CCA+, iFISH− iFISH+ CCA−, iFISH+ CCA+ and/or iFISH+
−5/5q−
−7/7q−
+8
−20/20q−
+1/1q
9 (10.3%) – 9 (10.3%) – 9 (10.3%)
7 (8.0%) – 9 (10.3%) 2 (2.3%) 9 (10.3%)
10 (11.5%) 1 (1.1%) 14 (16.1%) 5 (5.7%) 15 (17.2%)
5 (5.7%) 1 (1.1%) 6 (6.9%) 2 (2.3%) 7 (8.0%)
9 (10.3%) 1 (1.1%) 9 (10.3%) 1 (1.1%) 10 (11.5%)
men and 33 women) were included from the analysis. The patients’ age was between 19.6 and 83.0 years (mean = 57.6 years). The patient group included 25 cases of refractory anemia (RA), 5 cases of refractory anemia with ringed sideroblasts (RARS), 11 cases of refractory cytopenia with multilineage dysplasia (RCMD), 24 cases of refractory anemia with excess blasts-1 (RAEB-1), 19 cases of refractory anemia with excess blasts-2 (RAEB-2), and 3 cases of unclassifiable MDS (MDS-U), according to the WHO 2001 classification. Data concerning patient deaths and acute myeloid leukemia (AML) progression, obtained in May 2010, showed that 15 had progressed to AML, 21 died, and 66 survived. This study was approved by the Institutional Research Board of the Seoul National University Hospital (IRB No. H-1005-001-316). 2.2. Conventional cytogenetic analysis Cytogenetic studies using standard techniques were performed as a part of the diagnostic work-up. At least 20 metaphases were analyzed, whenever possible. Clonal abnormalities were defined as 2 or more cells with the same additional whole chromosome or chromosome rearrangements, or 3 or more cells with the same chromosome missing. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature 2005 [14].
found in 9 (10.3%), 9 (10.3%), 15 (17.2%), 7 (8.0%), and 10 (11.5%) of the subjects, respectively, by CCA and/or iFISH. The discrepancies between CCA and iFISH were observed in 12 (13.8%) patients, and their clinicopathological characteristics are detailed in Table 2. CCA detected clonal abnormalities of +8, −20/20q−, and +1/1q+ in 1 (1.1%), 1 (1.1%), and 1 (1.1%) patients who did not show those abnormalities in iFISH. The sizes of clones detected in CCA were variable (18.2–83.3%). iFISH detected submicroscopic clonal abnormalities of −7/7q−, +8, −20/20q−, and +1/1q+ in 2 (2.3%), 5 (5.7%), 2 (2.3%), and 1 (11%) patients, and the sizes of clones in iFISH ranged from 5.0 to 84.0%. Among them, 6 patients (010, 016, 019, 028, 046 and 067) showed normal karyotype in CCA (12.8% of 47 patients with normal karyotype). 3.2. Prognostic significance of qualitative results of conventional cytogenetic analysis and interphase fluorescence in situ hybridization
2.3. Interphase fluorescence in situ hybridization We used the iFISH technique to detect the abnormalities of chromosomes 5, 7, 8, 20, and 1 (−5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+). The following probes were used: LSI (locus-specific identifier) EGR1/D5S23, D5S721 Dual Color Probe for chromosome 5q; LSI D7S522/CEP 7 Probe for chromosome 7q; CEP (centromere enumeration probe) 8 DNA Probe for chromosome 8; LSI D20S108 Probe for chromosome 20q; and LSI p58 (1p36)/TelVysion 1p/LSI 1q25 Probe for chromosome 1; all probes were obtained from Vysis Inc. (Downers Grove, IL, USA). The normal cutoff values of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ based on the mean (3 SD) of 40 negative controls, were 2.0%, 2.0%, 2.0%, 2.0%, and 1.5%, respectively. No less than 200 nuclei of a sample were scored for deletion and rearrangement in each sample. Results were described according to the ISCN (2005) criteria [14]. 2.4. Statistical analysis All statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was applied for the comparison of categorical variables. Bivariate correlation analysis was used to evaluate the correlation between two continuous variables, and r (Pearson’s correlation coefficient) and r2 were considered in the evaluation. Mann–Whitney U-test was applied for the comparison of continuous variables. Survival curves were estimated using the Kaplan–Meier method. Log-rank tests were used to conduct univariate comparisons of overall survival and progression-free survival among subgroups. Multivariate analyses adjusted for significant prognostic factors were performed using Cox’s hazard regression model. Prognostic significance was evaluated in terms of 95% confidence intervals.
3. Results 3.1. Clonal abnormalities of chromosomes 5, 7, 8, 20 and 1 in conventional cytogenetic analysis and interphase fluorescence in situ hybridization Conventional cytogenetic analysis (CCA) detected clonal chromosomal abnormalities of any chromosomes in 40 (46.0%), and clonal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ in 31 (35.6%). Twelve (13.8%) had complex chromosomal abnormalities (≥3 abnormalities) in CCA. Interphase fluorescence in situ hybridization (iFISH) for −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ detected clonal chromosomal abnormalities in 36 (41.4%) patients. The incidence of clonal chromosomal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ observed in CCA and/or iFISH is summarized in Table 1. The clonal abnormalities were
Out of 12 with discrepancies between CCA and iFISH, the IPSS risk group changed in 1 case (8.3%: patient 003) from Int-2 to High due to the clonal abnormality detected only by CCA, and in 3 cases (25.1%: patient 016, 028, and 046) from Int-1 to Int-2 due to the submicroscopic clonal abnormalities detected by iFISH. In the total study population, the rate of evolution to AML, leukemia free survival and overall survival of each IPSS risk group are as follows: 0.0%, 654 days and 654 days in Low risk group; 10.2%, 617 days and 627 days in Int-1 risk group; 33.3% 382 days and 515 days in Int-2 risk group; 22.2%, 275 days and 300 days in High risk group. Patient 016 and 028 showed similar survival duration to Int-1 risk group, while patient 003 and 046 showed similar survival duration to High risk group and Int-2 group, respectively. Statistical comparison of survival could not be performed due to too small number of patients (4) who changed IPSS group. 3.3. Qualitative analysis of clonal chromosomal abnormality Clonal cells with chromosomal abnormalities were detected by CCA in 28.0%, 0.0%, 63.6%, 54.2%, 57.9% and 66.7% of the 25 RA, 5 RARS, 11 RCMD, 24 RAEB-1, 19 RAEB-2, and 3 MDS-U patients, respectively. The incidence was similar in iFISH analysis: the presence of any clonal abnormality of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ was detected in 32.0%, 20.0%, 45.5%, 45.8%, 42.1% and 66.7% of RA, RARS, RCMD, RAEB-1, RAEB-2 and MDS-U patients, respectively. There was no association between the incidence of clonal abnormality and the percentage of BM blasts, cytopenia, or MDS subtype (p > 0.05). 3.4. Quantitative analysis of clonal chromosomal abnormality When only the patients with clonal abnormalities detected by CCA and/or FISH are considered, the quantitative results of clonal cells detected in the 2 methods yielded a variable degree of agreement (poor to substantial, depending on the chromosomes involved): −5/5q−: r = 1.0577, r2 = 0.5022; −7/7q−: r = 0.0761, r2 = 0.0181; +8: r = 0.5164, r2 = 0.5179; −20/20q−: r = 0.6805, r2 = 0.8014; and +1/1q+: r = 0.2569, r2 = 0.1547 (Fig. 1). The
429 184 Int-1 (1.0) → Int-1 (1.0) Int-2 (2.0) → Int-2 (2.0)
3.5. Prognostic significance of quantitative results of conventional cytogenetic analysis and interphase fluorescence in situ hybridization In univariate analysis, the quantity of clonal cells in CCA > 50.0%, the presence of clonal cells in iFISH, the quantity of clonal cells in iFISH > 50.0%, isolated abnormalities of −5/5q− (detected by CCA and/or FISH), and the percentage of BM blasts >5% showed prognostic significance for overall survival (p < 0.05). In multivariate analysis, the quantity of clonal cells in CCA > 50.0% (p = 0.012) was the only independent prognostic factor for overall survival in this study population (Fig. 2). The following factors showed prognostic significance for the progression-free survival into AML cases in univariate analysis (p < 0.05): the quantity of clonal cells in CCA > 50.0%, the quantity of clonal cells in iFISH > 50.0%, isolated abnormalities of −5/5q− and +8, and the percentage of BM blasts >5%. In multivariate analysis, the percentage of BM blasts was the only independent prognostic factors for the progression-free survival into AML in this study group (p < 0.001). Individual cytogenetic abnormalities were not considered in the adjustment.
a
Not available due to inadequate quality of BM biopsy section. Leukemia free survival duration in the case evolved to AML.
4. Discussion
b
70–80 80–90 RAEB-1 RAEB-1 7.7–3170–1395–156 8.3–1980–530–19 067 (M/72) 082 (M/75)
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quantity of clonal cells from total metaphase cells in CCA was higher in the 43 patients with >5% BM blasts than in the 44 patients with <5% BM blasts in CCA (44.7% vs. 23.1%, p = 0.017; Fig. 2). The difference in the quantity of clonal cells in CCA between patients with >10% and those with 5–10% blasts was not statistically significant (p > 0.05). However, the quantity of clonal cells from the total interphase cells in iFISH was independent of the percentage of BM blasts: 44 patients had <5% BM blasts and 43 patients had >5% BM blasts (18.1% vs. 24.0%, p > 0.05). The difference in the quantity of clonal cells in iFISH between patients with >10% and those with 5–10% blasts also showed no statistically significance (p > 0.05).
−20/20q− (15.0%) −5/5q− (40.0%), −7/7q− (20.0%), +8 (16.0%)
452 (434b ) 511 57 Int-1 (1.0) → Int-2 (1.5) Int-1 (1.0) → Int-1 (1.0) High (2.5) → High (3.0) 10–1920–422–82 6.9–7230–4338–224 6.2–1100–231–3 046 (M/70) 048 (M/72) 065 (F/68)
RAEB-1 RAEB-1 RAEB-2
70–80 90–100 80–90
1592 1079 153 Int-1 (0.5) → Int-1 (1.0) Int-1 (0.5) → Int-2 (1.5) Int-2 (2.0) → Int-2 (2.0) 11.1–2390–1279–70 11.1–1640–1032–31 8.9–5730–1547–130 019 (F/44) 028 (M/20) 036 (F/77)
RA RA RAEB-1
20–30 20–30 80–90
6 1016 889 162 Int-2 (2.0) → High (3.0) Int-1 (0.5) → Int-1 (0.5) Int-1 (1.0) → Int-2 (1.5) High (3.0) → High (3.0)
No abnormality detected −20/20q− (27.5%) +8 (36.0%) −5/5q− (81.0%), −7/7q−(84.0%), +8 (5.3%) +8 (9.3%) −7/7q− (31.7%) −5/5q− (65.0%), −7/7q− (59.0%) +8 (11.0%) +8 (79.5%), +1/1q (5.0%) −5/5q (78.5%), +1/1q (35.5%)
47,XY,add(2)(p25),+mar[5]/47,idem,dup(1q)[25] 46,XY[20] 46,XX[20] 44,X,−5,i(8)(q10),der(8;9)(q10;q10),i(13)(q10),−16,+mar[3]/ 45,XY,idem[16]/46,XY[1] 46,XX[20] 46,XY[20] 45,XX,del(5)(q15q33),−7,der(12)t(7;12)(p11.2;p11.2),del(18) (q21)[18]/40–45,sl,del(7)(q22),−16,−17,−20,+1–2 mar[cp4] 46,XY[20] 47,XY,+8[20] 46,XX,del(5)(q13q33),inv(9)(p11q13),−13,der(18)t(13;18) (q14;q23),+ add(?22)(q?13),0–11dmin[5]/45–55,sl,+X,+1,+8,+11[cp7]/ 46,sl,add(16)(q?24)[4]/46,XX,inv(9)(p11q13)[4] 46,XY [20] 39–45,XY,+del(3)(q10),der(?5)t(?5;?12)(q?15;q13)?del(5(p?),− 7,−21[1]/39–42,X,sl,−Y,del(3)(p23),+add(9)(q?10),−12,−15,−17,− 22[13]/40–45,sl,add(19)(q13.1),+2mar[6]/46,XY[3] 50–60 NAa 20–30 NA 6.6–1990–1994–124 4.5–2830–2032–50 10.0–1170–246–85 8.0–9520–3903–57 003 (M/25) 010 (M/60) 016 (F/61) 017 (M/83)
RAEB-2 RARS RAEB-1 RAEB-2
Cellularity (%) Diagnosis Hb (g/dL)–WBC (/uL)–ANC (/uL)–PLT (×103 /L) Case no. (sex/age)
Table 2 Clinicopathological features, conventional cytogenetic analysis (CCA) results and interphase fluorescence in situ hybridization (iFISH) results of 12 patients with discrepancies between CCA and iFISH.
Overall survival (days) IPSS Group (Score) Either CCA or FISH → CCA & FISH iFISH Karyotype by CCA
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Little is known about the clinico-biological significance of the quantity of clonal cells in MDS. We performed both qualitative and quantitative analyses of the results of CCA and iFISH in MDS, and investigated their clinical significance. Qualitative analysis of CCA and iFISH data revealed a 13.8% (12/87) incidence of discrepancies between CCA and iFISH. This incidence could not be compared with others since too few studies have been performed. Among those classified with normal karyotypes by CCA, 12.8% (6/47) showed clonal abnormalities in iFISH, and they showed cytopenia, dysplasia and/or increased BM blasts as typical MDS does. Differences in the sensitivities of the two methods might have caused the discrepancy. Except for 1 case (patient 017, −7/7q−, 84%), the sizes of clones detected only by iFISH were relatively small (5.3–36.0%). It is in the same context with the previous studies with normal karyotype MDS which showed that the proportion of cells with submicroscopic abnormalities detected only in iFISH reached at most 30% [1,2,11]. The low mitotic index of the abnormal clone could be an explanation for the discrepancy. The clone in MDS is not limited to the blast, but is also present in the maturing trilineage cells, therefore the dividing ability of the clone must be variable. Another explanation could be that only a subset of clone is involved in chromosomal abnormalities. The previous studies with normal karyotype MDS suggested the subset of clone with chromosomal abnormalities are related to the disease progression as the size of clone in iFISH correlated with BM blast%, and it increases as the disease progressed to acute leukemia [1,2,11]. Further study is required to clarify this issue since the correlation with BM blast% and the quantity of clonal cells in iFISH were poor in our study with MDS with normal and abnormal karyotype.
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Fig. 1. Correlation of the proportion of clonal cells between conventional cytogenetic analysis (CCA) and interphase fluorescence in situ hybridization (iFISH) in myelodysplastic syndromes. x, the proportion of clonal cells in iFISH; and y, the proportion of clonal cells in CCA. (a) −5/5q−; (b) −7/7q−; (c) +8; (d) −20/20q−; and (e) +1/1q.
The IPSS risk grouping was changed only in a subset of patients with discrepancies (33.3%, 4/12), due to the fact that the remaining patients already presented a complex karyotype in CCA prior to iFISH analysis. Thus, the statistical significance of IPSS risk grouping before and after considering iFISH results could not be evaluated. Nevertheless, the combination of CCA and iFISH is beneficial for predicting the outcome for some MDS patients as shown in case 003 and 046 who have shorter overall survival and leukemia free survival estimated by IPSS determined only either by CCA or by iFISH.
The data from the quantitative analysis of CCA and iFISH in the present study is summarized as follows: (i) the quantity of clonal cells in CCA and iFISH did not correlate well with each other (r, range 0.0761–1.0577); (ii) the percentage of BM blasts correlated with the quantity of clonal cells in CCA (p = 0.017) but not with the quantity of clonal cells in iFISH (p > 0.05); (iii) the high quantity of clonal cells in CCA was a poor prognostic factor in predicting the survival of MDS patients either independently (overall survival, p = 0.012) or in relation to BM blast% (leukemia-free survival). To the best of our knowledge, no study has reported these findings.
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Fig. 2. Prognostic significance of the proportion of clonal cells in conventional cytogenetic analysis (CCA) in myelodysplastic syndromes. (a) Correlation between the proportion of clonal cells in CCA and the percentage of bone marrow blasts; and (b) correlation between the proportion of clonal cells in CCA overall survival; (c) correlation between the proportion of clonal cells in CCA leukemia-free survival.
The differences we observed could be attributed to the different types of cells used for the 2 techniques, metaphase cells for CCA and interphase cells for iFISH. Cells that are viable and able to proliferate tend to form metaphase cells more easily than quiescent cells in CCA; thus, the high percentage of blasts might have affected the CCA results. In contrast, the results of iFISH might be less affected by the percentage of blasts since the technique is performed on nondividing, interphase cells. It has been suggested that the increase in the size of abnormal clones, which was observed by iFISH, happens in all patients undergoing leukemic transformation [1]. However, Li et al. showed that almost all cases of MDS had higher clonal cells than blast quantity, and that some RA patients showed more than 90% clonal cells [15]. Our unpublished observations are concordant with the latter. It has been demonstrated that the presence of clonal cells is a poor prognostic factor in MDS [1,2]. Bernasconi et al. [2] and
Rigolin et al. [1] showed that MDS patients with a normal karyotype in CCA but with submicroscopic abnormalities in FISH had either a short disease progression-free survival or overall survival. The identity of the chromosomes involved in the clonal change was neither considered by these 2 studies nor in our study. A notable finding of our study is that a high quantity of clonal cells in CCA was an independent prognostic factor in predicting a short overall survival (p = 0.012), and it affects leukemia-free survival as well, in relation to BM blast%. Our study confirmed that CCA and iFISH analysis are complementary to each other by detecting cytogenetic abnormalities overlooked in either method. Quantitative analyses of CCA and iFISH results showed that the quantity of clonal cells in CCA and in iFISH do not correlate with each other, and that the high quantity of clonal cells in CCA could be used as a surrogate marker in predicting survival in MDS patients.
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Acknowledgements This work was supported in part by (i) the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of Education, Science, and Technology (20100020584). Tae Young Kim and Bora Oh are grateful for being awarded a BK21 fellowship, (ii) a grant (10172KFDA993) from Korea Food & Drug Administration in 2010, and (iii) Basic Science Research Program through the National Research Foundation of Korea (NRF) Funded by the Ministry of Education, Science and Technology (2010-0013651). Contributions. M.K. wrote the draft, M.K. and S.C. analyzed data, C.J.S. performed the cytogenetic analysis, S.S.Y. and B.K.K. clinically treated the MDS patients, H.K.K. and D.S.L. diagnosed the MDS patients, and D.S.L. designed the study, supervised the cytogenetic analysis, and reviewed the draft. Conflict of interest statement. Authors do not have any conflicts of interest to disclose. References [1] Rigolin GM, Bigoni R, Milani R, Cavazzini F, Roberti MG, Bardi A, et al. Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype. Leukemia 2001;15(December (12)):1841–7. [2] Bernasconi P, Cavigliano PM, Boni M, Calatroni S, Klersy C, Giardini I, et al. Is FISH a relevant prognostic tool in myelodysplastic syndromes with a normal chromosome pattern on conventional cytogenetics? A study on 57 patients. Leukemia 2003;17(11):2107–12. Nov. [3] Malcovati L, Nimer SD. Myelodysplastic syndromes: diagnosis and staging. Cancer Control 2008;October (15 Suppl.):4–13.
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