- Email: [email protected]
Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype
Leukemia Research 36 (2012) 51–58
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype Torsten Haferlach a,∗ , Ulrike Bacher b , Tamara Alpermann a , Claudia Haferlach a , Wolfgang Kern a , Susanne Schnittger a a b
MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany Department for Stem Cell Transplantation, University Cancer Center Hamburg, Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 9 March 2011 Received in revised form 26 April 2011 Accepted 28 April 2011 Available online 31 May 2011 Keywords: NPM1 mutation Founder mutation FLT3-ITD Blast percentages Normal karyotype AML
a b s t r a c t FLT3-ITDs are linked to higher leukocytes/blasts in acute myeloid leukemia. To evaluate the effect of NPM1 mutations, we correlated NPM1mut status with morphology in 805 adult normal karyotype AML. NPM1mut were found in 391/805 (48.6%), FLT3-ITD in 219/805 (27.2%). Frequencies of FLT3-ITD and NPM1mut cases were continuously increasing by blast decades: NPM1mut from 38/123 (30.9%) in 20–29% blast decade to 70/103 (68.0%) in 90–100% decade (p < 0.001), FLT3-ITDs from 15/123 (12.2%) to 58/103 (56.3%) (p < 0.001). Mean WBC count was highest in NPM1-mut/FLT3-ITD-positive and lowest in NPM1-wildtype/FLT3-ITD-negative patients (p < 0.0001); similar for BM blasts. Therefore, FLT3-ITD and NPM1mut might synergistically stimulate blast proliferation. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Internal tandem duplications of the FLT3 gene (FLT3-ITD) can be found in 35–45% of acute myeloid leukemia (AML) with a normal karyotype (nk-AML) and are prognostically adverse. The FLT3-ITDs result in increased autophosphorylation of the FLT3 receptor tyrosine kinase and thereby increase cell proliferation. Occurrence of the FLT3-ITD has been strongly associated with higher peripheral leukocytes and higher blast percentages [1–4]. Mutations of the nucleophosmin (NPM1) gene are found in 45–55% of nk-AML cases. In NPM1 mutant AML, the nucleophosmin protein (a chaperone protein) is aberrantly localized in the cytoplasm [5]. The NPM1 mutations are supposed to interfere with the ARF14 tumor suppressor pathway. In 40–45% of NPM1 mutated nk-AML also concomitant FLT3-ITDs were identified. Coincidence of the NPM1 mutations with the FLT3-ITD results in less favorable outcomes when compared to isolated occurrence of the NPM1 mutation [6–9]. Some authors mentioned higher peripheral leukocytes and marrow blasts also for NPM1 mutant AML [6,8]. Based on these observations, and to further study the impact of these mutations on blast proliferation in AML, we performed correlations of the frequency of NPM1 mutations and FLT3-ITD with cytomorphologic and other parameters in 805 adult patients
∗ Corresponding author. Tel.: +49 89 990 17 100; fax: +49 89 990 17 109. E-mail address: [email protected] (T. Haferlach). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.04.026
newly diagnosed with de novo nk-AML. We aimed to further clarify pathways and input of these genes for AML biology. Additionally, we included the prognostically adverse MLL-PTDs (partial tandem duplications of the MLL gene) [10], the FLT3-TKD mutations (involving the tyrosine kinase domain of the FLT3 gene) [11], IDH1 and IDH2 mutations (isocitrate dehydrogenase, conferring an adverse prognostic impact in FLT3-ITD negative AML) [12–14], and the prognostically favorable CEBPA mutations (in case of biallelic occurrence) [15] in the analysis, since these mutations are as well strongly associated to normal karyotype AML and have clinical relevance.
2. Patients The study cohort consisted of consecutive 805 patients (410 males, 395 females; median, 66.6 years; range, 20.0–93.3 years) who all had de novo nk-AML at diagnosis. Samples had been sent from different hematologic centers to the MLL Munich Leukemia Laboratory between 8/2005 and 6/2010. Prerequisite for inclusion in the study was a normal karyotype and the availability of bone marrow (BM) cytomorphology and analysis for the FLT3-ITD and the NPM1 mutation in parallel. Patients gave written informed consent for the use of laboratory data for research studies. The study was approved by the Bavarian Medical Association (“Bayerische Landesärztekammer”) and performed according to the Declaration of Helsinki.
52
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
3. Methods 3.1. Cytomorphology All cases were investigated by BM cytomorphology (May Gruenwald Giemsa staining combined with myeloperoxidase, MPO, and non-specific esterase, NSE, for cytochemistry). Cells were categorized as blasts following the guidelines of an International Working Group on Morphology published by Mufti et al. [16]. AML was classified according to FAB [17] and WHO [18] criteria. For correlation with molecular mutation analysis, cases were subdivided according to decades of marrow blasts (20–29%; 30–39%; → 80–89%, 90–100%). 3.2. Cytogenetics Chromosome banding analysis was done according to previously described standard procedures [19]. All patients selected for this study had normal karyotypes. 3.3. Multiparameter flow cytometry Immunophenotyping with multiparameter flow cytometry (MFC) was done by five color staining in 427 cases [20].
Table 1 Biologic parameters and FAB subtypes in 805 patients with normal karyotype AML. Parameter
Number of patients
Total cohort Biologic parameters Males Females Median age (range), years Laboratory parameters Mean WBC count × 109 /l (range) Mean bone marrow blasts (%) Mean thrombocyte count × 109 /l (range) Mean haemoglobin level, g/dl (range) FAB subtypes (n = 797) M0 M1 M2 M4 M5 M6 M7 Molecular markers NPM1 mutation FLT3-ITD MLL-PTD Biallelic CEBPA mutation FLT3-TKD IDH1/IDH2 mutation
805 410 395 66.6 (20.0–93.3) 37.7 (0.1–600.0) 60.6 (20.0–99.0) 97 (3–765) 9.4 (3.0–16.0) 27 (3.4%) 255 (32.0%) 312 (39.1%) 168 (21.1%) 21 (2.6%) 12 (1.5%) 2 (0.3%) 391/805 (48.6%) 219/805 (27.2%) 76/805 (9.4%) 30/620 (4.8%) 46/578 (8.0%) 101/312 (32.4%)
3.4. Molecular genetics Following isolation of mononucleated cells, mRNA extraction, and random primed cDNA synthesis, analysis for NPM1 mutations by LightCycler® based melting curve analysis was performed as described before [7,21]. Determination of the FLT3-ITD mutation status and of the mutation length and quantification of the FLT3ITD mutation load were realized by fragment analysis (GeneScan, 3130 sequence detection system, ABI) [3]. The FLT3-ITD load was defined as the ratio of the mutated compared to the FLT3 wildtype (wt) allele. Ratios ≥1 were indicative of loss or partial loss of the wt allele. Analysis for the MLL-PTD by real-time PCR [22] was done in all 805 cases. Screening for FLT3-TKD mutation (n = 578) [11], mutations of IDH1 and IDH2 (n = 312) [14], and CEBPA genes (n = 620) [23] was performed in different numbers of cases. Only patients with biallelic CEBPA mutations were included in the analysis since recent reports suggest that only this double mutated status confers a favorable prognostic impact in AML [15,24]. 3.5. Statistical analysis Overall survival (OS) and event free survival (EFS) were calculated according to Kaplan–Meier. Survival data was compared by two sided log rank test. Cox regression was performed for OS and EFS with different parameters as covariates. Parameters which were significant in univariate analysis were included into multivariate analysis. Dichotomous variables were compared between different subgroups using the 2 -test and continuous variables by Student’s t-test. Spearman rank correlation was used to analyze correlations between continuous parameters. SPSS (version 14.0.1) software (SPSS, Chicago, IL) was used for statistical analysis. 4. Results
AML. In the total cohort, mean WBC count was 37.7 × 109 /l (range, 0.1–600.0) and BM blasts were 60.6% (20.0–99.9%). 4.2. Molecular mutation rates and survival 4.2.1. NPM1 mutations and FLT3-ITDs In the total cohort, the mutation rate for NPM1 mutations (NPM1-mut) was 391/805 (48.6%) and for FLT3-ITD 219/805 (27.2%). Considering combinations of both markers, subgroups were distributed as follows: NPM1-mut/FLT3-ITD-pos.: n = 151 (18.8% of all cases); NPM1-wt/FLT3-ITD-positive: n = 68 (8.4%); NPM1-mut/FLT3ITD-negative: n = 240 (29.8%); NPM1-wt/FLT3-ITD-negative: n = 346 (43.0%). The 2-year OS rate was best in the NPM1-mut/FLT3ITD-negative patients (82.3%) when compared to all other subgroups (NPM1-wt/FLT3-ITD-neg.: 62.1%; NPM1-mut/FLT3-ITDpositive: 55.2%; NPM1-wt/FLT3-ITD-positive: 40.5%; p = 0.003). 4.2.2. Additional molecular markers IDH1 or IDH2 gene mutations were detected in 101/312 cases (32.4%). MLL-PTDs were found in 76/805 (9.4%), FLT3-TKDs in 46/578 (8.0%), and biallelic CEBPA mutations in 30/620 (4.8%). Considering marker composition with respect to the NPM1-mut and FLT3-ITDs, NPM1-mutated patients showed frequent coincidence with the IDH1 or IDH2-mut (56 IDH1 or IDH2-mut/174 NPM1-mut; 32.2%) and the FLT3-TKD (36 FLT3-TKD positive/313 NPM1-mut; 11.5%). The NPM1 mutations were not seen in combination with biallelic CEBPA mutations and the MLL-PTD. Regarding the FLT3ITD, IDH1/IDH2-mut cases were observed in 27/82 FLT3-ITD positive cases (32.9%), MLL-PTDs in 23/219 FLT3-ITD-positive cases (10.5%). Biallelic CEBPA-mut and FLT3-TKD-positive cases were rarely combined with the FLT3-ITDs. The different marker combinations are given in detail in the Supplemental Table S1 (see also Supplemental Figure S1).
4.1. Biological parameters and FAB subtypes 4.3. NPM1 mutations and FLT3-ITD Biological parameters and FAB subtypes are shown in Table 1. Median age of patients (66.6 years; range, 20.0–93.3) was corresponding to overall AML. The distribution of FAB subtypes (with a preponderance of M1, M2, and M4 subtypes) confirmed the expected pattern of morphologic subtypes in normal karyotype
4.3.1. Molecular mutation frequencies in the different blast decades Frequencies of both FLT3-ITD and NPM1 mutated cases, respectively, were continuously increasing by blast percentages from 20%
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
53
Table 2 Frequencies of the different molecular mutations in the different marrow blast categories given as decades. The p-value shows the comparison of the mutation rates in the different blast categories by chi square (* percentages of patients in relation to the total cohort). BM blasts
NPM1mut
FLT3-ITD
MLL-PTD
Biallelic CEBPAmut
FLT3-TKD
IDH1/IDH2mut
20–29% 30–39% 40–49% 50–59% 60–69% 70–79% 80–89% 90–100%
Number of patients 123 (15.3%)* 91 (11.3%) 69 (8.6%) 80 (9.9%) 102 (12.7%) 114 (14.2%) 123 (15.3%) 103 (12.8%)
38/123 (30.9%) 31/91 (34.1%) 20/69 (29.0%) 36/80 (45.0%) 51/102 (50.0%) 62/114 (54.4%) 83/123 (67.5%) 70/103 (68.0%)
15/123 (12.2%) 7/91 (7.7%) 11/69 (15.9%) 14/80 (17.5%) 25/102 (24.5%) 34/114 (29.8%) 55/123 (44.7%) 58/103 (56.3%)
10/123 (8.1%) 12/91 (13.2%) 12/69 (17.4%) 7/80 (8.8%) 6/102 (5.9%) 13/114 (11.4%) 8/123 (6.5%) 8/103 (7.8%)
0/97 (0.0%) 2/73 (2.7%) 2/56 (3.6%) 3/63 (4.8%) 10/75 (13.3%) 5/94 (5.3%) 5/86 (5.8%) 3/76 (3.9%)
2/75 (2.7%) 6/57 (10.5%) 3/48 (6.3%) 1/55 (1.8%) 3/73 (4.1%) 12/92 (13.0%) 11/92 (12.0%) 8/86 (9.3%)
8/42 (19.0%) 12/37 (32.4%) 4/20 (20.0%) 9/36 (25.0%) 19/52 (36.5%) 18/37 (48.6%) 16/50 (32.0%) 15/38 (39.5%)
Total
805 (100.0%)
391/805 (48.6%)
219/805 (27.2%)
76/805 (9.4%)
30/620 (4.8%)
46/578 (8.0%)
101/312 (32.4%)
<0.001
<0.001
n.s.
0.012
n.s.
n.s.
p
–
Fig. 1. Frequencies of the different mutations according to blast decades in the patients with normal karyotype AML (n = 805). The x-axis shows the blast decades; the y-axis demonstrates the percentages of cases carrying the respective mutations.
to 100%: The NPM1 mutations were detected in 38/123 (30.9%) of the 20–29% blast decade and in 31/91 (34.1%) of the 30–39% decade, increasing to 83/123 (67.5%) in the 80–89% decade and 70/103 (68.0%) in the 90–100% decade (p < 0.001). FLT3-ITDs were detected
in 15/123 (12.2%) of the 20–29% decade and in 7/91 (7.7%) of the 30–39% decade and were steadily increasing to 55/123 (44.7%) in the 80–89% decade and 58/103 (56.3%) in the 90–100% decade (p < 0.001) (Table 2, Fig. 1).
Table 3 Mean WBC count and mean marrow blasts depending on marker composition and the FLT3-ITD/wt mutant ratio (pos.: positive; neg.: negative; mut: mutated; wt: wildtype; * The p-values show comparison of the respective subgroup with all other subgroups by Student’s t-test. ** Ratio of FLT3-ITD mutated and wildtype alleles). Marker composition
Number of patients
NPM1-mut/FLT3-ITD-pos. 151/805 (18.8%) 240/805 (29.8%) NPM1-mut/FLT3-ITD-neg. 68/805 (8.4%) NPM1-wt/FLT3-ITD-pos. 346/805 (43.0%) NPM1-wt/FLT3-ITD-neg. FLT3-ITD/wt ratio** (FLT3-ITD-positive patients) 52/219 (23.7%) ≤0.25 45/219 (20.5%) >0.25 ≤ 0.5 39/219 (17.8%) >0.5 ≤ 0.75 38/219 (17.4%) >0.75 ≤ 1.0 45/219 (20.5%) >1.0
Mean WBC count, ×109 /l
p*
Mean bone marrow blasts, %
p*
76.4 38.6 50.9 16.7
<0.0001 n.s. n.s. <0.0001
75.3 62.5 70.3 50.9
<0.0001 n.s. <0.0001 <0.0001
48.9 71.1 85.8 50.8 87.3
0.053 n.s. n.s. n.s. n.s.
66.3 74.9 76.6 73.7 78.5
0.009 n.s. n.s. n.s. 0.043
54
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
Table 4 Proportions of cases with the different BM blast decades depending on the molecular subgroup (pos.: positive; neg.: negative; mut: mutated; wt: wildtype; n: number). Blast percentages
NPM1-mut/FLT3-ITDpos. (n = 151)
NPM1-wt/FLT3-ITDpos. (n = 68)
NPM1-mut/FLT3-ITDneg. (n = 240)
NPM1-wt/FLT3-ITDneg. (n = 346)
20–29% 30–39% 40–49% 50–59% 60–69% 70–79% 80–89% 90–100% p (comparison of all subgroups* )
9 (6.0%) 4 (2.6%) 7 (4.6%) 8 (5.3%) 18 (11.9%) 20 (13.2%) 40 (26.5%) 45 (29.8%) <0.001
6 (8.8%) 3 (4.4%) 4 (5.9%) 6 (8.8%) 7 (10.3%) 14 (20.6%) 15 (22.1%) 13 (19.1%) 0.074
29 (12.1%) 27 (11.3%) 13 (5.4%) 28 (11.7%) 33 (13.8%) 42 (17.5%) 43 (17.9%) 25 (10.4%) 0.064
79 (22.8%) 57 (16.5%) 45 (13.0%) 38 (11.0%) 44 (12.7%) 38 (11.0%) 25 (7.2%) 20 (5.8%) <0.001
*
chi square.
lower mean marrow blasts compared to those with ratios >0.25 (p = 0.009) (Table 3). 4.3.4. Marrow blast levels in the different molecular genetic subgroups Focusing on the marrow blast levels in the different molecular subgroups, the NPM1-mut/FLT3-ITD positive (double mutated) subgroup showed the highest proportion of cases with 90–100% blasts (45 of 151 cases; 29.8%), while low proportions of blasts were infrequent: only 9 and 4 cases (6.0% and 2.6%) from this molecular subgroup had 20–29% and 30–39% of blasts, respectively (p < 0.001). The NPM1-wt/FLT3-ITD-positive and NPM1-mut/FLT3-ITD negative subgroups peaked in the 80–89% blasts decade (15/68; 22.1%; and 43/240; 17.9%; respectively; n.s.). In contrast, the NPM1-wt/FLT3ITD-negative patients most frequently had low blast percentages of 20–29% (79/346; 22.8%), while ≥80% of blasts rarely were seen (p < 0.001) (Table 4, Fig. 3). 4.4. Additional molecular markers
Fig. 2. Mean WBC count and marrow blasts depending on NPM1-mut/FLT3-ITD composition (NPM1+: NPM1 mutated; NPM1−: NPM1 wildtype; FLT3+: FLT3-ITDpositive; and FLT3−: FLT3-ITD-negative.
4.3.2. WBC count and marrow blasts depend on NPM1-mut/FLT-ITD composition Subsequently, we compared mean WBC counts and mean marrow blasts in the different molecular subgroups considering the NPM1 mutations and the FLT3-ITD in combination: Mean WBC count was highest in the NPM1-mut/FLT3-ITD-positive subgroup followed by the NPM1-wt/FLT3-ITD-positive and the NPM1-mut/FLT3-ITD-negative subgroups, with the lowest values in the NPM1-wt/FLT3-ITD-negative patients (76.4 vs 50.9 vs 38.6 vs 16.7 × 109 /l; p < 0.0001). Mean BM blasts were also highest in the NPM1-mut/FLT3-ITD-positive subgroup with 75.3% (NPM1-wt/FLT3-ITD-pos.: 70.3%; NPM1-mut/FLT3-ITD-neg.: 62.5%; NPM1-wt/FLT3-ITD-neg.: 50.9%; p < 0.0001) (Table 3, Fig. 2).
4.4.1. Mutation frequencies according to blast decades The remaining mutations (MLL-PTDs, FLT3-TKDs, biallelic CEBPA, and IDH1/IDH2) were investigated for occurrence in the different blast decades. The MLL-PTDs (varying from 5.9% to 17.4%), FLT3TKD (1.8–13.0%), and IDH1/IDH2 mutations (19.0–48.6%) showed no significant differences across the different blast decades. The frequency of biallelic CEBPA mutations was heterogeneous among the different blast decade with a peak at the 60–69% decade, however, no overall trend for increase or decrease was present (Table 2, Fig. 1). 4.4.2. WBC count and marrow blasts in the different molecular subgroups Subsequently, we compared mean WBC counts and mean marrow blast percentages in dependence on the MLL-PTD, biallelic CEBPA, FLT3-TKD, and IDH1/IDH2 mutation status. None of these markers had a significant impact on WBC count. FLT3-TKD-positive cases had higher mean percentages of BM blasts compared to FLT3-TKD negative patients (69.7 vs 62.4%; p = 0.042), and CEBPA biallelically mutated cases showed higher mean percentages of marrow blasts (68.4 vs 59.4%; p = 0.006) when compared to cases without. The MLL-PTD or IDH1/IDH2 mutation status had no significant impact on marrow blasts (Table 5, Fig. 4). 4.5. Immunophenotyping
4.3.3. WBC count and marrow blasts depend on the FLT3-ITD/wt mutant ratio A higher FLT3-ITD/wildtype mutant ratio was correlated with a higher proportion of marrow blasts (Spearman; p < 0.001). When the FLT3-ITD positive cases were separated according to the FLT3ITD/wt ratio, patients with mutant ratios ≤0.25 had significantly
Cases with NPM1 mutations had a significantly lower expression of CD133 as compared to those without irrespective of the presence of FLT3-ITD (mean ± SD: NPM1mut/FLT3-ITD-positive, 9 ± 15%; NPM1mut/FLT3-ITD-negative, 10 ± 15%; NPM1wt/FLT3ITD-positive, 29 ± 26%; NPM1wt/FLT3-ITD-negative, 31 ± 25%;
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
55
Fig. 3. Proportions of cases with the different BM blast decades depending on NPM1-mut and FLT3-ITD composition (pos: positive; neg: negative; mut: mutated; and wt: wildtype).
p < 0.001 for NPM1mut vs NPM1wt). Higher percentages of blast counts were significantly related to a higher expression of CD117 (Spearman, r = 0.161, p = 0.001), CD135 (r = 0.241, p < 0.001), CD33 (r = 0.430, p < 0.001), and CD38 (r = 0.394, p < 0.001) as well as to a lower expression of CD14 (r = −0.237, p < 0.001).
4.6. Analysis of prognostic parameters In univariate analysis for prognostic parameters, OS was significantly superior in cases of younger age (p < 0.001; RR = 1.36 per 10 years of increase), lower WBC count (p < 0.001; RR = 1.06 per 10 × 109 /l), or lower BM blast percentage (p = 0.034). Also the NPMmut/FLT3-ITD-negative subgroup (p < 0.001; RR = 0.40) and a negative MLL-PTD mutation status (p = 0.007; RR = 2.135) were favorable with respect to OS in univariate analysis. No significant association to OS was found for gender, haemoglobin level, thrombocyte count, and FAB subtypes. In multivariate analysis, only age (p < 0.001), WBC count (p = 0.002), NPM1-mut/FLT3-ITD-negative status (p = 0.003), and MLL-PTD mutation status (p = 0.024) retained prognostic significance (Table 6).
5. Discussion Different mutation types are considered to interact in AML [25] which are either involved in proliferation and anti-apoptotic mechanisms (such as mutations of the FLT3 gene; “class I mutations”) or interfere with myeloid differentiation (e.g. mutations involving the MLL or CEBPA genes; “type II mutations”). Regarding the NPM1 mutations, a variety of functions has been discussed including interference with a tumor suppressor pathway and they were discussed to represent a distinct class of mutations [5,26]. AML at diagnosis can show highly heterogeneous clinical patterns, as it can be characterized by rapid blast proliferation and sudden onset of clinical symptoms, while in other cases, clinical courses may be determined by leukocytopenia and, eventually, by an oligo- or asymptomatic clinical prephase, e.g. in elderly patients. Normal karyotype AML as a cytogenetically homogeneous subgroup seemed best suitable to investigate the associations between the different molecular mutations and blast proliferation in the bone marrow. Early studies already found that the FLT3-ITDs were associated with higher peripheral leukocytes and marrow blasts [1–3].
Table 5 Mean WBC counts and bone marrow blasts in dependence on the different mutations. The p-values show the comparison between patients carrying the respective mutations and those without by Student’s t-test. Mutation
Number of pts
NPM1 mutation NPM1-mut NPM1-wt FLT3-ITD Positive Negative MLL-PTD Positive Negative Biallelic CEBPA mutation Mutated Wildtype FLT3-TKD Positive Negative IDH1/IDH2 mutation Positive Negative
805 391 414 805 219 586 805 76 729 620 590 30 578 46 532 312 101 211
Mean WBC count × 109 /l
p
53.7 22.1
<0.0001
67.5 54.1
<0.0001
69.0 25.7
<0.0001
73.7 55.7
<0.0001
29.3 38.5
n.s.
57.8 60.8
n.s.
34.5 33.0
n.s.
68.4 59.4
0.006
57.1 39.0
n.s.
69.7 62.4
0.042
39.4 42.9
n.s.
65.7 59.1
n.s.
Mean bone marrow blasts %
p
56
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
Table 6 Results of uni- and multivariate analysis for definition of prognostically relevant parameters regarding overall survival (OS). Parameter
Univariate (p)
Multivariate (p)
Age Gender WBC count Thrombocyte count Haemoglobin level FAB subtype Bone marrow blasts NPM1-mutated/FLT3-ITD-negative vs others MLL-PTD mutation status CEBPA mutation status FLT3-TKD mutation status IDH1/IDH2 mutation status
<0.001 n.s. <0.001 n.s. n.s. n.s. 0.034 <0.001 0.007 n.s. n.s. n.s.
<0.001 – 0.002 – – – n.s. 0.003 0.024 – – –
n.s.: non significant.
Considering the most frequent and prognostically most relevant markers in normal karyotype AML, we focused on the NPM1 mutations and the FLT3-ITD, performing correlations of the respective mutation status with cytomorphology in a large cohort of 805 cases with normal karyotype AML, and included other molecular markers with a strong association to this cytogenetic subgroup. The NPM1 mutations (48.6%) [7,8], FLT3-ITDs (27.2%) [3,4], and the other markers investigated in our cohort (MLL-PTDs: 9.4%; biallelic CEBPA: 4.8%; FLT3-TKD: 8.0%; IDH1/IDH2: 32.4%) were corresponding to the expected mutation rates in normal karyotype AML. Patients with an NPM1 mutation but no FLT3-ITD had a clear survival benefit when compared to all other molecular subgroups. These results confirmed the validity of our data set. First, increasing blast percentages were correlated with continuously increasing rates of both the NPM1 mutations (rising from 30.9% in the 20–29% to 68.0% in the 90–100% blast decade; p < 0.001) and the FLT3-ITDs (rising from 12.2% to 56.3%; p < 0.001). Also, combinations of both molecular mutations were relevant regarding marrow blast proportions: Mean WBC count and percentage of marrow blasts were lowest in patients with neither mutation and were continuously increasing to the NPM1-mut/FLT3-ITD-negative and NPM1-wt/FLT3-ITD-positive subgroups achieving the highest values in NPM1-mut/FLT3-ITD-positive patients. Patients with a
FLT3-ITD but no NPM1 mutation had higher mean WBC count and marrow blasts percentage than those with an NPM1 mutation alone. Our results are in accordance with Döhner et al. who investigated 300 patients with nk-AML aged 16–60 years. NPM1 mutant AML was associated with higher WBC counts (p < 0.001) and higher marrow blasts (p = 0.02). Marrow blasts were highest in the NPM1-mutated/FLT3-ITD positive subgroup, and lowest in the subgroup without either of these mutations [6]. In the study from Thiede et al. including 1485 patients with AML (of those, 709 with normal karyotypes), patients with an NPM1-mutated/FLT3ITD negative status had significantly higher mean WBC (26.3 vs 7.7 × 109 /l; p < 0.001) and marrow blasts (67.3 vs 56.0%; p < 0.001) when compared to patients without evidence of NPM1 or FLT3-ITD mutations. Mean WBC and marrow blasts showed highest levels in the double NPM1-mut/FLT3-ITD-positive patients, being followed by patients carrying an isolated FLT3-ITD or an isolated NPM1 mutation, respectively, and were lowest in patients carrying neither mutation [8]. The results from this and previous studies emphasize the cooperation of NPM1 and FLT3 mutations in normal karyotype AML and suggest that both mutations have a synergistic function in stimulating blast proliferation. Nucleophosmin shuttling had previously been identified to be relevant for cell cycle progression [27]. Second, these results should be considered in sight of the current interpretation of the NPM1 mutation as founder genetic lesion in AML. This theory had been based on the following: NPM1 mutations were shown to be stable at late relapses of AML [28]. In cases with a concomitant NPM1 mutation and FLT3-ITD, the NPM1 mutation seemed to precede the FLT3-ITD as higher proportions of alleles were affected by the NPM1 mutation. Cases with different FLT3-ITD mutants were observed to carry a single NPM1 mutation subtype suggesting that the different clones evolved from a single NPM1 mutated clone as reported by Thiede et al. [8]. Finally, NPM1 mutations were exclusive of distinct genetic alterations such as reciprocal rearrangements in a large study [29]. The potential of the NPM1 mutation to enhance blast proliferation as shown in this and previous studies adds a further argument to its role as founder genetic event. Regarding the FLT3-ITDs, their stimulating impact on cell proliferation is well established. FLT3-ITDs result in constitutive
Fig. 4. Mean WBC counts and marrow blasts depending on the mutation status regarding all markers investigated in this study (*significant difference according to statistical analysis; mut.: mutated; wt.: wildtype; pos.: positive; and neg.: negative).
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
activation of FLT3 kinase [30]. Transfection of IL-3 dependent cell lines with mutant FLT3-transfected cells resulted in autonomous cell growth [31] and presence of the FLT3-ITD has been associated with strong constitutive activation of the STAT pathway [32]. In mice, FLT-ITDs induced a myeloproliferative disorder characterized by leukocytosis and splenomegaly [33]. Lee et al. engineered a human FLT3-ITD into exon 14 of the murine FLT3 locus. Correctly targeted embryonic stem cells were used to produce viable mice carrying mutant FLT3-ITD alleles and presenting with a myeloproliferative disease resembling chronic myelomonocytic leukemia (CMML) [34]. Many studies confirmed the association of the FLT-ITD with increased blast percentages and WBC count [1–3]. Subsequently, we investigated the influence of additional mutations – FLT3-TKD, (biallelic) CEBPA, IDH1/IDH2, and MLL-PTDs – on peripheral leukocyte and marrow blast proliferation in our cohort. Only a positive FLT3-TKD and biallelic CEBPA mutation status showed significance for higher marrow blasts (p = 0.042 and p = 0.006, respectively), and none of these additional markers had a significant impact on WBC counts. Therefore, these mutations apparently had a weaker proliferative effect (FLT3-TKDs, CEBPAmut) when compared to the NPM1-mut and FLT3-ITDs or no proliferative effect at all (MLL-PTDs, IDH-mut) in our study which was in accordance with previous observations: In a study from Steudel et al., occurrence of an isolated MLL-PTD was linked to lower WBC count and marrow blasts when compared to MLL-PTDnegative patients (without a FLT3-ITD) [35]. Dufour et al. found no significant impact of mono- or biallelic CEBPA mutations on WBC count or marrow blasts [24]. Green et al. identified no significant correlation between IDH1 mutations and WBC count [13], while Paschka et al. reported an association of IDH1/IDH2 mutations to a lower WBC count (p = 0.04) but higher marrow blasts (attributed to IDH1 mutations; p = 0.06) [12]. In an earlier study, we found the FLT3-TKD associated to higher WBC when compared to FLT3 wildtype cases, but to lower levels when compared to FLT3-ITD mutated cases [11]. Furthermore in our study, NPM1 mutations and the MLL-PTD were mutually exclusive, and showed no coincidence with biallelic CEBPA mutations corresponding to previous observations [24], leading to the conclusion that these genetic alterations do not cooperate. In conclusion, both the NPM1 mutations and the FLT3-ITDs are strongly associated to increased blast proliferation in normal karyotype AML. As coincidence of both markers is associated to higher WBC and blast levels when compared to cases carrying one of these mutations only, these results further emphasize the synergism of the NPM1 mutations and FLT3-ITD. In contrast, other mutations, such as the IDH1/IDH2, CEBPA, or MLL-PTD, confer no or only a weaker proliferative effect on blast proliferation. These results contribute to the understanding of the phenotype heterogeneity of normal karyotype AML and confirm the hypothesis that the NPM1 mutations may be founder genetic lesions in AML. Conflict of interest statement TH, CH, WK, and SS declare part ownership of the MLL Munich Leukemia Laboratory GmbH. TA is employed by the MLL Munich Leukemia Laboratory GmbH. UB has nothing to disclose. No additional funding was received for this study. Acknowledgements Funding source: None. Contributions. TH and UB performed data analysis and wrote the manuscript. TH was responsible for cytomorphology. TA and WK performed statistical analysis; WK in addition was responsible for
57
immunophenotyping. CH did cytogenetics, and SS was responsible for molecular genetics. All authors contributed to writing of the manuscript and approved the final version. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2011.04.026. References [1] Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001;98:1752–9. [2] Fröhling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K, et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 2002;100:4372–80. [3] Thiede C, Steudel C, Mohr B, Schaich M, Schäkel U, Platzbecker U, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002;99:4326–35. [4] Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics. FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002;100:59–66. [5] Falini B, Bolli N, Liso A, Martelli MP, Mannucci R, Pileri S, et al. Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications. Leukemia 2009;23:1731–43. [6] Döhner K, Schlenk RF, Habdank M, Scholl C, Rücker FG, Corbacioglu A, et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 2005;106:3740–6. [7] Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF, et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005;106:3733–9. [8] Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M, et al. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 2006;107:4011–20. [9] Gale RE, Green C, Allen C, Mead AJ, Burnett AK, Hills RK, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood 2008;111:2776–84. [10] Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia 2000;14:796–804. [11] Bacher U, Haferlach C, Kern W, Haferlach T, Schnittger S. Prognostic relevance of FLT3-TKD mutations in AML: the combination matters—an analysis of 3082 patients. Blood 2008;111:2527–37. [12] Paschka P, Schlenk RF, Gaidzik VI, Habdank M, Kronke J, Bullinger L, et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol 2010;28:3636–43. [13] Green CL, Evans CM, Hills RK, Burnett AK, Linch DC, Gale RE. The prognostic significance of IDH1 mutations in younger adult patients with acute myeloid leukemia is dependent on FLT3/ITD status. Blood 2010;116:2779–82. [14] Schnittger S, Haferlach C, Ulke M, Alpermann T, Kern W, Haferlach T. IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status. Blood 2010;116:5486–96. [15] Wouters BJ, Loewenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood 2009;113:3088–91. [16] Mufti GJ, Bennett JM, Goasguen J, Bain BJ, Baumann I, Brunning R, et al. Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts. Haematologica 2008;93:1712–7. [17] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French–American–British Cooperative Group. Ann Intern Med 1985;103:620–5. [18] Swerdlow S, Campo E, Lee Harris N, Jaffe E, Pileri S, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. IARC Press; 2008.
58
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
[19] Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U, et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: a study on 350 cases. Leukemia 2002;16:53–9. [20] Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;104:3078–85. [21] Schnittger S, Kern W, Tschulik C, Weiss T, Dicker F, Falini B, et al. Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic information in AML. Blood 2009;114:2220–31. [22] Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S. Risk assessment by monitoring expression levels of partial tandem duplications in the MLL gene in acute myeloid leukemia during therapy. Haematologica 2005;90:881–9. [23] Snaddon J, Smith ML, Neat M, Cambal-Parrales M, Dixon-McIver A, Arch R, et al. Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2. Genes Chromosomes Cancer 2003;37:72–8. [24] Dufour A, Schneider F, Metzeler KH, Hoster E, Schneider S, Zellmeier E, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol 2010;28:570–7. [25] Gilliland DG. Hematologic malignancies. Curr Opin Hematol 2001;8:189–91. [26] Renneville A, Roumier C, Biggio V, Nibourel O, Boissel N, Fenaux P, et al. Cooperating gene mutations in acute myeloid leukemia: a review of the literature. Leukemia 2008;22:915–31. [27] Brady SN, Yu Y, Maggi Jr LB, Weber JD. ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway. Mol Cell Biol 2004;24:9327–38.
[28] Meloni G, Mancini M, Gianfelici V, Martelli MP, Foa R, Falini B. Late relapse of acute myeloid leukemia with mutated NPM1 after eight years: evidence of NPM1 mutation stability. Haematologica 2009;94:298–300. [29] Falini B, Mecucci C, Saglio G, Lo Coco F, Diverio D, Brown P, et al. NPM1 mutations and cytoplasmic nucleophosmin are mutually exclusive of recurrent genetic abnormalities: a comparative analysis of 2562 patients with acute myeloid leukemia. Haematologica 2008;93:439–42. [30] Gilliland DG, Griffin JD. Role of FLT3 in leukemia. Curr Opin Hematol 2002;9:274–81. [31] Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura T, Saito H, et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000;19:624–31. [32] Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Müller C, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 2000;96:3907– 14. [33] Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL, Gilliland DG. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 2002;99:310–8. [34] Lee BH, Tothova Z, Levine RL, Anderson K, Buza-Vidas N, Cullen DE, et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell 2007;12:367–80. [35] Steudel C, Wermke M, Schaich M, Schäkel U, Illmer T, Ehninger G, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer 2003;37:237–51.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype Torsten Haferlach a,∗ , Ulrike Bacher b , Tamara Alpermann a , Claudia Haferlach a , Wolfgang Kern a , Susanne Schnittger a a b
MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany Department for Stem Cell Transplantation, University Cancer Center Hamburg, Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 9 March 2011 Received in revised form 26 April 2011 Accepted 28 April 2011 Available online 31 May 2011 Keywords: NPM1 mutation Founder mutation FLT3-ITD Blast percentages Normal karyotype AML
a b s t r a c t FLT3-ITDs are linked to higher leukocytes/blasts in acute myeloid leukemia. To evaluate the effect of NPM1 mutations, we correlated NPM1mut status with morphology in 805 adult normal karyotype AML. NPM1mut were found in 391/805 (48.6%), FLT3-ITD in 219/805 (27.2%). Frequencies of FLT3-ITD and NPM1mut cases were continuously increasing by blast decades: NPM1mut from 38/123 (30.9%) in 20–29% blast decade to 70/103 (68.0%) in 90–100% decade (p < 0.001), FLT3-ITDs from 15/123 (12.2%) to 58/103 (56.3%) (p < 0.001). Mean WBC count was highest in NPM1-mut/FLT3-ITD-positive and lowest in NPM1-wildtype/FLT3-ITD-negative patients (p < 0.0001); similar for BM blasts. Therefore, FLT3-ITD and NPM1mut might synergistically stimulate blast proliferation. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Internal tandem duplications of the FLT3 gene (FLT3-ITD) can be found in 35–45% of acute myeloid leukemia (AML) with a normal karyotype (nk-AML) and are prognostically adverse. The FLT3-ITDs result in increased autophosphorylation of the FLT3 receptor tyrosine kinase and thereby increase cell proliferation. Occurrence of the FLT3-ITD has been strongly associated with higher peripheral leukocytes and higher blast percentages [1–4]. Mutations of the nucleophosmin (NPM1) gene are found in 45–55% of nk-AML cases. In NPM1 mutant AML, the nucleophosmin protein (a chaperone protein) is aberrantly localized in the cytoplasm [5]. The NPM1 mutations are supposed to interfere with the ARF14 tumor suppressor pathway. In 40–45% of NPM1 mutated nk-AML also concomitant FLT3-ITDs were identified. Coincidence of the NPM1 mutations with the FLT3-ITD results in less favorable outcomes when compared to isolated occurrence of the NPM1 mutation [6–9]. Some authors mentioned higher peripheral leukocytes and marrow blasts also for NPM1 mutant AML [6,8]. Based on these observations, and to further study the impact of these mutations on blast proliferation in AML, we performed correlations of the frequency of NPM1 mutations and FLT3-ITD with cytomorphologic and other parameters in 805 adult patients
∗ Corresponding author. Tel.: +49 89 990 17 100; fax: +49 89 990 17 109. E-mail address: [email protected] (T. Haferlach). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.04.026
newly diagnosed with de novo nk-AML. We aimed to further clarify pathways and input of these genes for AML biology. Additionally, we included the prognostically adverse MLL-PTDs (partial tandem duplications of the MLL gene) [10], the FLT3-TKD mutations (involving the tyrosine kinase domain of the FLT3 gene) [11], IDH1 and IDH2 mutations (isocitrate dehydrogenase, conferring an adverse prognostic impact in FLT3-ITD negative AML) [12–14], and the prognostically favorable CEBPA mutations (in case of biallelic occurrence) [15] in the analysis, since these mutations are as well strongly associated to normal karyotype AML and have clinical relevance.
2. Patients The study cohort consisted of consecutive 805 patients (410 males, 395 females; median, 66.6 years; range, 20.0–93.3 years) who all had de novo nk-AML at diagnosis. Samples had been sent from different hematologic centers to the MLL Munich Leukemia Laboratory between 8/2005 and 6/2010. Prerequisite for inclusion in the study was a normal karyotype and the availability of bone marrow (BM) cytomorphology and analysis for the FLT3-ITD and the NPM1 mutation in parallel. Patients gave written informed consent for the use of laboratory data for research studies. The study was approved by the Bavarian Medical Association (“Bayerische Landesärztekammer”) and performed according to the Declaration of Helsinki.
52
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
3. Methods 3.1. Cytomorphology All cases were investigated by BM cytomorphology (May Gruenwald Giemsa staining combined with myeloperoxidase, MPO, and non-specific esterase, NSE, for cytochemistry). Cells were categorized as blasts following the guidelines of an International Working Group on Morphology published by Mufti et al. [16]. AML was classified according to FAB [17] and WHO [18] criteria. For correlation with molecular mutation analysis, cases were subdivided according to decades of marrow blasts (20–29%; 30–39%; → 80–89%, 90–100%). 3.2. Cytogenetics Chromosome banding analysis was done according to previously described standard procedures [19]. All patients selected for this study had normal karyotypes. 3.3. Multiparameter flow cytometry Immunophenotyping with multiparameter flow cytometry (MFC) was done by five color staining in 427 cases [20].
Table 1 Biologic parameters and FAB subtypes in 805 patients with normal karyotype AML. Parameter
Number of patients
Total cohort Biologic parameters Males Females Median age (range), years Laboratory parameters Mean WBC count × 109 /l (range) Mean bone marrow blasts (%) Mean thrombocyte count × 109 /l (range) Mean haemoglobin level, g/dl (range) FAB subtypes (n = 797) M0 M1 M2 M4 M5 M6 M7 Molecular markers NPM1 mutation FLT3-ITD MLL-PTD Biallelic CEBPA mutation FLT3-TKD IDH1/IDH2 mutation
805 410 395 66.6 (20.0–93.3) 37.7 (0.1–600.0) 60.6 (20.0–99.0) 97 (3–765) 9.4 (3.0–16.0) 27 (3.4%) 255 (32.0%) 312 (39.1%) 168 (21.1%) 21 (2.6%) 12 (1.5%) 2 (0.3%) 391/805 (48.6%) 219/805 (27.2%) 76/805 (9.4%) 30/620 (4.8%) 46/578 (8.0%) 101/312 (32.4%)
3.4. Molecular genetics Following isolation of mononucleated cells, mRNA extraction, and random primed cDNA synthesis, analysis for NPM1 mutations by LightCycler® based melting curve analysis was performed as described before [7,21]. Determination of the FLT3-ITD mutation status and of the mutation length and quantification of the FLT3ITD mutation load were realized by fragment analysis (GeneScan, 3130 sequence detection system, ABI) [3]. The FLT3-ITD load was defined as the ratio of the mutated compared to the FLT3 wildtype (wt) allele. Ratios ≥1 were indicative of loss or partial loss of the wt allele. Analysis for the MLL-PTD by real-time PCR [22] was done in all 805 cases. Screening for FLT3-TKD mutation (n = 578) [11], mutations of IDH1 and IDH2 (n = 312) [14], and CEBPA genes (n = 620) [23] was performed in different numbers of cases. Only patients with biallelic CEBPA mutations were included in the analysis since recent reports suggest that only this double mutated status confers a favorable prognostic impact in AML [15,24]. 3.5. Statistical analysis Overall survival (OS) and event free survival (EFS) were calculated according to Kaplan–Meier. Survival data was compared by two sided log rank test. Cox regression was performed for OS and EFS with different parameters as covariates. Parameters which were significant in univariate analysis were included into multivariate analysis. Dichotomous variables were compared between different subgroups using the 2 -test and continuous variables by Student’s t-test. Spearman rank correlation was used to analyze correlations between continuous parameters. SPSS (version 14.0.1) software (SPSS, Chicago, IL) was used for statistical analysis. 4. Results
AML. In the total cohort, mean WBC count was 37.7 × 109 /l (range, 0.1–600.0) and BM blasts were 60.6% (20.0–99.9%). 4.2. Molecular mutation rates and survival 4.2.1. NPM1 mutations and FLT3-ITDs In the total cohort, the mutation rate for NPM1 mutations (NPM1-mut) was 391/805 (48.6%) and for FLT3-ITD 219/805 (27.2%). Considering combinations of both markers, subgroups were distributed as follows: NPM1-mut/FLT3-ITD-pos.: n = 151 (18.8% of all cases); NPM1-wt/FLT3-ITD-positive: n = 68 (8.4%); NPM1-mut/FLT3ITD-negative: n = 240 (29.8%); NPM1-wt/FLT3-ITD-negative: n = 346 (43.0%). The 2-year OS rate was best in the NPM1-mut/FLT3ITD-negative patients (82.3%) when compared to all other subgroups (NPM1-wt/FLT3-ITD-neg.: 62.1%; NPM1-mut/FLT3-ITDpositive: 55.2%; NPM1-wt/FLT3-ITD-positive: 40.5%; p = 0.003). 4.2.2. Additional molecular markers IDH1 or IDH2 gene mutations were detected in 101/312 cases (32.4%). MLL-PTDs were found in 76/805 (9.4%), FLT3-TKDs in 46/578 (8.0%), and biallelic CEBPA mutations in 30/620 (4.8%). Considering marker composition with respect to the NPM1-mut and FLT3-ITDs, NPM1-mutated patients showed frequent coincidence with the IDH1 or IDH2-mut (56 IDH1 or IDH2-mut/174 NPM1-mut; 32.2%) and the FLT3-TKD (36 FLT3-TKD positive/313 NPM1-mut; 11.5%). The NPM1 mutations were not seen in combination with biallelic CEBPA mutations and the MLL-PTD. Regarding the FLT3ITD, IDH1/IDH2-mut cases were observed in 27/82 FLT3-ITD positive cases (32.9%), MLL-PTDs in 23/219 FLT3-ITD-positive cases (10.5%). Biallelic CEBPA-mut and FLT3-TKD-positive cases were rarely combined with the FLT3-ITDs. The different marker combinations are given in detail in the Supplemental Table S1 (see also Supplemental Figure S1).
4.1. Biological parameters and FAB subtypes 4.3. NPM1 mutations and FLT3-ITD Biological parameters and FAB subtypes are shown in Table 1. Median age of patients (66.6 years; range, 20.0–93.3) was corresponding to overall AML. The distribution of FAB subtypes (with a preponderance of M1, M2, and M4 subtypes) confirmed the expected pattern of morphologic subtypes in normal karyotype
4.3.1. Molecular mutation frequencies in the different blast decades Frequencies of both FLT3-ITD and NPM1 mutated cases, respectively, were continuously increasing by blast percentages from 20%
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
53
Table 2 Frequencies of the different molecular mutations in the different marrow blast categories given as decades. The p-value shows the comparison of the mutation rates in the different blast categories by chi square (* percentages of patients in relation to the total cohort). BM blasts
NPM1mut
FLT3-ITD
MLL-PTD
Biallelic CEBPAmut
FLT3-TKD
IDH1/IDH2mut
20–29% 30–39% 40–49% 50–59% 60–69% 70–79% 80–89% 90–100%
Number of patients 123 (15.3%)* 91 (11.3%) 69 (8.6%) 80 (9.9%) 102 (12.7%) 114 (14.2%) 123 (15.3%) 103 (12.8%)
38/123 (30.9%) 31/91 (34.1%) 20/69 (29.0%) 36/80 (45.0%) 51/102 (50.0%) 62/114 (54.4%) 83/123 (67.5%) 70/103 (68.0%)
15/123 (12.2%) 7/91 (7.7%) 11/69 (15.9%) 14/80 (17.5%) 25/102 (24.5%) 34/114 (29.8%) 55/123 (44.7%) 58/103 (56.3%)
10/123 (8.1%) 12/91 (13.2%) 12/69 (17.4%) 7/80 (8.8%) 6/102 (5.9%) 13/114 (11.4%) 8/123 (6.5%) 8/103 (7.8%)
0/97 (0.0%) 2/73 (2.7%) 2/56 (3.6%) 3/63 (4.8%) 10/75 (13.3%) 5/94 (5.3%) 5/86 (5.8%) 3/76 (3.9%)
2/75 (2.7%) 6/57 (10.5%) 3/48 (6.3%) 1/55 (1.8%) 3/73 (4.1%) 12/92 (13.0%) 11/92 (12.0%) 8/86 (9.3%)
8/42 (19.0%) 12/37 (32.4%) 4/20 (20.0%) 9/36 (25.0%) 19/52 (36.5%) 18/37 (48.6%) 16/50 (32.0%) 15/38 (39.5%)
Total
805 (100.0%)
391/805 (48.6%)
219/805 (27.2%)
76/805 (9.4%)
30/620 (4.8%)
46/578 (8.0%)
101/312 (32.4%)
<0.001
<0.001
n.s.
0.012
n.s.
n.s.
p
–
Fig. 1. Frequencies of the different mutations according to blast decades in the patients with normal karyotype AML (n = 805). The x-axis shows the blast decades; the y-axis demonstrates the percentages of cases carrying the respective mutations.
to 100%: The NPM1 mutations were detected in 38/123 (30.9%) of the 20–29% blast decade and in 31/91 (34.1%) of the 30–39% decade, increasing to 83/123 (67.5%) in the 80–89% decade and 70/103 (68.0%) in the 90–100% decade (p < 0.001). FLT3-ITDs were detected
in 15/123 (12.2%) of the 20–29% decade and in 7/91 (7.7%) of the 30–39% decade and were steadily increasing to 55/123 (44.7%) in the 80–89% decade and 58/103 (56.3%) in the 90–100% decade (p < 0.001) (Table 2, Fig. 1).
Table 3 Mean WBC count and mean marrow blasts depending on marker composition and the FLT3-ITD/wt mutant ratio (pos.: positive; neg.: negative; mut: mutated; wt: wildtype; * The p-values show comparison of the respective subgroup with all other subgroups by Student’s t-test. ** Ratio of FLT3-ITD mutated and wildtype alleles). Marker composition
Number of patients
NPM1-mut/FLT3-ITD-pos. 151/805 (18.8%) 240/805 (29.8%) NPM1-mut/FLT3-ITD-neg. 68/805 (8.4%) NPM1-wt/FLT3-ITD-pos. 346/805 (43.0%) NPM1-wt/FLT3-ITD-neg. FLT3-ITD/wt ratio** (FLT3-ITD-positive patients) 52/219 (23.7%) ≤0.25 45/219 (20.5%) >0.25 ≤ 0.5 39/219 (17.8%) >0.5 ≤ 0.75 38/219 (17.4%) >0.75 ≤ 1.0 45/219 (20.5%) >1.0
Mean WBC count, ×109 /l
p*
Mean bone marrow blasts, %
p*
76.4 38.6 50.9 16.7
<0.0001 n.s. n.s. <0.0001
75.3 62.5 70.3 50.9
<0.0001 n.s. <0.0001 <0.0001
48.9 71.1 85.8 50.8 87.3
0.053 n.s. n.s. n.s. n.s.
66.3 74.9 76.6 73.7 78.5
0.009 n.s. n.s. n.s. 0.043
54
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
Table 4 Proportions of cases with the different BM blast decades depending on the molecular subgroup (pos.: positive; neg.: negative; mut: mutated; wt: wildtype; n: number). Blast percentages
NPM1-mut/FLT3-ITDpos. (n = 151)
NPM1-wt/FLT3-ITDpos. (n = 68)
NPM1-mut/FLT3-ITDneg. (n = 240)
NPM1-wt/FLT3-ITDneg. (n = 346)
20–29% 30–39% 40–49% 50–59% 60–69% 70–79% 80–89% 90–100% p (comparison of all subgroups* )
9 (6.0%) 4 (2.6%) 7 (4.6%) 8 (5.3%) 18 (11.9%) 20 (13.2%) 40 (26.5%) 45 (29.8%) <0.001
6 (8.8%) 3 (4.4%) 4 (5.9%) 6 (8.8%) 7 (10.3%) 14 (20.6%) 15 (22.1%) 13 (19.1%) 0.074
29 (12.1%) 27 (11.3%) 13 (5.4%) 28 (11.7%) 33 (13.8%) 42 (17.5%) 43 (17.9%) 25 (10.4%) 0.064
79 (22.8%) 57 (16.5%) 45 (13.0%) 38 (11.0%) 44 (12.7%) 38 (11.0%) 25 (7.2%) 20 (5.8%) <0.001
*
chi square.
lower mean marrow blasts compared to those with ratios >0.25 (p = 0.009) (Table 3). 4.3.4. Marrow blast levels in the different molecular genetic subgroups Focusing on the marrow blast levels in the different molecular subgroups, the NPM1-mut/FLT3-ITD positive (double mutated) subgroup showed the highest proportion of cases with 90–100% blasts (45 of 151 cases; 29.8%), while low proportions of blasts were infrequent: only 9 and 4 cases (6.0% and 2.6%) from this molecular subgroup had 20–29% and 30–39% of blasts, respectively (p < 0.001). The NPM1-wt/FLT3-ITD-positive and NPM1-mut/FLT3-ITD negative subgroups peaked in the 80–89% blasts decade (15/68; 22.1%; and 43/240; 17.9%; respectively; n.s.). In contrast, the NPM1-wt/FLT3ITD-negative patients most frequently had low blast percentages of 20–29% (79/346; 22.8%), while ≥80% of blasts rarely were seen (p < 0.001) (Table 4, Fig. 3). 4.4. Additional molecular markers
Fig. 2. Mean WBC count and marrow blasts depending on NPM1-mut/FLT3-ITD composition (NPM1+: NPM1 mutated; NPM1−: NPM1 wildtype; FLT3+: FLT3-ITDpositive; and FLT3−: FLT3-ITD-negative.
4.3.2. WBC count and marrow blasts depend on NPM1-mut/FLT-ITD composition Subsequently, we compared mean WBC counts and mean marrow blasts in the different molecular subgroups considering the NPM1 mutations and the FLT3-ITD in combination: Mean WBC count was highest in the NPM1-mut/FLT3-ITD-positive subgroup followed by the NPM1-wt/FLT3-ITD-positive and the NPM1-mut/FLT3-ITD-negative subgroups, with the lowest values in the NPM1-wt/FLT3-ITD-negative patients (76.4 vs 50.9 vs 38.6 vs 16.7 × 109 /l; p < 0.0001). Mean BM blasts were also highest in the NPM1-mut/FLT3-ITD-positive subgroup with 75.3% (NPM1-wt/FLT3-ITD-pos.: 70.3%; NPM1-mut/FLT3-ITD-neg.: 62.5%; NPM1-wt/FLT3-ITD-neg.: 50.9%; p < 0.0001) (Table 3, Fig. 2).
4.4.1. Mutation frequencies according to blast decades The remaining mutations (MLL-PTDs, FLT3-TKDs, biallelic CEBPA, and IDH1/IDH2) were investigated for occurrence in the different blast decades. The MLL-PTDs (varying from 5.9% to 17.4%), FLT3TKD (1.8–13.0%), and IDH1/IDH2 mutations (19.0–48.6%) showed no significant differences across the different blast decades. The frequency of biallelic CEBPA mutations was heterogeneous among the different blast decade with a peak at the 60–69% decade, however, no overall trend for increase or decrease was present (Table 2, Fig. 1). 4.4.2. WBC count and marrow blasts in the different molecular subgroups Subsequently, we compared mean WBC counts and mean marrow blast percentages in dependence on the MLL-PTD, biallelic CEBPA, FLT3-TKD, and IDH1/IDH2 mutation status. None of these markers had a significant impact on WBC count. FLT3-TKD-positive cases had higher mean percentages of BM blasts compared to FLT3-TKD negative patients (69.7 vs 62.4%; p = 0.042), and CEBPA biallelically mutated cases showed higher mean percentages of marrow blasts (68.4 vs 59.4%; p = 0.006) when compared to cases without. The MLL-PTD or IDH1/IDH2 mutation status had no significant impact on marrow blasts (Table 5, Fig. 4). 4.5. Immunophenotyping
4.3.3. WBC count and marrow blasts depend on the FLT3-ITD/wt mutant ratio A higher FLT3-ITD/wildtype mutant ratio was correlated with a higher proportion of marrow blasts (Spearman; p < 0.001). When the FLT3-ITD positive cases were separated according to the FLT3ITD/wt ratio, patients with mutant ratios ≤0.25 had significantly
Cases with NPM1 mutations had a significantly lower expression of CD133 as compared to those without irrespective of the presence of FLT3-ITD (mean ± SD: NPM1mut/FLT3-ITD-positive, 9 ± 15%; NPM1mut/FLT3-ITD-negative, 10 ± 15%; NPM1wt/FLT3ITD-positive, 29 ± 26%; NPM1wt/FLT3-ITD-negative, 31 ± 25%;
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
55
Fig. 3. Proportions of cases with the different BM blast decades depending on NPM1-mut and FLT3-ITD composition (pos: positive; neg: negative; mut: mutated; and wt: wildtype).
p < 0.001 for NPM1mut vs NPM1wt). Higher percentages of blast counts were significantly related to a higher expression of CD117 (Spearman, r = 0.161, p = 0.001), CD135 (r = 0.241, p < 0.001), CD33 (r = 0.430, p < 0.001), and CD38 (r = 0.394, p < 0.001) as well as to a lower expression of CD14 (r = −0.237, p < 0.001).
4.6. Analysis of prognostic parameters In univariate analysis for prognostic parameters, OS was significantly superior in cases of younger age (p < 0.001; RR = 1.36 per 10 years of increase), lower WBC count (p < 0.001; RR = 1.06 per 10 × 109 /l), or lower BM blast percentage (p = 0.034). Also the NPMmut/FLT3-ITD-negative subgroup (p < 0.001; RR = 0.40) and a negative MLL-PTD mutation status (p = 0.007; RR = 2.135) were favorable with respect to OS in univariate analysis. No significant association to OS was found for gender, haemoglobin level, thrombocyte count, and FAB subtypes. In multivariate analysis, only age (p < 0.001), WBC count (p = 0.002), NPM1-mut/FLT3-ITD-negative status (p = 0.003), and MLL-PTD mutation status (p = 0.024) retained prognostic significance (Table 6).
5. Discussion Different mutation types are considered to interact in AML [25] which are either involved in proliferation and anti-apoptotic mechanisms (such as mutations of the FLT3 gene; “class I mutations”) or interfere with myeloid differentiation (e.g. mutations involving the MLL or CEBPA genes; “type II mutations”). Regarding the NPM1 mutations, a variety of functions has been discussed including interference with a tumor suppressor pathway and they were discussed to represent a distinct class of mutations [5,26]. AML at diagnosis can show highly heterogeneous clinical patterns, as it can be characterized by rapid blast proliferation and sudden onset of clinical symptoms, while in other cases, clinical courses may be determined by leukocytopenia and, eventually, by an oligo- or asymptomatic clinical prephase, e.g. in elderly patients. Normal karyotype AML as a cytogenetically homogeneous subgroup seemed best suitable to investigate the associations between the different molecular mutations and blast proliferation in the bone marrow. Early studies already found that the FLT3-ITDs were associated with higher peripheral leukocytes and marrow blasts [1–3].
Table 5 Mean WBC counts and bone marrow blasts in dependence on the different mutations. The p-values show the comparison between patients carrying the respective mutations and those without by Student’s t-test. Mutation
Number of pts
NPM1 mutation NPM1-mut NPM1-wt FLT3-ITD Positive Negative MLL-PTD Positive Negative Biallelic CEBPA mutation Mutated Wildtype FLT3-TKD Positive Negative IDH1/IDH2 mutation Positive Negative
805 391 414 805 219 586 805 76 729 620 590 30 578 46 532 312 101 211
Mean WBC count × 109 /l
p
53.7 22.1
<0.0001
67.5 54.1
<0.0001
69.0 25.7
<0.0001
73.7 55.7
<0.0001
29.3 38.5
n.s.
57.8 60.8
n.s.
34.5 33.0
n.s.
68.4 59.4
0.006
57.1 39.0
n.s.
69.7 62.4
0.042
39.4 42.9
n.s.
65.7 59.1
n.s.
Mean bone marrow blasts %
p
56
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
Table 6 Results of uni- and multivariate analysis for definition of prognostically relevant parameters regarding overall survival (OS). Parameter
Univariate (p)
Multivariate (p)
Age Gender WBC count Thrombocyte count Haemoglobin level FAB subtype Bone marrow blasts NPM1-mutated/FLT3-ITD-negative vs others MLL-PTD mutation status CEBPA mutation status FLT3-TKD mutation status IDH1/IDH2 mutation status
<0.001 n.s. <0.001 n.s. n.s. n.s. 0.034 <0.001 0.007 n.s. n.s. n.s.
<0.001 – 0.002 – – – n.s. 0.003 0.024 – – –
n.s.: non significant.
Considering the most frequent and prognostically most relevant markers in normal karyotype AML, we focused on the NPM1 mutations and the FLT3-ITD, performing correlations of the respective mutation status with cytomorphology in a large cohort of 805 cases with normal karyotype AML, and included other molecular markers with a strong association to this cytogenetic subgroup. The NPM1 mutations (48.6%) [7,8], FLT3-ITDs (27.2%) [3,4], and the other markers investigated in our cohort (MLL-PTDs: 9.4%; biallelic CEBPA: 4.8%; FLT3-TKD: 8.0%; IDH1/IDH2: 32.4%) were corresponding to the expected mutation rates in normal karyotype AML. Patients with an NPM1 mutation but no FLT3-ITD had a clear survival benefit when compared to all other molecular subgroups. These results confirmed the validity of our data set. First, increasing blast percentages were correlated with continuously increasing rates of both the NPM1 mutations (rising from 30.9% in the 20–29% to 68.0% in the 90–100% blast decade; p < 0.001) and the FLT3-ITDs (rising from 12.2% to 56.3%; p < 0.001). Also, combinations of both molecular mutations were relevant regarding marrow blast proportions: Mean WBC count and percentage of marrow blasts were lowest in patients with neither mutation and were continuously increasing to the NPM1-mut/FLT3-ITD-negative and NPM1-wt/FLT3-ITD-positive subgroups achieving the highest values in NPM1-mut/FLT3-ITD-positive patients. Patients with a
FLT3-ITD but no NPM1 mutation had higher mean WBC count and marrow blasts percentage than those with an NPM1 mutation alone. Our results are in accordance with Döhner et al. who investigated 300 patients with nk-AML aged 16–60 years. NPM1 mutant AML was associated with higher WBC counts (p < 0.001) and higher marrow blasts (p = 0.02). Marrow blasts were highest in the NPM1-mutated/FLT3-ITD positive subgroup, and lowest in the subgroup without either of these mutations [6]. In the study from Thiede et al. including 1485 patients with AML (of those, 709 with normal karyotypes), patients with an NPM1-mutated/FLT3ITD negative status had significantly higher mean WBC (26.3 vs 7.7 × 109 /l; p < 0.001) and marrow blasts (67.3 vs 56.0%; p < 0.001) when compared to patients without evidence of NPM1 or FLT3-ITD mutations. Mean WBC and marrow blasts showed highest levels in the double NPM1-mut/FLT3-ITD-positive patients, being followed by patients carrying an isolated FLT3-ITD or an isolated NPM1 mutation, respectively, and were lowest in patients carrying neither mutation [8]. The results from this and previous studies emphasize the cooperation of NPM1 and FLT3 mutations in normal karyotype AML and suggest that both mutations have a synergistic function in stimulating blast proliferation. Nucleophosmin shuttling had previously been identified to be relevant for cell cycle progression [27]. Second, these results should be considered in sight of the current interpretation of the NPM1 mutation as founder genetic lesion in AML. This theory had been based on the following: NPM1 mutations were shown to be stable at late relapses of AML [28]. In cases with a concomitant NPM1 mutation and FLT3-ITD, the NPM1 mutation seemed to precede the FLT3-ITD as higher proportions of alleles were affected by the NPM1 mutation. Cases with different FLT3-ITD mutants were observed to carry a single NPM1 mutation subtype suggesting that the different clones evolved from a single NPM1 mutated clone as reported by Thiede et al. [8]. Finally, NPM1 mutations were exclusive of distinct genetic alterations such as reciprocal rearrangements in a large study [29]. The potential of the NPM1 mutation to enhance blast proliferation as shown in this and previous studies adds a further argument to its role as founder genetic event. Regarding the FLT3-ITDs, their stimulating impact on cell proliferation is well established. FLT3-ITDs result in constitutive
Fig. 4. Mean WBC counts and marrow blasts depending on the mutation status regarding all markers investigated in this study (*significant difference according to statistical analysis; mut.: mutated; wt.: wildtype; pos.: positive; and neg.: negative).
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
activation of FLT3 kinase [30]. Transfection of IL-3 dependent cell lines with mutant FLT3-transfected cells resulted in autonomous cell growth [31] and presence of the FLT3-ITD has been associated with strong constitutive activation of the STAT pathway [32]. In mice, FLT-ITDs induced a myeloproliferative disorder characterized by leukocytosis and splenomegaly [33]. Lee et al. engineered a human FLT3-ITD into exon 14 of the murine FLT3 locus. Correctly targeted embryonic stem cells were used to produce viable mice carrying mutant FLT3-ITD alleles and presenting with a myeloproliferative disease resembling chronic myelomonocytic leukemia (CMML) [34]. Many studies confirmed the association of the FLT-ITD with increased blast percentages and WBC count [1–3]. Subsequently, we investigated the influence of additional mutations – FLT3-TKD, (biallelic) CEBPA, IDH1/IDH2, and MLL-PTDs – on peripheral leukocyte and marrow blast proliferation in our cohort. Only a positive FLT3-TKD and biallelic CEBPA mutation status showed significance for higher marrow blasts (p = 0.042 and p = 0.006, respectively), and none of these additional markers had a significant impact on WBC counts. Therefore, these mutations apparently had a weaker proliferative effect (FLT3-TKDs, CEBPAmut) when compared to the NPM1-mut and FLT3-ITDs or no proliferative effect at all (MLL-PTDs, IDH-mut) in our study which was in accordance with previous observations: In a study from Steudel et al., occurrence of an isolated MLL-PTD was linked to lower WBC count and marrow blasts when compared to MLL-PTDnegative patients (without a FLT3-ITD) [35]. Dufour et al. found no significant impact of mono- or biallelic CEBPA mutations on WBC count or marrow blasts [24]. Green et al. identified no significant correlation between IDH1 mutations and WBC count [13], while Paschka et al. reported an association of IDH1/IDH2 mutations to a lower WBC count (p = 0.04) but higher marrow blasts (attributed to IDH1 mutations; p = 0.06) [12]. In an earlier study, we found the FLT3-TKD associated to higher WBC when compared to FLT3 wildtype cases, but to lower levels when compared to FLT3-ITD mutated cases [11]. Furthermore in our study, NPM1 mutations and the MLL-PTD were mutually exclusive, and showed no coincidence with biallelic CEBPA mutations corresponding to previous observations [24], leading to the conclusion that these genetic alterations do not cooperate. In conclusion, both the NPM1 mutations and the FLT3-ITDs are strongly associated to increased blast proliferation in normal karyotype AML. As coincidence of both markers is associated to higher WBC and blast levels when compared to cases carrying one of these mutations only, these results further emphasize the synergism of the NPM1 mutations and FLT3-ITD. In contrast, other mutations, such as the IDH1/IDH2, CEBPA, or MLL-PTD, confer no or only a weaker proliferative effect on blast proliferation. These results contribute to the understanding of the phenotype heterogeneity of normal karyotype AML and confirm the hypothesis that the NPM1 mutations may be founder genetic lesions in AML. Conflict of interest statement TH, CH, WK, and SS declare part ownership of the MLL Munich Leukemia Laboratory GmbH. TA is employed by the MLL Munich Leukemia Laboratory GmbH. UB has nothing to disclose. No additional funding was received for this study. Acknowledgements Funding source: None. Contributions. TH and UB performed data analysis and wrote the manuscript. TH was responsible for cytomorphology. TA and WK performed statistical analysis; WK in addition was responsible for
57
immunophenotyping. CH did cytogenetics, and SS was responsible for molecular genetics. All authors contributed to writing of the manuscript and approved the final version. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2011.04.026. References [1] Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001;98:1752–9. [2] Fröhling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K, et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 2002;100:4372–80. [3] Thiede C, Steudel C, Mohr B, Schaich M, Schäkel U, Platzbecker U, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002;99:4326–35. [4] Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics. FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002;100:59–66. [5] Falini B, Bolli N, Liso A, Martelli MP, Mannucci R, Pileri S, et al. Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications. Leukemia 2009;23:1731–43. [6] Döhner K, Schlenk RF, Habdank M, Scholl C, Rücker FG, Corbacioglu A, et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 2005;106:3740–6. [7] Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF, et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005;106:3733–9. [8] Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M, et al. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 2006;107:4011–20. [9] Gale RE, Green C, Allen C, Mead AJ, Burnett AK, Hills RK, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood 2008;111:2776–84. [10] Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia 2000;14:796–804. [11] Bacher U, Haferlach C, Kern W, Haferlach T, Schnittger S. Prognostic relevance of FLT3-TKD mutations in AML: the combination matters—an analysis of 3082 patients. Blood 2008;111:2527–37. [12] Paschka P, Schlenk RF, Gaidzik VI, Habdank M, Kronke J, Bullinger L, et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol 2010;28:3636–43. [13] Green CL, Evans CM, Hills RK, Burnett AK, Linch DC, Gale RE. The prognostic significance of IDH1 mutations in younger adult patients with acute myeloid leukemia is dependent on FLT3/ITD status. Blood 2010;116:2779–82. [14] Schnittger S, Haferlach C, Ulke M, Alpermann T, Kern W, Haferlach T. IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status. Blood 2010;116:5486–96. [15] Wouters BJ, Loewenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood 2009;113:3088–91. [16] Mufti GJ, Bennett JM, Goasguen J, Bain BJ, Baumann I, Brunning R, et al. Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts. Haematologica 2008;93:1712–7. [17] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French–American–British Cooperative Group. Ann Intern Med 1985;103:620–5. [18] Swerdlow S, Campo E, Lee Harris N, Jaffe E, Pileri S, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. IARC Press; 2008.
58
T. Haferlach et al. / Leukemia Research 36 (2012) 51–58
[19] Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U, et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: a study on 350 cases. Leukemia 2002;16:53–9. [20] Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;104:3078–85. [21] Schnittger S, Kern W, Tschulik C, Weiss T, Dicker F, Falini B, et al. Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic information in AML. Blood 2009;114:2220–31. [22] Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S. Risk assessment by monitoring expression levels of partial tandem duplications in the MLL gene in acute myeloid leukemia during therapy. Haematologica 2005;90:881–9. [23] Snaddon J, Smith ML, Neat M, Cambal-Parrales M, Dixon-McIver A, Arch R, et al. Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2. Genes Chromosomes Cancer 2003;37:72–8. [24] Dufour A, Schneider F, Metzeler KH, Hoster E, Schneider S, Zellmeier E, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol 2010;28:570–7. [25] Gilliland DG. Hematologic malignancies. Curr Opin Hematol 2001;8:189–91. [26] Renneville A, Roumier C, Biggio V, Nibourel O, Boissel N, Fenaux P, et al. Cooperating gene mutations in acute myeloid leukemia: a review of the literature. Leukemia 2008;22:915–31. [27] Brady SN, Yu Y, Maggi Jr LB, Weber JD. ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway. Mol Cell Biol 2004;24:9327–38.
[28] Meloni G, Mancini M, Gianfelici V, Martelli MP, Foa R, Falini B. Late relapse of acute myeloid leukemia with mutated NPM1 after eight years: evidence of NPM1 mutation stability. Haematologica 2009;94:298–300. [29] Falini B, Mecucci C, Saglio G, Lo Coco F, Diverio D, Brown P, et al. NPM1 mutations and cytoplasmic nucleophosmin are mutually exclusive of recurrent genetic abnormalities: a comparative analysis of 2562 patients with acute myeloid leukemia. Haematologica 2008;93:439–42. [30] Gilliland DG, Griffin JD. Role of FLT3 in leukemia. Curr Opin Hematol 2002;9:274–81. [31] Hayakawa F, Towatari M, Kiyoi H, Tanimoto M, Kitamura T, Saito H, et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000;19:624–31. [32] Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Müller C, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 2000;96:3907– 14. [33] Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL, Gilliland DG. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 2002;99:310–8. [34] Lee BH, Tothova Z, Levine RL, Anderson K, Buza-Vidas N, Cullen DE, et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell 2007;12:367–80. [35] Steudel C, Wermke M, Schaich M, Schäkel U, Illmer T, Ehninger G, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer 2003;37:237–51.