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Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML)

Christian Thiede, Sina Koch, Eva Creutzig, Christine Steudel, Thomas Illmer, Markus Schaich and Gerhard Ehninger for the Deutsche Studieninitiative Leukämie (DSIL)

Article Figures & Data

Figures

  • Figure 1.

    Mutations of NPM1 found in 229 sequenced patients. Comparison of the nucleotide and deduced amino acid sequences of mutations identified in AML patients. Amino acids are given in single-letter code. The blue boxes denote the 2 tryptophan residues at amino acid positions 288 and 290 important for nuclear transport.34 (A) Mutations described by Falini et al.15 (B) Novel mutations. *Also described by Suzuki et al.35

  • Figure 2.

    Confocal laser scanning microscopy of NPM1 wt and mutant cells. Cells of the NPM1-wt cell line MV4-11 (A) or mononuclear cells from patient samples with mutations DD-3 (B), DD-4 (C), or DD-1 (D) were stained with a monoclonal antibody against NPM1, Cy-3 labeled with a secondary antibody, and analyzed using confocal laser scanning microscopy. Nuclei were stained with DAPI; original magnification, × 240.

  • Figure 3.

    Analysis of NPM1 and FLT3-mutant/wt ratio in patient samples. (A) Genescan analysis for NPM1 and FLT3-ITD. Lanes 1 and 2 show examples of an NPM1-wt– and FLT3-ITD–negative case. Lanes 3 through 6 illustrate two patients with mutant NPM1 and FLT3-ITD mutations. Note that in both cases, two independent FLT3-ITD mutations were found. (B) Comparison of NPM1 and FLT3-ITD mutant/wt ratio in 149 samples analyzed for both abnormalities. (C) Comparison of NPM1 and FLT3 in 3 different groups according to the difference between NPM1-mut/wt and FLT3-ITD mut/wt ratio.

  • Figure 4.

    Kaplan-Meier analysis of the actuarial probability of OS and DFS in all patients analyzed. Comparison of OS (A) and DFS (B) in all patients according to the presence or absence of NPM1 mutations.

  • Figure 5.

    Kaplan-Meier analysis of NPM1 and FLT3-ITD mutations in patients with AML and normal karyotype. Kaplan-Meier analysis of OS (A) and DFS (B) in AML patients with normal karyotype according to NPM1 mutations; OS (C) and DFS (D) according to NPM1 and FLT3-ITD groups. Analysis was done in the 4 groups defined in the text. Patients in group A (NPM1-mut alone) had a significantly higher probability of OS than group B (double mutants; P = .001), group C (FLT3-ITDpos only; P = .032), and group D (wt for both; P = .03). Also, the DFS was significantly higher in group A than group B (P = .04), group C (P < .001), and group D (P = .04).

  • Figure 6.

    OS, DFS, and CIR in AML patients (60 years or younger) with normal karyotype. (A) Comparison of the probability of OS according to the NPM1 and FLT3-ITD mutational status in the 4 defined groups. Group A (NPM1-mut alone) had a significantly higher actuarial probability of OS than group B (double mutants; P = .003) and D (all wt; P = .02), and trend was seen versus group C (FLT3-ITD alone; P = .11). (B) Probability of DFS in the 4 groups. Group A had a significantly higher probability of disease-free survival than group B (P = .02), group C (P < .001), and group D (P = .006). (C) CIR in the 4 groups. Analysis was done using the Gray k-sample algorithm in patients with normal karyotype showing a significantly reduced CIR for group A (CIR at 40 months, 25%) compared with the other groups (P = .004). Highest relapse rates were seen in group B (57%) and group C (51%); the CIR was 32.7% in group D.

Tables

  • Table 1.

    Cytogenetic risk groups

    Risk groupAbnormalities
    Low risk t(8;21) with or without additional abnormalities
    Intermediate risk All other abnormalities
    High risk -5/del(5q); -7/del(7q); other monosomies; inv(3q); t(3;3); abnl 12p; abnl 11q; +11; +1 +21; +22; t(6;9); t(9;22); multiple aberrations (3 or more structural or numerical abnormalities)
  • Table 2.

    Clinical features and NPM1 and FLT3 mutations in AML and RAEB-t patients at diagnosis

    NPM1-mut/FLT3-ITDnegNPM1-mut/FLT3-ITDposNPM1-wt/FLT3-ITDposNPM1-wt/FLT3-ITDneg
    No. 244 164 148 929
    Median BM blasts, % (range) 67.3 (6-95)* 75.5 (29.5-100)* 73 (11-96) 56 (0.5-98.5)
    Median WBC count, × 109/L (range) 26.3 (0.5-380)* 50 (1.1-372)* 36 (0.8-465) 7.7 (0.3-450)
    Median platelet count, × 109/L (range) 68 (7-302)* 56 (3-514) 47 (5-372) 46 (4-1430)
    Median LDH level, U/mL (range) 449 (27-4065)* 544 (23-7250)* 607 (16-7096) 369 (4.5-5274)
    Median age, y (range) 60 (18-83) 57.5 (19-81) 54 (17-83) 58 (15-87)
    Female, % 55.3 62.2 49.3 43.3
    De novo AML, no. (%) 223 (91.4)* 150 (91.5) 129 (87.2) 719 (77.4)
    Prior MDS, no. (%) 16 (6.6)* 12 (7.3) 12 (8.1) 149 (16.0)
    TAML, no. (%) 2 (0.8)* 2 (1.2) 5 (3.4) 46 (5.0)
    FAB
        M0, no. (%) 0 (0)* 2 (1.2) 2 (1.4) 57 (6.1)
        M1, no. (%) 50 (20.5) 47 (28.7) 43 (29.1) 180 (19.4)
        M2, no. (%) 79 (32.4) 50 (30.5) 37 (25) 290 (19.4)
        M3, no. (%) 0 (0)* 0 (0) 12 (8.1) 43 (4.6)
        M4, no. (%) 37 (15.2) 20 (12.2) 27 (18.2) 87 (9.4)
        M4eo, no. (%) 1 (0.4) 0 (0) 4 (2.7) 66 (7.1)
        M5a, no. (%) 41 (16.8) 25 (15.2) 11 (7.4) 78 (8.4)
        M5b, no. (%) 21 (8.6)* 14 (8.5) 1 (0.7) 14 (1.5)
        M6, no. (%) 2 (0.8) 0 (0) 2 (1.4) 46 (5.0)
        M7, no. (%) 0 (0) 2 (1.2) 0 (0) 10 (1.1)
    RAEB-t, no. (%) 7 (2.9) 1 (0.6) 2 (1.4) 37 (4.0)
    RAEB1, no. (%) 1 (0.4) 0 (0) 0 (0) 5 (0.5)
    RAEB2, no. (%) 2 (0.8) 0 (0) 2 (1.4) 10 (1.1)
    • RAEB-t indicates refractory anemia with excess of blasts in transformation; t-AML, therapy-related AML.

    • * P < .001.

    • P < .01.

    • Patients with FAB M6.

  • Table 3.

    Comparison of cytogenetic aberrations and NPM1 and FLT3-ITD mutations in patients with AML (n = 1485)

    NPM1-mut/FLT3-ITDnegNPM1-mut/FLT3-ITDposNPM1-wt/FLT3-ITDposNPM1-wt/FLT3-ITDneg
    No. 244 164 148 929
    Unknown, no. (%) 14 (5.7) 12 (7.3) 7 (4.7) 57 (6.1)
    Normal, no. (%) 182 (79.1) 142 (93.4) 76 (53.9) 309 (35.4)
    Abnormal, no. (%) 48 (20.9) 10 (6.6) 65 (46.1) 563 (64.6)
    t(8;21), no. 2 0 3 52
    inv(16)/t(16;16), no. 2 0 5 60
    t(15;17), no. 0 0 11 36
    +8, no. 11 1 12 100
    -7/7q-, no. (%) 5 0 3 114
    -5/5q-, no. (%) 3 0 0 91
    t(6;9), no. 0 0 7 0
    Complex, no.* 4 0 5 176
    -X/-Y, no. 7 2 2 48
    FLT3D835/836, no. (%) 31/204 (15.2) 8/133 (6.0) 9/124 (7.3) 42/772 (5.4)
    MLL-PTD, no. (%) 0/126 (0) 0/81 (0)§ 11/73 (15.1)§ 28/476 (5.9)
    • The number of patients with individual aberrations adds to a higher number than the absolute number of patients with aberrations because several patients had more than one aberration.

    • * Three or more structural or numerical chromosomal aberrations.

    • P < .01.

    • P < .001.

    • § P < .05.

    • No. of positive cases/total cases analyzed.

  • Table 4.

    Multivariate analysis for cytogenetics and clinical and biologic variables (all patients)

    All patients within studyPatients 60 y or younger
    OSDFSOSDFS
    POR (95% CI)POR (95% CI)POR (95% CI)POR (95% CI)
    Age, 60 y or younger vs older than 60 y < .001 1.90 (1.65-2.15) < .001 1.91 (1.50-2.42)
    Cytogenetics*
        Low < .001 0.48 (0.33-0.69) .002 0.48 (0.30-0.77) .005 0.55 (0.36-0.84) .039 0.56 (0.32-0.97)
        High < .001 1.86 (1.57-2.2) < .001 2.08 (1.51-2.87) < .001 1.80 (1.42-2.80) .001 1.98 (1.35-2.91)
    WBC count
        Intermediate (> 9.2 ≤ 21.7 GPT/L) < .005 1.30 (1.08-1.55) .024 1.43 (1.05-1.96) .082 1.26 (0.97-1.64) NS
        Highest, 21.7 or more GPT/L < .001 1.48 (1.25-1.75) < .001 1.88 (1.41-2.51) .002 1.48 (1.16-1.89) .005 1.69 (1.17-2.44)
    De novo AML vs sAML .001 0.73 (0.58-0.91) .013 0.63 (0.44-0.91) .002 0.65 (0.49-0.85) .041 0.60 (0.36-0.98)
    NPM1-FLT3 group
        NPM1-mut/FLT3-ITDneg .005 0.73 (0.58-0.91) .086 0.75 (0.54-1.042) .037 0.71 (0.51-0.98) .082 0.67 (0.43-1.05)
        NPM1-mut/FLT3-ITDpos NS NS .089 1.33 (0.96-1.87) NS
        NPM1-wt/FLT3-ITDpos NS < .001 1.97 (1.38-2.82) NS .014 1.70 (1.12-2.59)
    • For all patients, n = 1328; for patients 60 years old or younger, n = 737.

      Not significant (NS) were the following: platelets, LDH (log), sex, MLL-PTD, FLT3-TKD, BM blasts.

      GPT/L indicates gigaparticles per liter; —, not applicable.

    • * Cytogenetics were as defined by the Medical Research Council (MRC) study group; if more than two groups were built, a reference group was used.

    • For cytogenetics, the intermediate group was was set as the reference group; for WBC counts, lowest WBC count (9.2 or fewer GPT/L) was set as the reference group; and for de novo AML versus sAML NPM1-FLT3 group, NPM1-wt/FLT3-ITDneg was set as the reference group.

  • Table 5.

    Multivariate analysis of outcome in patients with normal karyotype

    All patients with normal karyotypePatients with normal karyotype, 60 y or younger
    OSDFSOSDFS
    POR (95% Cl)POR (95% Cl)POR (95% Cl)POR (95% Cl)
    Age, 60 y or younger vs older than 60 y <.001 1.69 (1.38-2.07) <.001 1.94 (1.42-2.66)
    NPM1-FLT3 group*
        NPM1-mut/FLT3-ITDneg .043 0.76 (0.59-0.99) .036 0.66 (0.45-0.97) .019 0.63 (0.43-0.93) .009 0.49 (0.29-0.84)
        NPM1-mut/FLT3-ITDpos NS NS NS NS
        NPM1-wt/FLT3-ITDpos NS .006 2.02 (1.22-3.34) NS NS
    WBC count*
        Intermediate (>9.2 ≤ 21.7 GPT/L) NS NS NS NS
        Highest, 21.7 or more GPT/L .002 1.87 (1.25-2.79) .001 2.25 (1.40-3.63) .006 1.66 (1.15-2.34) .019 1.86 (1.11-3.13)
    De novo AML versus sAML NS .039 0.53 (0.29-0.97) NS NS
    • For all patients with normal karyotype, n = 701; for those 60 years old or younger, n = 387.

      Not significant (NS) were the following: platelets, LDH (log), sex, MLL-PTD, FLT3-TKD, BM blasts.

      — indicates not applicable.

    • * Reference group for NPM1-FLT3 was NPM1-wt/FLT3-ITDneg; for WBC count, those with 9.2 or fewer GPT/L.

  • Table 6.

    Comparison of SCT procedures performed in first CR in patients who had de novo AML, were 60 years old or younger and had normal karyotype (N = 154)

    NPM1-mut/FLT3-ITDnegNPM1-mut/FLT3-ITDposNPM1-wt/FLT3-ITDposNPM1-wt/FLT3-ITDneg
    No. 44 28 25 57
    Autologous, no. 29 18 13 25
    Allogeneic, family, no. 15 8 10 27
    Allogeneic, unrelated, no. 0 2 2 5