Letter to the Editor
Prognosis in patients with MDS or AML and bone marrow blasts between
10% and 30% is not associated with blast counts but depends on
cytogenetic and molecular genetic parameters
- Online Supplement -
ADDITIONAL REFERENCES RELATED TO METHODS
Cytomorphology
1. Löffler H, Raststetter J, Haferlach T. Atlas of Clinical Hematology. 6th edition. Springer, Berlin,
2004.
2. 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 FrenchAmerican-British Cooperative Group. Ann Intern Med 1985; 103: 620-5.
3. 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 edition. IARC press, Lyon, 2008.
Chromosome banding analysis
4. 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.
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Molecular mutation analysis
5. 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.
6. 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.
7. 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.
8. 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.
9. Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S. Implications of NRAS mutations in
AML: a study of 2502 patients. Blood 2006; 107: 3847-53.
10. 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.
11. Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T, et al. RUNX1
mutations are frequent in de novo AML with non complex karyotype and confer an
unfavourable prognosis. Blood 2010; epub ahead of print 9 Dec 2010; doi: 10.1182/blood2009-11-255976.
12. 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.
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Immunophenotyping with multiparameter flow cytometry (MFC)
13. 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.
14. Kern W, Haferlach C, Schnittger S, Haferlach T. Clinical utility of multiparameter flow
cytometry in the diagnosis of 1013 patients with suspected myelodysplastic syndrome:
correlation to cytomorphology, cytogenetics, and clinical data. Cancer 2010; 116: 4549-63.
Microarray gene expression profiling
15. Kohlmann A, Kipps TJ, Rassenti LZ, Downing JR, Shurtleff SA, Mills KI, et al. An international
standardization programme towards the application of gene expression profiling in routine
leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase. Br J
Haematol 2008; 142: 802-7.
16. Kohlmann A, Haschke-Becher E, Wimmer B, Huber-Wechselberger A, Meyer-Monard S,
Huxol H, et al. Intraplatform reproducibility and technical precision of gene expression profiling
in 4 laboratories investigating 160 leukemia samples: the DACH study. Clin Chem 2008; 54:
1705-15.
17.
Miesner M, Haferlach C, Bacher U, Weiss T, Macijewski K, Kohlmann A, Klein HU, Dugas M,
Kern W, Schnittger S, Haferlach T. Multilineage dysplasia (MLD) in acute myeloid leukemia
(AML) correlates with MDS-related cytogenetic abnormalities and a prior history of MDS or
MDS/MPN but has no independent prognostic relevance: a comparison of 408 cases
classified as "AML not otherwise specified" (AML-NOS) or "AML with myelodysplasia-related
changes" (AML-MRC). Blood 2010; 116: 2742-51.
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SUPPLEMENTARY TABLES
Table S1: Overall survival (OS) according to cytomorphology, cytogenetics, and molecular
genetics.
Parameter
Number of
patients
Median OS
(months)
10%-19% BM blasts (= MDS cohort)
20%-30% (= AML cohort)
96
180
34.4
43.7
Cytogenetic risk group (MRC criteria)
intermediate karyotype
p
Separation in two blast categories
n.s.
213
43.7
unfavorable karyotype
Molecular subgroups
63
13.9
NPM1 mutated
NPM1 wild-type
50
226
n.r.
27.3
0.005
FLT3-ITD positive
FLT3-ITD negative
12
264
34.9
14.7
n.s.
NPM1-mutated/FLT3-ITD-negative
all other NPM1/FLT3-ITD compositions
41
235
n.r.
27.3
0.002
MLL-PTD positive
MLL-PTD negative
18
258
34.9
n.r.
n.s.
RUNX1-mutated
RUNX1 wild-type
34
82
14.1
n.r.
n.s.
4
<0.001
Table S2: Uni- and multivariate analyses for definition of prognostically relevant parameters with
regard to overall survival (OS). *Cytogenetic risk stratification followed revised MRC criteria.
**NPM1mut/FLT3-ITD-negative status was associated with a better prognosis when compared to all
other molecular subgroups. Due to overlapping features, only NPM1mut/FLT3-ITD-negative status, but
not the NPM1mut status considered as single parameter, was included in the multivariate analysis.
Parameter
Univariate (p)
Multivariate (p)
age
WBC count
<0.001
n.s.
0.048
-
platelets
hemoglobin
0.018
n.s.
0.033
-
10%-19% vs 20%-30% BM blasts
cytogenetic risk group*
n.s.
<0.001
0.010
FLT3-ITD mutation status alone
NPM1mut status alone
n.s.
0.008
-
NPM1mut/FLT3ITD-negative status vs
all others**
0.005
n.s.
MLL-PTDmut status alone
RUNX1mut status alone
n.s.
n.s.
-
CD34+ cells (multiparameter flow
cytometry)
n.s.
-
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Legend to Supplemental Table S3A-C
Table S3: Results of gene expression analysis from 26 patients of the cohort. A: comparing
the MDS (n=11) versus AML (n=15) cases; B: comparing cases with intermediate (n=16) and
with unfavorable karyotypes (n=10); C: comparing cases with normal (n=9) and abnormal
(n=17) karyotypes.
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SUPPLEMENTARY FIGURES
Supplemental Figure S1: Frequency of cytogenetic and molecular alterations in MDS and AML
cohorts. The y-axis shows the percentages of cases carrying the respective genetic alteration. The
grey columns visualize the MDS cases, the red columns the AML patients, respectively (unfav.:
unfavorable; KTs: karyotypes; complex: complex aberrant karyotypes).
Supplemental Figure S2: Hierarchical clustering analysis of global gene expression profiles. For
supervised statistical analyses of the gene expression data, the samples were grouped accordingly
and for each respective category differentially expressed genes were calculated by means of t-tests.
To assess significance, false discovery rates (FDR) were calculated. Results of gene expression
profiling are shown in a total of 26 cases (15 AML, 11 MDS) by hierarchical clustering using Euclidean
distance and complete linkage parameters. The 26 cases are shown in columns; the rows depict the
gene expression intensity for the genes from the top differentially expressed genes. Each heatmap
depicts the gene expression signature for the top-500 differentially detected probe sets. A: Results of
gene expression analysis comparing cases with 10%-19% (group 0) and cases with 20%-30% of BM
blasts (group 1). B: Gene expression profiles of cases with intermediate (group 1) versus unfavorable
karyotypes (group 2). C: Gene expression profiles of cases with normal (group 0) versus abnormal
karyotypes (group 1). Detailed information on the differentially expressed probe sets and their
functional annotation is available online. Note: the raw p-value did not remain significant after strict
adjustment for multiple testing.
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