Tumour samples
Immunohistochemical studies were carried out in paraffin-embedded bone marrow
biopsies from 20 NPMc+ AML patients. All bone marrow biopsies were fixed in B5 and
decalcified in EDTA, as previously described1. All cases showed a normal karyotype and
CD34 negativity; 17 cases carried NPM mutation A, 2 cases mutation B and one case
mutation D.
Immunohistochemistry: The following antibodies were used to detect NPM in bone marrow
paraffin sections: monoclonal anti-NPM antibody (clone 376) recognizing both wild-type and
mutant NPM2; polyclonal antibody Sil-A that detects the mutated but not wild-type NPM2.
Other antibodies used in this study were: a mouse monoclonal antibody directed against a
fixative-resistant epitope of the CD20 molecule (Dako, Glostrup, Denmark) and a rabbit
monoclonal antibody directed a fixative-resistant epitope of the CD3 molecule (Lab Vision
Corporation, Fremont, CA, USA).
Western blotting: The following antibodies were used for Western blot analysis: monoclonal
anti-NPM antibody (clone 376); polyclonal antibody Sil-A specific for NPM mutants; and a
mouse monoclonal antibody that recognizes the wild-type but not the mutated NPM proteins
(Invitrogen, Carlsbad, CA, USA)
Immunohistochemical studies
Paraffin sections from all bioptic samples were subjected to antigen retrieval
(microwaving in 1 mM EDTA buffer, pH 8.0) and incubated with primary antibodies. The
antigen-antibody reaction was revealed using the immuno-alkaline phosphatase (APAAP)
technique; double immunoenzymatic staining for CD3/NPM and CD20/NPM was performed
using a sequential /Immunoperoxidase/ APAAP procedure.
Double immuno-fluorescence staining for CD3/NPM was carried out in paraffin
sections using the rabbit monoclonal antibody anti-CD3 in combination with the mouse
monoclonal anti-NPM (clone 376). The rabbit monoclonal antibody against CD3 was revealed
with a secondary goat anti-rabbit antibody IgG (H + L) (Alexa 568); the mouse monoclonal
antibody against NPM was revealed with a secondary goat anti-mouse IgG (H + L) (Alexa
Purification of lymphoid cells from AML patients. Peripheral blood from patients with AML
was collected at diagnosis upon patient informed consensus and subjected to separation on
Ficoll-Hypaque. In each sample, representation of leukaemic cells and lymphoid populations
(B or T cells) was analyzed by FACS scan using anti-CD19 or CD20, anti-CD3, and antiCD33 and CD117 monoclonal antibodies, according to standard procedures. Samples
containing >1% of either T or B-cells were subjected to T and B-cells purification. T lymphoid
cells were purified using E-rosetting technique according to standard procedure. T-cell purity
was evaluated by FACS scan analysis with anti-CD3, anti-CD19 and anti-CD33 monoclonal
antibodies. Only samples with T-cell representation >70% and leukaemic blast cells
percentage less than 10% were included in our study. Successful T-cell purification was
obtained in seven NPMc+ AML patients.
B lymphoid cells were purified using anti-CD19 specific immunomagnetic beads
according to manufacturer’s protocol (Miltenyi Biotec, Bergish Gladbach, Germany), and
purity evaluated by anti-CD20 immunostaining of a single cytospin preparation.
lymphoid B cells for Western blot analysis of NPM mutant proteins could be obtained only in
two NPMc+ AML samples (90% purity).
Western Blot analysis. Either 1 x 106 leukaemic blast cells or 0.2-1 x 106 lymphoid cells
isolated from peripheral blood of patients with NPMc+ AML were dissolved directly in 30 l of
Laemmli sample buffer 1X and boiled at 95°C for 5 minutes. Proteins were separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% acrylamide-bisacrylamide
electrophoresis gel, transferred to polyvinylidene difluoride membrane (PVDF; Millipore,
Billerica, MA), and probed with a rabbit polyclonal antibody anti-NPM mutant (anti-NPMm)
and a mouse monoclonal anti-NPM wild-type antibody (clone FC-61991, Invitrogen, Carlsbad,
CA) followed by horseradish peroxidase-conjugated secondary antibodies (GE Healthcare
Lifesciences, Uppsala, Sweden). Polypeptides were visualized using either the enhanced
chemiluminescence (ECL) or ECL-Plus western blot detection system according to the
manufacturer’s instruction (GE Healthcare Lifesciences).
The human OCI/AML3 leukaemic cell line which carries an exon-12 NPM1 mutation A3
was used as positive control for NPM mutant A protein expression.
1. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio
D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi
P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF.
Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.
N Engl J Med. 2005;352:254-266.
2. Pasqualucci L, Liso A, Martelli MP, Bolli N, Pacini R, Tabarrini A, Carini M, Bigerna B,
Pucciarini A, Mannucci R, Nicoletti I, Tiacci E, Meloni G, Specchia G, Cantore N, Di
Raimondo F, Pileri S, Mecucci C, Mandelli F, Martelli MF, Falini B. Mutated
nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia:
Impact on WHO classification. Blood. 2006;108:4146-4155.
3. Quentmeier H, Martelli MP, Dirks WG, Bolli N, Liso A, MacLeod RAF, Nicoletti
I, Mannucci R, Pucciarini A, Bigerna B, Martelli MF, Mecucci C, Drexler HG, Falini B.
Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression
of nucleophosmin. Leukemia. 2005;19:1760-1767.
Supplementary Figure 1 legend
Supplementary Figure 1. Immunohistochemical analysis of T cells in NPMc+ AML. A)
Bone marrow biopsy (paraffin section) from a NPMc+ AML patient with FAB-M4 morphology
(hematoxylin-eosin; x 800). B) Paraffin section from the same case double stained for CD3
(rabbit monoclonal antibody) by immunoperoxidase (brown) and NPM (mouse monoclonal
antibody 376) by the APAAP technique (blue). As expected, leukaemic cells are CD3negative and show nuclear plus aberrant cytoplasmic positivity for NPM (blue) (double
arrows); in contrast, CD3 cells display surface CD3-positivity (brown) and nucleus-restricted
positivity for NPM (blue) (single arrows). No aberrant cytoplasmic positivity indicative of NPM1
mutations is observed in CD3-positive T cells (no counterstaining; x 800).
Supplementary Figure 1
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