12th Congress of the European Hematology ... - Haematologica

0576
WESTERN BLOT IDENTIFICATION OF NPM1 LEUKEMIC MUTANTS IN CYTOLOGICAL AML
SAMPLES: A POWERFUL DIAGNOSTIC TECHNIQUE
M.P. Martelli, N. Manes, A Liso, B Verducci Galletti, V. Pettirossi,
M.T. Pallotta, N. Bolli, R. Pacini, A. Tabarrini, F. Di Raimondo,
F. Fabbiano, G. Specchia, M.F. Martelli, B. Falini
Hematology, BARI, Italy
Nucleophosmin (NPM1) mutations occur in 50-60% of adult acute
myeloid leukemia (AML) patients with normal karyotype and are predictors
of favourable prognosis. For these reasons, analysis of NPM1
mutations has recently become a major new step in the diagnosis and
prognostic stratification of AML patients. About 40 different molecular
NPM1 mutation variants have been so far identified, with about 90%
represented by mutation A (about 80%) and B (about 10%), other NPM1
mutations being extremely rare. Mutations of the NPM1 gene can be reliably
identified by molecular biology techniques or by immunohistochemistry
through detection of aberrant cytoplasmic NPM positivity
(NPMc+). Mutational analysis is carried out only in specialized laboratories;
immunohistochemistry overcome this problem but it is only
applicable to paraffin sections from bone marrow biopsies. Hereby, we
describe a highly sensitive and specific Western blot method that allows
the easy identification of NPM1 leukemic mutants in cytological samples
from AML patients. Rabbit polyclonal antibodies were generated
against the altered C-terminal portion of the most common NPM mutant
protein (type A). Western blot analysis of a selected number of AML
samples proved that these antibodies reacted specifically with the NPM
mutant but not with the wild-type NPM protein. These findings prompted
us to use this method to analyze systematically cytological leukemic
samples and to compare blindly the results obtained by Western Blot
with those derived from immunohistochemical studies and, when available,
from NPM1 mutational analysis. A total of 114 AML patients classified
by immunohistochemistry into NPMc + (cytoplasmic-positive;
n=57) and NPMc- (cytoplasmic-negative; n=57) were enclosed in the
study. Western Blot analysis was performed retrospectively in 83 AML
cases and prospectively in 32 cases. We investigated a total of 174 cytological
preparations (82 from NPMc + and 92 from NPMc- AMLs) of various
types, including frozen dry cell pellets of Ficoll-isolated leukemic
cells; 1 to 2 drops of fresh whole bone marrow or peripheral blood; or
even cells obtained scraping the surface of leukemic cytospins or smears.
Western Blot analysis was performed according to standard procedures.
Seventy-two out of 82 (88%) NPMc + AML samples resulted positive at
Western Blot analysis. Results obtained in cytological material of different
types were comparable, indicating the high flexibility of the method.
However, retrospective Western Blot analysis of badly preserved
leukemic smears sometimes gave negative Results. In addition to NPM
mutant A, the specific anti-NPM mutant antibodies recognized NPM
mutant proteins of type B, D, E, and L. For patients studied prospectively,
Western Blot analysis predicted NPM1 mutation in all (16 out of 16)
NPMc + AML patients investigated. Importantly, no false-positive results
were registered. In conclusion, Western Blot analysis represents a new
highly sensitive, specific and low cost assay for detecting the most common
NPM1 mutations in AML cytological samples processed in different
ways. This also represents the first example of employment of Western
Blot for identification of a specific genetic lesion in AML, that should
turn out to be very useful not only for leukemia diagnosis but also for
research purposes.
12 th Congress of the European Hematology Association
0577
HLA AND IMMUNOGENIC MARKERS IN PATIENTS WITH LGL-CD3+ PHENOTYPE
P. Garrido, 1 J. Almeida, 2 J. Canton, 1 M.A. Lopez-Nevot, 1 Y. Sandberg, 3
A. Langerak, 3 F. Garrido, 1 A. Orfao, 2 F. Ruiz-Cabello1 1 Hospital Virgen de las Nieves, GRANADA, Spain; 2 Cancer Investigation Center
( CSIC-USAL), SALAMANCA, Spain; 3 Erasmus Medical Center, ROT-
TERDAM, Netherlands
Background. T - Large granular lymphocyte lymphocytosis (LGL) represents
a clonal expansion of both CD3 + cells and natural killer (NK)
cells. The most common phenotype typical for this condition is T α/β
CD3 + , CD8 + , CD57 + . Autoimmune diseases (arthritis, erithroblastopenia)
are frequently associated with LGL, although, these conditions frequently
have a benign course. Recent studies support an idea that a subtype
of LGL, TCR α-β+/CD4 + T-large granular lymphocyte (LGL) lymphocytosis,
represents a clonal expansion with a CDR3 sequence homology in
TCR and a common HLA DRB1*0701 genotype, and has an antigendriven
mechanism underlying its origin ( Garrido et al. Blood. 2007. February.
PMID: 17303697). Considering that HLA DRB1*0701 is a frequent
allele in the Caucasian population, a possible defect in the immune regulation
could be relevant in the development of this clonal expansion.
OBJECTIVES: To analyse the possible implications of functional polymorphisms
in IFN- γ, CTLA - 4, IL10, IL4, TNF-α, FAS, FASL, and MICA
genes in association with autoimmune diseases and persistent viral infections
predisposing development of LGL CD4 + , CD8 + . Methods. In 41 cases
with LGL - T- CD3 + , CD8 + and in 40 cases with LGL- CD4 ++ , CD8+
we performed a single nucleotide polymorphism (SNPs) genotyping
using a Taqman 5´ allelic discrimination assay. PCR reactions were carried
out in a total reaction volume of 5 microlitres with the following
amplification protocol: denaturation at 92ºC for 15 seconds, followed by
an annealing and extension at 58ªC for 1 min. Post-PCR, the genotype
of each sample was assigned automatically by measuring the allele -specific
fluorescence on the ABI FAST 7500 Sequence Detection Systems
using the SDS 1.3.1 software for allelic discrimination (Applied Biosystems,
Foster City, CA, USA). The results were compared with 170 DNA
samples from a healthy population obtained from a blood bank. Results.
the frequencies of the studied allelic polymorphisms in the LGL CD4 + ,
CD8 + population were similar to that found in control group. Conclusions.
The results obtained suggest that the analyzed polymorphisms do not
seem to play a major role in the LGLs susceptibility.
haematologica/the hematology journal | 2007; 92(s1) | 215