Material and Method: Morphological, haematological and immunophenotypic characteristics of NPM1 and FLT3 mutations in 126 patients of de novo AML including adults and children were studied. Apart from the French American British (FAB) method for classification, blasts were assessed for cuplike morphology as per strict definition for cuplike nuclei, ≥10% blasts with nuclear invaginations ≥25% of the nuclear area.
Results: FLT3 mutation in 31/126 (25%) and NPM1 mutation was found in 17/126 (13.4%) of the AML patients. 6 (5%) samples were positive for both NPM1 and FLT3/ITD mutations. Associations between the FLT3 and NPM1 gene mutations with haematological and immunophenotypic characteristics are reported.
Conclusion: The results suggest that presence of distinct morphology and haematological and immunophenotypic characteristics together may serve as important indicators and surrogate for NPM1 and FLT3/ITD mutations. Further, comprehensive studies on the biological effects of NPM1 and FLT3/ITD mutations and their interactions with other genetic alterations are needed to gain insight into the molecular mechanism of these mutations involved in the pathogenesis of AML.
Morphology and Cytochemical Stains
Peripheral blood and bone marrow smears were stained
with Leishman and Giemsa stain and cytochemistry was
performed by standard methods (Myeloperoxidase-MPO; Periodic Acid Schiff-PAS; non specific esterase-NSE).
Apart from the French American British (FAB) method for
classification, blasts were assessed for cuplike morphology
as per strict definition for cuplike nuclei, ≥10% blasts with
nuclear invaginations ≥25% of the nuclear area.
Immunophenotyping
Flow cytometry was performed on P.B/B.M on a 4 colour
Flowcytometer (FACSCalibur, Becton Dickinson, San Jose,
CA,USA) using a standard protocol. Monoclonal antibodies
used in this study included fluorescein isothiocyanate
(FITC), phycoerythrin (PE), peridinin chlorophyll-protein
(Per-CP) or allophycocyanin (APC) labelled CD45, CD2,
CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD19,
CD22, CD33, CD34, CD38, CD56, CD117, HLA-DR, Tdt,
Cyto MPO and isotype control IgGs.
Molecular Tests
PCR for FLT3 and NPM1 mutations was carried out at an
NABL accredited lab. FLT3/ITD mutations were assessed by
using specific primers for exon 11, 12 and D835 mutation
by Restriction Fragment Length Polymorphism (RFLP)
mediated assay using primers flanking the mutation site.
For NPM1 mutation, amplification of exon 12 of the NPM1
gene was carried out followed by analysis of PCR products
using automated DNA sequencing.
Statistical Analysis
The data was subjected to statistical analysis using unpaired
students t-test, Chi square test and linear regression
analysis.
Figure 1: Peripheral smear showing blasts with cup like morphology (Leishman stain; x400) .
Table I: Comparison between FLT3/NPM1 positive and negative cases
Table II: Immunophenotypic characteristics of FLT3 and NPM1 positive groups
Cuplike morphology of blasts was seen in 81% of the cases positive of FLT3 and/or NPM1 mutation. This distinct morphology has been described in the literature to be associated with FLT3 and NPM1 mutations [7], however significant conclusions in this regard could not be drawn in the present study because of the lower number of cases.
Immunophenotypically, combination detection of CD117/ CD7 was found to be significantly associated with FLT3 mutations in AML cases. Recent studies have found CD7 as an important marker and predictor of FLT3 mutations in AML cases [8]. High frequency of FLT3 mutations had been described in CD117 positive T-ALL cases [9]. We suggest that expression of CD7 along with CD117 may be taken as a surrogate profile for FLT3 mutation.
NPM1 mutation was significantly associated with the lack of CD34 (23.5% vs. 73%) and expression of CD14 (52.9% vs. 14.8%), compared to the group without NPM1 mutation. On regression analysis, CD14 and CD34 were found to be most important indicators of NPM1 mutation. The NPM1 mutation was inversely associated with the expression of CD34 in several European studies [3,10] and those from South Asian countries on AML [4]. Chauhan et al. reported the similar lack of CD34 expression in AML cases with NPM1 mutation [5].
In conclusion, the results suggest that presence of distinct morphology together with haematological and immunophenotypic characteristics may serve as important indicators and surrogate for NPM1 and FLT3/ ITD mutations. Further, comprehensive studies on the biological effects of NPM1 and FLT3/ITD mutations and their interactions with other genetic alterations are needed to gain insight into the molecular mechanisms of these mutations involved in the pathogenesis of AML.
CONFLICT of INTEREST
The authors declare no conflict of interest
1) Gilliland DG1, Griffin JD. The roles of FLT3 in hematopoiesis
and leukemia. Blood. 2002;100:1532-42.
2) Suzuki T, Kiyoi H, Ozeki K, Tomita A, Yamaji S, Suzuki R, Kodera
Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki C,
Akiyama H, Nishimura M, Motoji T, Shinagawa K, Takeshita A,
Ueda R, Kinoshita T, Emi N, Naoe T. Clinical characteristics and
prognostic implications of NPM1 mutations in acute myeloid
leukemia. Blood. 2005;106:2854-61.
3) Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich
M, Ehninger G. Prevalence and prognostic impact of NPM1
mutations in 1485 adult patients with acute myeloid leukemia
(AML). Blood. 2006;107:4011-20.
4) Boonthimat C, Thongnoppakhun W, Auewarakul CU.
Nucleophosmin mutation in Southeast Asian acute myeloid
leukemia: Eight novel variants, FLT3 coexistence and prognostic
impact of NPM1/FLT3 mutations. Haematologica. 2008;93:1565-9.
5) Chauhan PS, Ihsan R, Singh LC, Gupta DK, Mittal V, Kapur S.
Mutation of NPM1 and FLT3 genes in acute myeloid leukemia
and their association with clinical and immunophenotypic
features. Dis Markers. 2013;35:581-8.
6) Kiyoi H, Ohno R, Ueda R, Saito H, Naoe T. Mechanism of
constitutive activation of FLT3 with internal tandem duplication
in the juxtamembrane domain. Oncogene. 2002;21:2555-63.
7) Park BG, Chi HS, Jang S, Park CJ, Kim DY, Lee JH, Lee JH, Lee
KH. Association of cup-like nuclei in blasts with FLT3 and NPM1
mutations in acute myeloid leukemia. Ann Hematol. 2013;92:451-7.
8) Rausei-Mills V, Chang KL, Gaal KK, Weiss LM, Huang Q.
Aberrant expression of CD7 in myeloblasts is highly associated
with de novo acute myeloid leukemias with FLT3/ITD mutation.
Am J Clin Pathol. 2008;129:624-9.