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2018, Volume 34, Number 2, Page(s) 171-174
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DOI: 10.5146/tjpath.2017.01415 |
Acute Myeloid Leukaemia with Gene Mutation: A Correlation with Haematological and Immunophenotypic Characteristics and Our Experience in a Tertiary Care Cancer Center in South India |
Rachna KHERA1, Faiq AHMED1, Manasi MUNDADA1, Lavanya NAMBARU1, Sudha S MURTHY1, Sandhya DEVÝG1, Senthil J RAJAPPA2, Krishna Mohan MALLAVARAPU2, A SANTA2, Pavan KUMAR2 |
1Department of Lab Medicine, Basavatarakam Indo American Cancer Hospital and Research Institute, HYDERABAD, INDIA
2Department of Medical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, , HYDERABAD, INDIA |
Keywords: Acute myeloid leukemia, FLT3, NPM1, South India |
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Objective: Molecular genetic analysis of FLT3, NPM1, and CEBPA is already the standard of care in patients with acute myeloid leukaemia
(AML) and represents the most frequent genetic alterations and important diagnostic and prognostic indicators. This study was undertaken to
determine the frequency of FLT3 and NPM1 gene mutations in our institution and to characterize the association between gene mutations and
haematological parameters as well as immunophenotypic features.
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. |
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Acute Myeloid Leukemia (AML) develops from malignant
transformation of immature haematopoietic cells through
a complex multistep process that requires co-operation of
different types of genetic alterations 1. Several molecular
abnormalities have been shown to have prognostic
importance in patients with AML. Genetic testing for FLT3,
NPM1, and CEBPA is already the standard of care in patients
with Acute Myeloid Leukemia (AML) and represents the
most frequent genetic alterations and important diagnostic
and prognostic indicators. The study presented here was
conducted to determine the frequencies of FLT3, NPM1
gene mutations in patients of de novo AML. Associations of
these two gene mutations with haematological parameters
and immunophenotypic markers were also evaluated. |
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Abstract
Introduction
Methods
Results
Disscussion
References
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Retrospective observational study carried out in the
Department of Laboratory Medicine, Basavatarakam
Indo-American Cancer Hospital and Research Institute
(BIACH&RI), Hyderabad. 126 cases (113 adults, 13
children) with confirmed diagnosis of AML on morphology,
cytochemistry, immunophenotyping and tested for FLT3
and NPM1 gene mutations during Jan 2008 - July 2014
were included in the study. Cases diagnosed as AML on
morphology, cytochemistry and/or immunophenotyping
but not worked up further for genetic abnormalities were
excluded from the study.
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
student’s t-test, Chi square test and linear regression
analysis. |
Top
Abstract
Introduction
Methods
Results
Disscussion
References
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A total of 126 AML cases were tested for gene mutations
of FLT3 and NPM1 by PCR. The male-to-female ratio was
1.5:1 (76 males: 50 females). The age of diagnosis ranged
from 12 to 62 years with a mean of 37.5 years. 113 (89.6%)
patients were adults (18 years or older) while the remaining
13 (10.4%) patients were children (18 years or younger).
A distinct cuplike morphology of the blasts (Figure 1) was
seen in 81% of the cases positive for FLT3 and/or NPM1
mutation. FLT3 mutation was found in 31/126 (25%) and
NPM1 mutation in 17/126 (13.4%) of the AML patients. 6
(5%) samples were positive for both NPM1 and FLT3/ITD
mutations. FLT3/ITD mutation was associated with higher
white blood count (P= 0.008) and higher blast count in the
peripheral blood (P= 0.003) (Table I). Cuplike morphology
of the blasts was seen in 81% of the cases positive for
FLT3 and/or NPM1 mutation. Immunophenotypically,
the NPM1 mutation was associated with the lack of CD34 (P= 0.009), while the FLT3/ITD mutation was positively
associated with the expression of CD117 and CD7 (P=
0.04). CD34 and CD14 were found to be most important
markers for diagnosis in NPM1 cases while CD117 and
CD7 co-expression was found to be predictive for FLT3
mutations by regression analysis. Immunophenotypic
expression of markers in FLT3 and NPM1 group has been
summarized in Table II.
Click Here to Zoom |
Figure 1: Peripheral smear showing blasts with cup like
morphology (Leishman stain; x400) . |
Click Here to Zoom |
Table II: Immunophenotypic characteristics of FLT3 and
NPM1 positive groups |
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Top
Abstract
Introduction
Methods
Results
Disscussion
References
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Genetic alterations in AML are known to be major
determinants of the patients’ response to therapy and
outcome besides their role in the pathogenesis of the
disease. FLT3 and NPM1 mutations have been shown to be
the most prevalent somatic alterations in AML, particularly
in cytogenetically normal AML. In the current study,
the frequency of NPM1 and FLT3/ITD mutations was
25% (31/126) and 13.4% (17/126) in adults, respectively.
Frequency of FLT3/ITD mutation is similar to that
reported in Japanese (22.6%) 2 and in German adult AML
population (21%) 3. However, NPM1 mutations seem to
occur at much lower frequency in our setup as compared
to Japanese (24.9%) and German cohort (27.4%). The
frequency of both FLT3/ITD and NPM1 mutations was
higher in Southeast Asian region as compared to our study
(33% vs. 25% and 26% vs. 13.4% respectively) 4. Chauhan
et al. from North India reported the same frequency of
FLT3/ITD mutation as in our study, however NPM1
mutation was found to be higher (21% vs. 13.4%) 5.
Patients with FLT3/ITD mutation had significantly higher
white blood counts compared to patients without FLT3/ITD mutations (P= 0.008) in our study, similar to that reported
by Chauhan et al. FLT3/ITD mutations were also associated
with significantly higher blast counts as compared to
patients without FLT3/ITD mutations (P=0.003). Higher
WBC counts in patients with FLT3/ITD mutations can be
attributed to ligand independent constitutive activation of
FLT3 induced by ITD mutation, thus activating downstream
signal molecules that contribute to cell proliferation and
survival advantages 6. This may be the reason for the
higher WBC and blast count in these cases.
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 |
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Abstract
Introduction
Methods
Results
Discussion
References
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Abstract
Introduction
Methods
Results
Discussion
References
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