Breast response to treatment as well as disease

Breast Cancer Resistance Protein (BCRP) gene expression  in a
cohort of adult Egyptian patients with acute myeloid leukemia

 

 

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Acute myeloid
leukemia (AML) is a genetically heterogeneous clonal disorder. The multidrug
transporter Breast Cancer Resistance Protein (BCRP)
gene expression is a prognostic  marker
in adult AML.; BCRP mRNA
expression was measured  by quantitative
real time RT-PCR in 50 de novo cytogenetically normal adult AML patients and 20 healthy  normal controls. The  expression was evaluated in relation to  other clinical and prognostic factors, response
to treatment and survival. BCRP mRNA was over expressed in AML
patients. There was a positive correlation
between BCRP gene expression and the percent of CD34expression . There was no statistically significant
relation between BCRP mRNA and response to treatment as well as disease free
survival. BCRP is over expressed in AML ,but did not affect the disease
prognosis.  The biology of BCRP expression and function in
AML is more complex and needs more standardized 
clinical studies.

Key
words; BCRP; AML;  Gene expression;
prognosis.

1. Introduction;

     
Acute myeloid leukemia (AML) is a genetically heterogeneous clonal
disorder characterized by the accumulation of acquired genetic
alterations in hematopoietic progenitor cells 1, with different
categories according to  cytogenetic or
molecular abnormalities 2. Several prognostic factors has been described in
AML, including molecular markers such as gene mutations or changes in levels of
gene expression  3.

    
One of these prognostic factors which may affect the disease outcome is
the presence of transmembrane transporter proteins; which confer multidrug
resistance (MDR); and mutations or overexpression of specific genes 4.

    The human Breast Cancer Resistance Protein
(BCRP), also known as ATP-binding-cassette protein (ABCG2), coded by a
gene  located on chromosome 4q22,
encoding a 655 amino acid long protein, was confirmed to create an “atypical”
drug resistance phenotype when overexpressed in tumor cell lines 5. This
characteristic is attributed to the transport function of this transmembrane
protein that expels a wide range of chemicals from cells, including typical
chemotherapy agents, giving a particular relevance to the expression of this
protein in cancer treatment 6.

BCRP expression is a characteristic feature of cancer  cell lines which are able to self-renew and
differentiate 5. These cancer cells have been postulated to play an important
role in MDR, where their inherent BCRP expression may allow them to survive
chemotherapy and re-populate the tumor after exposure to chemotherapeutics.
This observation raises the exciting possibility that by inhibiting BCRP,
cancer cells may be targeted and eradicated 5,7.

Limited number
of clinical  prognostic studies of BCRP m-RNA
expression in AML  is available, with
different selection criteria and interpretation methodology 8-13.The aim of
the current study is to measure the level of mRNA expression of BCRP gene by
quantitative real time RT-PCR in 50 de novo cytogenetically normal adult
Egyptian AML patients in addition to  20
age and sex comparable healthy controls. The BCRP gene mRNA expression was
evaluated in relation to other prognostic factors, initial response to
induction treatment and  overall disease
free survival to verify its prognostic significance in adult Egyptian patients
with AML.

2.Subjects and methods:

2.1.Study
population;

The present
study  included 50 newly diagnosed
cytogenetically normal adult acute AML patients. Patients were
stratified morphologically according to FAB classification. Patients diagnosed as
promyelocytic leukemia(M3) , were excluded from the study due to different
therapeutic modalities and prognostic criteria. Informed consent was obtained
from all patients for the research use of their specimens. The research was
approved by the Regional Ethical Committee of National Cancer Institute.
All procedures performed were in accordance with
the  World Medical Association
Declaration of Helsink  and
its later amendments . They were  26 males (52%) and 24 females (48 %). Their
ages ranged from 18 to 71 years with a mean of 41.2±15.9 years. Twenty age and
sex-matched normal volunteers; as a control group; were included.

   Patients were subjected to thorough clinical
assessment , bone marrow examination. Immunophenotyping to confirm the
diagnosis and proper classification  of
AML with a wide panel of myeloid markers (MPO, CD13, CD33, CD117, CD14 and
CD15) and CD34 as well as CD56 and HLA-DR on routine basis. Conventional
karyotyping ,cytogenetic analysis for t(8;21) and inversion of chromosome 16
were done. In addition to the study of Nucleophosmin (NPM) gene mutations and FMS-like
tyrosine kinase 3—internal tandem duplication (FLT3-ITD).

2.2.BCRP m-RNA expression;

Total RNA was extracted from cases and control samples using QIAamp RNA blood
Mini Kit (Qiagen) according to the
manufacturer’s instructions. Then the cDNA
reaction preparation had done using High capacity cDNA Archive kit (Applied
Biosystems)
according to the manufacturer’s instructions. Primer and Taqman probe sequences
were designed with the Primer Express Software. BCRP forward primer:
5′-CAGTACTTCAGCATTCCACGAT-3′, Reverse primer: 5′-GGCAGAAGTTTTGTCCCAAA-3′. BCRP
probe: 5′-FAM-CATTATGCTGCAAAGCCGTAAATCCA-TAMRA-3′ were used. Each amplification
reaction (25 ?) contained 1 ? of the cDNA product, 12.5 ? TaqMan
universal PCR mastermix (Applied Biosytems, Inc.), 1 ? of
each primer and 0.5 ? Taqman probe. The reaction protocol
involved heating for 10 min at 95 °C, followed by 40 cycles of amplification
(15 s at 95 °C and 1min at 60 °C). Each assay included negative control without
template and the standard curve for BCRP. All tests were carried out in
triplicate. Detection of the fluorescent product was carried
out at the end of the 60 °C extension period. The “comparative threshold method” ( 2 -??
CT method) is used to calculate the relative expression levels of a
target relative to a reference control using the Ct data. The amount of target,
normalized to an endogenous housekeeping gene (GAPDH) (A gene that has
consistent expression levels in all cell types) and relative to the calibrator,
is then given by 2 -?? CT, where ?? Ct = ? Ct (sample)
– ? Ct (calibrator), and ? Ct is the Ct of the target gene subtracted from the
Ct of the housekeeping gene.

 

2.3 Treatment
regimen and response to therapy

The  patients were treated according to the
adopted protocol of the Department of Medical Oncology, Cairo University. AML
patients were subjected to 7-3 protocol for induction of remission. Treatment
of AML depends on the fitness of the patient. Fit patients (5% blasts
or evidence of leukemia in other sites
(14).

            The
disease free survival (DSF) was calculated
from the time of remission to relapse or death or loss to follow. The overall
and disease free survival functions were estimated by the Kaplan and Meier
method and compared by the log rank test. All p-values
are two-sided. P-values