This study was conducted on 48 breast cancer female
patients. Their ages ranged from 25 to 60 years. They
were referred to the surgery clinics of Ain Shams University
Hospital. The diagnosis of breast cancer was based
on radiology, mammography, tissue biopsy for histopathological
examination, ultrasonography and bone scan for
detection of secondaries. The carcinoma cases were of
infiltrating ductal carcinoma type and 10 of them showed
associated areas of intraductal carcinoma. The patient
group was classified into two groups:
1. Localized breast cancer group: This included 26 patients
with localized breast cancer, with no evidence
of distant metastases.
2. Metastatic group: This group included 22 patients suffering
from metastatic breast carcinoma. This group
was subdivided into:
a. Those with axillary lymph node metastases (9 patients).
b. Those with distant metastases (13 patients).
To study the effect of treatment, 10 patients of the
metastatic group who have MAG m-RNA positive cells
were selected prospectively after receiving their course of
Histopathologically, all breast cancer patients were
graded according to the system of Bloom and Richardson
 which was recommended by WHO  into:
- Grade I (Well differentiated): It included 8 patients.
- Grade II (Moderately differentiated): It included 26
- Grade III (Poorly differentiated): It included 14 patients.
This group included 28 subjects: 10 healthy volunteers
served as healthy control group and 18 patients served as
patient control group (6 patients with fibroadenoma, 4
patients with uterine carcinoma, 4 patients with ovarian
carcinoma and 4 patients with colon cancer).
For histopathological study, the healthy control group
included the normal breast tissue adjacent to fibroadenomas.
Under complete aseptic conditions, 5 mL of venous
blood was collected by venipuncture in sterile EDTAtreated
tubes (Bekton Dickenson, OK) from all controls
and breast cancer patients before initiation of the first line
chemotherapy and/or hormonal treatment. In case of metastatic
group, another blood sample was collected from
10 patients after receiving chemotherapy and hormonal
Breast tissue samples were fixed in 10% formaldehyde,
routinely processed to paraffin blocks, then 5 µm
thick sections were prepared and stained with hematoxylin
and eosin stain for histopathological examination.
Nested RT-PCR for detection of circulating MAGmRNA
was performed as follows:
1) RNA extraction from venous blood sample was immediately
performed after sample collection using
QIAmp RNA blood kit (QIAGEN Inc., USA). The
extracted RNA was diluted prior to its assay. The
concentration and purity of RNA extracts were determined
by measuring their absorbance at 260 nm
(A260) and 280 nm (A280) using a spectrophotometer.
Pure RNA has an A260/A280 ratio of 1.6-1.9.
An absorbance of 1 µ unit at 260 nm corresponds to
40 µg RNA/mL. The concentration of RNA stock was
then determined (concentration of RNA stock = 40
RNA µg/mL x A260 x dilution factor), then the total
yield was calculated by multiplying concentration by
volume of stock in mL.
The primer and reaction parameters for nested RTPCR
were chosen according to:
The first primer pair was:
The second primer pair used in nested PCR reaction
included, the inner upstream primer, which
(5`-AGCACTGCTACGCAGGCTCT-3`) and the
downstream primer, which was:
2) The RNA samples were subjected to reverse transcription
and the RNA samples amplification using
QIAGEN one-step RT-PCR Enzyme Mix Kit (QIAGEN
Inc., USA). Nested PCR was done using Taq
PCR Master Mix Kit (QIAGEN Inc., USA).
3) The amplified products were analyzed by electrophoresis
on 2% agarose gel stained with ethidium bromide.
A DNA molecular weight marker XIII was also
run to identify the site of bands. The band if present
was compared to the DNA marker for the site of target
DNA that was 202 bp products (Figure 1).
Fig 1. Photomicrograph of 2% argarose gell showing:
- The DNA molecular weight marker showing the band of 202 bp (lane 1),
- The positive results (lane 2,3,4,5,6,8&11),
- The negative results (lane 7,9,10&11)
Streptavidin-biotin technique was used to investigate
mammaglobin (MAG), estrogen receptors (ER) and Ki-
67 expression. Three slides from each case were deparaf-
finized, hydrated and incubated in 3% hydrogen peroxide
for 30 minutes to block the internal peroxidase activity.
Antigen retrieval was done by microwave pretreatment
for 10 minutes in 0.01 citrate buffer. For each case, one
slide was incubated with anti-mammaglobin monoclonal
antibody (Dako Corporation) at a dilution 1:100, the second
slide was incubated with mouse monoclonal antibody
to ER at a dilution 1:50 (Dako Corporation) and the third
one was incubated with MIB1 (Ki-67) at 4 ºC overnight.
Sections were then washed twice for 5 minutes with PBS
and incubated for 10 minutes with biotinylated secondary
antibody (Dako Cytomation). The slides were washed
twice for 5 minutes in PBS and incubated for 10 minutes
in performed avidin-biotin-peroxidase complex (Dako
Cytomation). Chromogen development was accomplished
by immersion of the sections in 2, 3-Diaminobenzidin
tetrahydrochloride (BAB) (Dako Cytomatin) for 5 minutes.
The nuclei were counterstained with hematoxylin, dehydrated, cleared and mounted. For negative controls,
the primary antibody was omitted and replaced with PBS.
Mammaglobin gives cytoplasmic staining while ER and
Ki67 appear as nuclear staining (Figures 2, 3).
Fig 2 (A,B). (A): Infiltrating ductal carcinoma of the
breast showing positive staining for mammaglobin; (B)
Negative staining for mammaglobin in uterine carcinoma
Fig 3: Infiltrating ductal carcinoma showing positive staining
for Ki-67 (A); and estrogen receptors (B) (Immunoperoxidase,
According to Han et al. , the intensity of mammaglobin
expression were scored as no staining, weak, moderate
and strong staining.
Ki-67 immunostaining was evaluated using Leica Image
Processing and Analysis System. In each case, the
analysis was done on areas expressing quantitatively the
highest number of immunoreactive nuclei (10-20 microscopic
fields at x400 magnification were measured for
each case). The results were expressed as Ki-67 proliferation
index which is defined as the percentage of positively
stained nuclei divided by the total number of the counted
nuclei. Ki-67 proliferation index was either ≤20 or >20.
According to Eerola et al. , estrogen receptor was
considered positive when ≥ 10% of cells were stained.
Blood and breast tissue MAG expression were correlated
with tissue estrogen receptors and Ki-67 expression.
Statistical analysis was done using SPSS software
package. Results were expressed as number and percentages.
The comparison of the frequency of positivity
of MAG between the different groups was done by Chi-
Square test (÷2 test) or Fisher exact test where necessary.
A p-value of less than 0.05 was considered statistically
The diagnostic performance of MAG mRNA was
evaluated. The diagnostic sensitivity, specificity, positive
predictive value, negative predictive value and the diagnostic
efficiency were calculated.