الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Breast cancer (BC) is a multi-factorial disease and various factors contribute to its
occurrence. The incidence, mortality and survival rates of BC vary considerably among
different parts of the world, which could be due to changing in risk factors. Classification of
breast cancer based on risk factors is effective in improving risk free methods and designing
targeted breast cancer screening programs. BC classification is important for proper diagnosis
and prognosis for disease to choose an approriae treatment, and the treatment can be higly
effective when the disease is identified early.
Imaging techniques are main diagnosis approaches which could provide valuable data
on patients with breast cancer.In the case of solid tumors, the development of cancer is hard to
understand at the cellular level because obtaining solid-tumor biopsies can be difficult,
dangerous, and unfeasible or, whatever happens, results in locally and timely restricted
information on cancer composition. This results in a lack of access to molecular and
phenotypic information on cancer evolution that could be crucial for treatment success. So
beside the imaging techniques, it is important that use of techniques enabling molecular
subtyping in clinical practice would provide more accurate information about patient-specific
diagnosis, prognosis, risk of relapse and probability for pathological complete response.
Utilization of biochemical biomarkers such as proteins, DNAs, mRNAs, microRNAs, and
circulatory tumor cells (CTCs) could be employed as new diagnosis tools for patients with
breast cancer.
Circulation tumor cells (CTCs) in the blood stream represent a motivating window on the
progression of cancers, the metastatic process and therapeutic response. Therefore, the ability to
accurately detect and analyze CTCs in peripheral blood of donors is of importance.
Quantitative real time polymerase chain reaction (qRT-PCR) is one of the most widely
used techniques in the CTC-research field. In which, the viable CTCs detection by a detection
of epithelial or tumour specific mRNAs. Also, the Flow cytometry (FACS) technique is
considered as one of the essential methods for CTCs diagnosis. It is a fast, sensitive and
affordable technique, ideal for rare-cell detection.
The objective of this study; is establish a sensitive and specific techniques for detection
of “potential” CTCs from as little as one mL of blood moreover, the procedures reduce the
volume of sample. For this comparative study, qRT-PCR and Flow cytometry methods were
used to set up a better method for early diagnosis to help in predicting recurrence and
planning appropriate therapies to improve survival.
The design of experiment; this comparative study enrolled 80 clinical samples of breast
cancer women with different clinicopathological data and 10 healthy volunteers’ women of
matched age who had no previous history of malignancy especially breast cancer.
The methodology; Cytokeratins and vimentin were chosen as the main markers to
identify the potential CTCs. The methods were first validated on cell lines with known
expression for selected markers to show their specificity and accuracy (Lopresti et al.,2019)
Summary, Conclusion & Recommendations
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because their accurate detection is necessary to enable valid assessment of the diagnostic
relevance of these findings in the clinical setting. MDA-MB-231 breast cancer cell lines
which contain vimentin gene as positive control and the HCT 116 colon cancer cell line
which contain cytokeratins genes as positive control were used for spiking experiments to
validate the both methods.
Peripheral blood samples from breast cancer and healthy women were collected in a 5-
mL vacutainer tubes containing EDTA-K2 as an anticoagulant. To avoid endothelial cell
contamination during the skin puncture, the first milliliters of blood were discarded:
For molecular investigations, RNA was isolated from peripheral blood samples of
healthy and BC women then reverse transcribed to cDNA, which was then used in qRT-
PCR against cytokeratins (CK14, CK19) and vimentin (VIM). Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) was used as endogenous control.
For cytological investigations, Alexa Fluor® 647 Mouse Anti-Human Cytokeratin 14,
15, 16 and 19 (antibody Cocktail, BD Bioscience) and Alexa Fluor® 488 Mouse Anti-
Human Vimentin (BD Bioscience) were selected as the main markers to identify
potential CTCs. This combination was complemented with propedium iodide (PI) dye
to select nucleated cells, and CD45-APC (BD Bioscience) to exclude cells from the
hematopoietic lineage.
Statistical analysis; The measurement data was expressed as mean ± standard error (S.E),
the count data were expressed in the form of percentage. Also, the measurement data among
groups were compared with the Mann-Whitney test. The ROC curves were plotted and analyzed
for each assay to detect cut-off value, sensitivity and specificity of the test. p<0.05 was considered
as a statistically significant difference.Data analyses were performed using SPSS software
version21. Finally, the correlations between the parameters were analyzed by suitable correlation
test.The diagnostic values of each method and the recovery curves using spiked different cell lines
in peripheral blood were analyzed.
The results of this study were summarized as following:
1. Based on qRT-PCR results, there was a strong significant direct correlation between
the expression levels of CK14 (rs = 0.648, p<0.001) and vimentin. There was a strong
statistically significant indirect correlation between the clinical stages and CK19
expression levels (rs = -0.501, p<0.001). On the other hand, it reported a moderate
statistically significant direct correlation between the clinical stages and VIM
expression levels (rs = 0.366, p<0.05).
2. Based on the FACS results, there was a significant strong direct correlation between
the clinical stages and both VIM+CTCs (rs = 0.679, p<0.001) and VIM+CK+CTCs (rs =
0.534, p<0.001). Also, there was a significant strong direct correlation between the
VIM+CTCs and VIM+CK+CTCs (rs = 0.766, p<0.001).
3. The diagnostic values of ROC curves of both techniques (qRT-PCR and FACS), to
identify BC from healthy women and TNBC from NTNBC women showed that
vimentin was the highest accuracy with highest sensitivity and specificity with specific
cut-off value, followed by CK19.