Comparative, Molecular, Immunohistochemical and Tissue Microarray Study on Iraqi and Italian Breast Cancer Patients

number: 
2452
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Farooq Ibrahem Mohammad
Supervisor: 
Dr. Saleh A. Waheeb
Dr. Walid Kh. Al Hadithy
year: 
2010
Abstract:

Breast cancer is the most common cancer of women worldwide. It is a heterogeneous disease. The incidence rate of this disease increased in the last years in Iraq. The current study was conducted to study the immunohistochemical and molecular parameters for the breast tumors by using tissue microarray (TMA) technique on Iraqi and Italian patients with breast carcinoma. Histopathological data for the patients were obtained. Formalin-fixed, paraffin-embedded (FFPE) samples were used in this study. Three TMAs recipient blocks were designed and constructed for a first time on Iraqi samples. Fifty-five Iraqi breast cancer cases used as the first TMA block; thirty Italian breast cancer cases were used to construct the second TMA block and fifteen Iraqi benign breast lesions, which used as a control and to construct the third TMA. For TMAs construction, each donor sample were represented in triplicate, cores were transferred from each donor patient sample to the recipient block. Hematoxylin and Eosin (H and E) stained then immunohistochemically (IHC) studied for estrogen receptor (ER), progesterone receptor (PR), Ki67, p53 and HER-2 for the three TMAs. The mean age of the Iraqi breast cancer patients in this study 49.38 ±8.29 years old; ranging from 36-69 years old. The mean age of the Italian patients 57.00 ±8.52; the range 39-73 years old. The mean age of the Iraqi patients was lower than the Italian breast cancer cases used in this study. ER positive expression was found in 26 (47.3%) out of 55 cases of the Iraqi breast cancer cases, In the Italian TMA, 23 (76%) cases out of 30 sample showed positively expressed ER; ER expression in the Iraqi samples were lower than the Italian samples. PR expression was observed in 35 (63.6%) out of 55 samples of the Iraqi breast cancer. For the Italian breast cancer TMA, 23 out of 30 cases had positively expressed PR (76%). Ki67 showed positive expression in 30 out of 55 of the Iraqi breast cancer (54.5%), while in the Italian breast cancer TMA 23 out of 30 cases were positively expressed (76%). The positive immunohistochemical expression of p53 in the Iraqi malignant breast TMA were observed in 48 out of 55 samples (87.2%). For the Italian TMA, 23 out of 30 samples were positively expressed of p53 (76%). Overexpression of HER-2 (score 2+ and 3+) was observed in 30 of the Iraqi breast cancer samples (54.5%) and in 16 out of 30 of the Italian breast cancer patients (53.3%). Correlation between the five markers were studied statistically and the study revealed that there was a significant relationship between the HER-2 expression and expression of ER and p53 (P value <0.01), but there was no significant relationship between HER-2 expression with the expression of PR and Ki67 (P value >0.01). Twelve cases out of 55 Iraqi samples were triple negative (negative for ER, PR and HER-2 overexpression) (21.8%), whereas only one case out of 30 cases of the Italian samples was triple negative (3.3%). The results indicated that the Iraqi breast cancer cases used in this study were phenotypicaly more aggressive than the Italian breast cancer samples. We utilized TMA sections for the validation of HER-2 molecular testing. This study reviewed the validity and reliability of the two most commonly used tests for HER-2 detection: IHC and FISH. Depending on a statistical analysis of studies comparing IHC and FISH, FISH assay revealed that there was amplification in the HER-2 gene in (88%) of IHC2+ cases, and were amplified in (98.2%) of the IHC3+. For the Italian samples, positive amplification was observed in (81.80%) of the IHC2+, and amplified in (100%) for IHC3+. DNA extraction was performed successfully from the formalin fixed, paraffin embedded (FFPE) sections. After DNA quantification, only the DNA samples extracted from the Italian samples were successfully used for polymerase chain reaction (PCR) amplification of BRCA1 gene exon 11. Agarose gel electrophoresis of the PCR product showed the presence of a band with size of 185 bp, which represents the correct expected band in 24 out of 30 samples used in this study. PCR products were purified, and their DNA sequencing was performed, and analyzed by National Center for Biotechnology Information (NCBI) and the results revealed 4 samples with deletion mutation and 3 samples with point mutation. In conclusion, the mean age of the Iraqi of the Iraqi breast cancer patients was lower than that of the Italian breast cancer patients. TMA was successfully used as a rapid, economic and effective method for IHC and FISH studies. There were a differences in the ER, PR, Ki67, p53 and HER-2 expression between the Iraqi and the Italian samples. ER and PR expression in the Italian cases were higher than the Iraqi cases which means that the Iraqi breast cancer was more aggressive than the Italian samples and the negative cases not responding to the hormonal therapy. Triple negative cases were higher in the Iraqi samples than the Italian samples. The Iraqi breast cancer samples were phenotypicaly more aggressive than the Italian breast cancer samples. FISH is the gold standard method to assess the response to the therapy for HER-2 positive cases. DNA was successfully extracted from FFPE sections and used for BRCA1 exon11 to determine the mutations in the sporadic breast cancer cases, the DNA sequencing results and analysis showed 7 mutations in BRCA1 exon11.