الفهرس 
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Abstract
Neoplastic diseases have been defined as proliferative disorders characterized by an uncoordinated cell growth. Cellular proliferation follows an orderly progression through the cell cycle, which is controlled by protein complexes composed of cyclins and cyclin-dependent kinases (CDKs). Their peak of synthesis and activity during the G1 phase and apparently regulate the transition from G l to S phase. Flow cytometric method of assessing cell proliferation provides prognostic information; also, its result may be particularly closed with histological tumor grade.
The present work aimed to evaluate the prognostic role of cyclin D1 and AgNORs in invasive breast cancer and its correlation with other established prognostic parameters of breast cancer.
The current study was carried out on 40 prospective biopsies taken from patients with breast tumor. They were divided into benign (10 cases) and malignant (30 cases) groups as well as control (10 cases taken from tissue parenchyma adjacent to the tumor). All biopsies were obtained from Department of Pathology, Medical Research Institute, Alexandria University during the period from February to August (2015).
Gross examination for surgically excised samples was done with the measurement of the size of the tumor and the auxiliary lymph nodes that were dissected. Parts from the tumors and from the tissue adjacent to the tumor were subjected to the standard procedure of fixation, dehydration, paraffin embedding and sectioning. Sections have been stained with haematoxylin and eosin (H&E) for histological and histopathological structure examination, Feulgen staining for DNA demonstration and AgNORs silver-staining for demonstrating the argyrophilic nucleolar organizer regions as well as immunohistochemical staining for cyclin D1 protein as a prognostic marker. In addition, homogenized fresh breast tumor samples have been used for flow cytometric analysis to assess the cell cycle analysis.
The histopathological types of the present study revealed (50%, 5/10) fibroadenoma and fibrocystic diseases breast lesion of the benign group, and the selected invasive ductal carcinoma (IDC) of malignant group was diagnosed as grade II (60%, 18/30) and grade III (40%, 12/30).
The pathological parameters represented the majority of tumor affected site was the left tumor mass (LT) (56.7 %) in the malignant group and (50.0 %) in the benign group, the majority of tumor size was among 2 - ≤5 cm in both malignant (75 %) and benign cases (70 %). The age was distributed at 35-45 years in benign cases (60 %) and 45-55 years in malignant cases (33.3 %). All the malignant cases represented positive in vascular invasion (VI) with 93.3% and positive lymph node metastasis (LNM) with 63.3%.
In addition, ER immunostaining was distributed as positive in 66.7% of all malignant cases. PR immunostaining was distributed as positive in grade II (61.1%) and grade III (66.7%). Also, the immunostaining of HER2 was distributed as positive in grade II (77.8%) and grade III (41.7%).
The main aim of this study was to assessment the proliferation induce of the breast cancer cell under different histo-morphological technique as follow: According to the histochemical Feulgen staining for assessing DNA content in the ductal breast cells nuclei, the results evaluated deep magenta color in normal breast tissue and pale magenta color in malignant ones indicating the decreased DNA content.
For assessing the nucleolar organizer regions (AgNORs) in the ductal breast cells, by silver stain, the results dependent on counting the number of the dotes and revealed, increased number of dark brown dots in nucleoli and nuclear matrix in the malignant group. There was a statistical increase significant (P ≤ 0.001) in the AgNORs staining in malignant and benign groups comparing with control.
In addition, the immunohistochemical staining technique was used to detect the cyclin D1 protein in breast cancer through the ABC and DAB stain. The result appeared as brown nuclei and some regular brown granules in the cytoplasm around the positive and negative nuclei, the benign group revealed 60% positivity, week staining, for the protein, but in the malignant group there was 24 out of 30 cases were judged positive for cyclin D1 expression, of which 3 (10%) were classified as weak, 9 (30%) as moderate and 12 (40%) as strong expressions. The remaining 6 tumors (20%) did not show any appreciable staining for cyclin D1 in the nucleus and were classed as negative. The immunostaining of cyclin D1 was statistically significantly correlated with ER-positivity (P= 0.012), hence cyclin D1-staining in all malignant cases scored 66.3 % ER-positive compared to 33.3 % ER-negative. In addition, cyclin D1 was statistically significantly correlated with PR-positivity (P= 0.047), hence there was 63.3 % of the all malignant cases were PR-positive compared to 36.3 % were PR-negative. Also the present assessment depend on the nuclei count, there was a high positive expression nuclei counts in both grades of IDC malignant cases compared to control cases with significant difference (P < 0.001). Also, the positive nuclear counts of cyclin D1 expression of the IDC cases were noticed in the grade II more than grade III with a significant difference (P < 0.001). Furthermore, there was a statistically significant difference (P <0.001) between the mean count of cyclin D1 and AgNORs, as general, in the total cases.
Moreover, the flow cytometry analysis of the individual groups revealed that, the percentage of G0/1 was in G II 57.68 % and in G III 44.92 %, while S phase percentage was 16.78 % in G II compared to 9.69 % in G III. The statistical analysis for these results showed that, there was no any significant correlation between cyclin D1 staining score and increased S-phase % at any of the individual groups.
The new concept of this work was the assessment of the histochemical and immunohistochemical stains quantitatively and carried out for calculating the density of stains by Integrated Optical Density (IOD) using digital image analysis software. The mean values of each reaction were based on the mean of pixel digit. The IOD intensity color based on Gray value-level differentiated in digitized images according to different reaction from dark to light (124.76 down to 43.65) in Feulgen and (195.54 down to 100.11) in cyclin D1.
The present results of the (IOD) analysis revealed that:
• The possibility of quantifying DNA by the Feulgen stain is proportional to the DNA concentration associated to the histone protein (chromosomes), which present evaluation a high mean of IOD in the control than in the tumor individuals with a significant difference (P < 0.001).
• The cyclin D1 antibody IOD evaluation represent that, a high significant difference (P < 0.001) was noticed in malignant tumor IDC compare to benign, also, the IOD that evaluated in GII was more than GIII with a significant difference (P < 0.001). The cyclin D1 protein IOD evaluation was noticed in malignant tumor IDC compare to benign breast tissue with high significant difference (P < 0.001). Also, the IOD was evaluated in GII more than GIII with a significant difference (P < 0.001).
• The comparison between Feulgen stain IOD and cyclin D1 stain IOD, showed that, there was a statistically significance difference between all of the individual groups
(P <0.001).
• There was no statistically association between cyclin D1-IOD and flow cytometry assessment of cell cycle phases (G1 phase % or S phase %) in the all individual groups.
CONCLUSION
• The study revealed that grade II was the common cases of IDC in our country, these incidences may be related to the environmental cofactors due to changes in reproductive factors and lifestyle.
• There was a statistically significant correlation of cyclin D1 with (ER & PR) positivity; this may be provided cyclin D1acts as significant prognostic factor and a marker of disease response for treatment.
• The proliferation indices assessment by AgNORs silver stain and immunostaining of cyclin D1 protein revealed the role of both as favorable prognostic markers.
• AgNORs stain is easy to work and low in cost, thus it offers a good available method can be used in any laboratory.
• The immunostaining evaluation using image analysis for counting and density of stain (IOD) reflected the role of the image analysis as a valuable method for assessing tumor cell proliferation, as well as, the pathological markers or immunoprotein staining.