الفهرس 
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Abstract
Breast carcinoma is the most frequently occurring cancer in women worldwide and the second common cause of malignancy-associated deaths. It is estimated that more than 5 million new cases will affect women over the next decade; approximately 1 in 10 women in western countries will develop breast cancer during their life time.’ it is one of the most common cancers among American women and world worldwide. In 2007, about 178.480 new cases of invasive breast cancer were diagnosed and approximately 40,460 women died from the disease nationwide.
The prediction of clinical outcome in patients is difficult and usually based on clinico-pathological variables, such as tumor stage, histologic grade, and the presence of mctastases to regional lymph nodes and distant organs. Despite improvements in treatment modalities, there is still a high failure rate mainly due to tumor invasion and metastasis.
Breast cancer invasion is a three-step process characterized by altered cellular adhesion, cell motility and invasion of the extracellular matrix. Signals transmitted to the tumor cells from the pcritumoral stroma will influence their activity, proliferation, and motility. This is accomplished in part through transmembranc receptors that interact with stromal extracellular matrix molecules. One of these receptors is CD44, which binds to the extracellular matrix component HA. The engagement of CD44 with HA results in intraccllular signaling that has been linked to diverse cellular functions such as adhesion, migration, and invasion, which are all important in cancer progression and metastasis. They may play a role in tumor cell adhesion to the vascular cndothclium, which precedes extravasation of malignant cells and the development of mctastases.
The current study seeks to evaluate the immunohistochemical expression of the adhesion molecule sCD44 variant 6 in both breast cancer and benign breast lesions and its correlation with the different Clinicopathological parameters. Moreover, the influence of the presence of CD44v6 expression in the primary breast tumor and the melastatic disease on serum levels of CD44v6 and HA was investigated, by means of quantitative estimation of serum sCD44\6, and HA in the groups under study using EL1SA technique. Furthermore, on the same series of patients, the hyaluronidases en/ymcs activities were determined chemically according to the colorimetric method of Reissig et al. in the sera of the patients aiming to throw some light on their role in relation to the HA and CD44v6 in breast cancer and their relation to the different Clinicopathological parameters and breast cancer prognosis.
The study was undertaken on 20 cases of benign breast lesion and 50 breast cancer patients The mean age of breast cancer patients was (52±I1.83) while in benign bieast lesion patients it was (33.1±1 1.2), as regard the menstrual status of breast cancer patients most of them were post- menopause 60% while 40% were premenopause. H&E staining and histopathological examination revealed that 47 out of the 50 patients had infiltrating duct carcinoma (IDC) (94%) Concerning the tumor grade. 78% of the eases were grade II, 18% grade III and 4% were Grade i. The majority of patients (24) had tumor si/e T2 (2-5cm) ,15 patients had Tl (<2cm), and 11,were T3 ’<>5cm).Concerning the lymph node involvement and the hormonal receptors expression of the patients results showed that 34% had regional positive lymphnodcs while 16 had negative lymphnode, 20% of the examined tumors were negative for ER expression and 80% were positive and in case of PR status 26% were negative and 74% were positive for PR expression, According to the pTNM classification, at the time of diagnosis 12% of patients were stage I, 42% stage II, 30% stage 111 and 8 (16%) patients were stage IV had metastases at different sites (mainly to the bone or the liver), the histo-pathological examination of the breast cancer tumors also showed that 31 specimens had no vascular invasion while the remaining 19 were positive for vascular invasion.
The immunohistochcmical expression of the adhesion molecule CD44v6 revealed that it was expressed in both benign breast lesion (and in breast cancer patients with different degree of positivity (weak, moderate or strong). The expression of CD44\6 increased with malignant transformation, where 36% of the cases were strongly positive, while weak and moderate expression, were 24% and 32% respectively and 8% were negative. It was noticed that the intensity of CD44vo staining is higher in breast cancer than in benign tumors but this difference was not statistically significant.
The expression of CD44v6 in breast cancer patients in the study was found to be independent of the age, tumor size, menstrual or hormonal status but a significant association was found between CD44v6 expression and tumor grade as well as the stage of breast cancer, CD44v6 expression was closely related to the degree of differentiation, where strong intensity (+++) was noticed in tumors grade III and patients in stage IV.
Moreover a positive correlation was present between the expression of CD44v6 and the lymphnode involvement as well as vascular invasion. In respect of the disease free and overall survival of the breast cancer patients our results showed that CD44v6 expression correlated with the overall survival of the patients and not with the relapse free survival.
As regard the scrum CD44v6 levels, the present results revealed that the mean scrum level in breast cancer patients was (104.46±45.3) (ng/ml) while in benign breast lesion patients it was (94.3±26.25) (ng/ml), its level in breast caneer was higher but this difference was not statistically significant. The present study showed an association between Scrum CD44v6 in breast cancer patients and tumor grade, vascular invasion, regional lymphnode involvement, ER and stage of the disease where P<0.05.
Meanwhile, by the end of the follow up period (48 months), scrum CD44v6 level was significantly correlated to the disease free and overall survival of the patients, where it was found that patients with higher level of serum CD44v6 developed metastasis.
AS regard the HA serum level its mean in breast cancer was (79.54±53.1) (ng/ml) and in benign breast lesion group (39.7±I8.76) (ng/ml) with a higher concentration in breast cancer patients and this was statistically significant where (P=0.00001). Concerning the relation of HA serum concentration with the different clinicopathologieal factors, results revealed that the HA concentration positively correlated with the tumor grade, lymphnode involvement and ER expression, tumor vascular invasion (P<~0.05). a significant relation was also present between Jhe HA serum level and the patient’s breast cancer stage where patients in stage IV who had metastasis at the time of diagnosis in comparison to the non metastatic patients (P<0.05). Moreover, our results revealed that there is a positive correlation between serum MA and the disease free and overall survival of the patients where PO.05.
On the other hand . in case of the hyaluronidascs, results revealed that the activity of HAE in the sera of breast cancer patients (6.75±2.7) (umol/ NAG min ’ !”’) was much lower than in the benign breast lesion group (I0.67±3.56) (umol/ NAG min ’ 1”’) and this difference was statistically significant where P^O.OOOl. No correlation was found between the serum hyaluronidases activity and any of the clinicopathological factors, neither with the tumor si/e, grade or the patient’s menstrual status, age, lymphnodc status, hormonal receptors expression, vascular invasion or stage of the breast cancer in breast cancer patients (P>0.05).
Surprisingly inspitc of that, patients with lower level of hyaluronidases had a poorer prognosis and it was found that metastatic patients had a significant lower mean level of HAS (5.51±3.58) (umol/ NAG min”1 1” ) in comparison to non metastatic group (6.7Si2.94) (umol/ NAG min ! 1”’) and this was statistically significant where P-0.012. Consequently, the serum HAS level is affecting the disease free and overall survival of the patients (PO.05).
from these findings we can conclude that adhesion molecule CD44\6, Hyaluronic acid and hyaluronidases can be considered as additional potential predictors for the clinical course of breast cancer. However our sample size is small and larger scale research in this field is needed in order to team more about their role in breast cancer development, progression and metastasis.
SUMMARY
Breast carcinoma is the most frequently occurring cancer in women worldwide and the second common cause of malignancy-associated deaths. It is estimated that more than 5 million new cases will affect women over the next decade; approximately 1 in 10 women in western countries will develop breast cancer during their life time.’ it is one of the most common cancers among American women and world worldwide. In 2007, about 178.480 new cases of invasive breast cancer were diagnosed and approximately 40,460 women died from the disease nationwide.
The prediction of clinical outcome in patients is difficult and usually based on clinico-pathological variables, such as tumor stage, histologic grade, and the presence of mctastases to regional lymph nodes and distant organs. Despite improvements in treatment modalities, there is still a high failure rate mainly due to tumor invasion and metastasis.
Breast cancer invasion is a three-step process characterized by altered cellular adhesion, cell motility and invasion of the extracellular matrix. Signals transmitted to the tumor cells from the pcritumoral stroma will influence their activity, proliferation, and motility. This is accomplished in part through transmembranc receptors that interact with stromal extracellular matrix molecules. One of these receptors is CD44, which binds to the extracellular matrix component HA. The engagement of CD44 with HA results in intraccllular signaling that has been linked to diverse cellular functions such as adhesion, migration, and invasion, which are all important in cancer progression and metastasis. They may play a role in tumor cell adhesion to the vascular cndothclium, which precedes extravasation of malignant cells and the development of mctastases.
The current study seeks to evaluate the immunohistochemical expression of the adhesion molecule sCD44 variant 6 in both breast cancer and benign breast lesions and its correlation with the different Clinicopathological parameters. Moreover, the influence of the presence of CD44v6 expression in the primary breast tumor and the melastatic disease on serum levels of CD44v6 and HA was investigated, by means of quantitative estimation of serum sCD44\6, and HA in the groups under study using EL1SA technique. Furthermore, on the same series of patients, the hyaluronidases en/ymcs activities were determined chemically according to the colorimetric method of Reissig et al. in the sera of the patients aiming to throw some light on their role in relation to the HA and CD44v6 in breast cancer and their relation to the different Clinicopathological parameters and breast cancer prognosis.
The study was undertaken on 20 cases of benign breast lesion and 50 breast cancer patients The mean age of breast cancer patients was (52±I1.83) while in benign bieast lesion patients it was (33.1±1 1.2), as regard the menstrual status of breast cancer patients most of them were post- menopause 60% while 40% were premenopause. H&E staining and histopathological examination revealed that 47 out of the 50 patients had infiltrating duct carcinoma (IDC) (94%) Concerning the tumor grade. 78% of the eases were grade II, 18% grade III and 4% were Grade i. The majority of patients (24) had tumor si/e T2 (2-5cm) ,15 patients had Tl (<2cm), and 11,were T3 ’<>5cm).Concerning the lymph node involvement and the hormonal receptors expression of the patients results showed that 34% had regional positive lymphnodcs while 16 had negative lymphnode, 20% of the examined tumors were negative for ER expression and 80% were positive and in case of PR status 26% were negative and 74% were positive for PR expression, According to the pTNM classification, at the time of diagnosis 12% of patients were stage I, 42% stage II, 30% stage 111 and 8 (16%) patients were stage IV had metastases at different sites (mainly to the bone or the liver), the histo-pathological examination of the breast cancer tumors also showed that 31 specimens had no vascular invasion while the remaining 19 were positive for vascular invasion.
The immunohistochcmical expression of the adhesion molecule CD44v6 revealed that it was expressed in both benign breast lesion (and in breast cancer patients with different degree of positivity (weak, moderate or strong). The expression of CD44\6 increased with malignant transformation, where 36% of the cases were strongly positive, while weak and moderate expression, were 24% and 32% respectively and 8% were negative. It was noticed that the intensity of CD44vo staining is higher in breast cancer than in benign tumors but this difference was not statistically significant.
The expression of CD44v6 in breast cancer patients in the study was found to be independent of the age, tumor size, menstrual or hormonal status but a significant association was found between CD44v6 expression and tumor grade as well as the stage of breast cancer, CD44v6 expression was closely related to the degree of differentiation, where strong intensity (+++) was noticed in tumors grade III and patients in stage IV.
Moreover a positive correlation was present between the expression of CD44v6 and the lymphnode involvement as well as vascular invasion. In respect of the disease free and overall survival of the breast cancer patients our results showed that CD44v6 expression correlated with the overall survival of the patients and not with the relapse free survival.
As regard the scrum CD44v6 levels, the present results revealed that the mean scrum level in breast cancer patients was (104.46±45.3) (ng/ml) while in benign breast lesion patients it was (94.3±26.25) (ng/ml), its level in breast caneer was higher but this difference was not statistically significant. The present study showed an association between Scrum CD44v6 in breast cancer patients and tumor grade, vascular invasion, regional lymphnode involvement, ER and stage of the disease where P<0.05.
Meanwhile, by the end of the follow up period (48 months), scrum CD44v6 level was significantly correlated to the disease free and overall survival of the patients, where it was found that patients with higher level of serum CD44v6 developed metastasis.
AS regard the HA serum level its mean in breast cancer was (79.54±53.1) (ng/ml) and in benign breast lesion group (39.7±I8.76) (ng/ml) with a higher concentration in breast cancer patients and this was statistically significant where (P=0.00001). Concerning the relation of HA serum concentration with the different clinicopathologieal factors, results revealed that the HA concentration positively correlated with the tumor grade, lymphnode involvement and ER expression, tumor vascular invasion (P<~0.05). a significant relation was also present between Jhe HA serum level and the patient’s breast cancer stage where patients in stage IV who had metastasis at the time of diagnosis in comparison to the non metastatic patients (P<0.05). Moreover, our results revealed that there is a positive correlation between serum MA and the disease free and overall survival of the patients where PO.05.
On the other hand . in case of the hyaluronidascs, results revealed that the activity of HAE in the sera of breast cancer patients (6.75±2.7) (umol/ NAG min ’ !”’) was much lower than in the benign breast lesion group (I0.67±3.56) (umol/ NAG min ’ 1”’) and this difference was statistically significant where P^O.OOOl. No correlation was found between the serum hyaluronidases activity and any of the clinicopathological factors, neither with the tumor si/e, grade or the patient’s menstrual status, age, lymphnodc status, hormonal receptors expression, vascular invasion or stage of the breast cancer in breast cancer patients (P>0.05).
Surprisingly inspitc of that, patients with lower level of hyaluronidases had a poorer prognosis and it was found that metastatic patients had a significant lower mean level of HAS (5.51±3.58) (umol/ NAG min”1 1” ) in comparison to non metastatic group (6.7Si2.94) (umol/ NAG min ! 1”’) and this was statistically significant where P-0.012. Consequently, the serum HAS level is affecting the disease free and overall survival of the patients (PO.05).
from these findings we can conclude that adhesion molecule CD44\6, Hyaluronic acid and hyaluronidases can be considered as additional potential predictors for the clinical course of breast cancer. However our sample size is small and larger scale research in this field is needed in order to team more about their role in breast cancer development, progression and metastasis.