COMPARATIVE STUDY OF IMMUNOHISTOCHEMICAL EXPRESSION OF GP88 AND Cath-D AS PROGNOSTIC MARKERS AND CORRELATION WITH PATHOLOGICAL AND HISTOLOGICAL PARAMETERS IN HUMAN BREAST CANCER

Progranulin or acrogranin is an 88-kDa glycoprotein identified by a biological screen for protein targets associated with high tumorigenicity. Cathepsin D (Cath-D) is a soluble lysosomal aspartyl glycoprotease that can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion, and angiogenesis. The present work aimed to compare the expression of GP88 and Cath-D as novel prognostic biomarkers in human invasive ductal carcinoma (IDC) versus benign tumors and normal breast tissues as well as their correlation with different pathological and histological parameters. The immunohistochemical technique was used to examine the expression of GP88 and Cath-D in normal, benign, and IDC. Present results showed higher expression of GP88, and Cath-D in IDC compared to normal and benign breast tissues.


Introduction
Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in women worldwide (1) .Breast cancer represents a major scientific, clinical, and societal problem.It is the most common malignancy and the second leading cause of cancer death in females following lung cancer (2) with more than 1,000,000 new cases and 370,000 deaths yearly worldwide (3).
Progranulin is an 88-kDa glycoprotein known as GP88, PC-cell derived growth factor or acrogranin, GP88 gene is located on the 21q portion of chromosome 17, while the mouse gene was found on chromosome 11.The autocrine growth factor GP88 is abundantly expressed in epithelial cells, immune cells, neurons, and chondrocytes (4) .
Cathepsin D (Cath-D) is a soluble lysosomal aspartyl glycoprotease that can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion, and angiogenesis.The aspartic protease Cath-D, a poor prognostic indicator of breast cancer, is abundantly secreted as pro-Cath-D by human breast cancer cells and self-activates at low pH in vitro, giving rise to catalytically active Cath-D (5) .
In the present study, the expression of GP88 and Cath-D in an invasive ductal carcinoma (IDC) was investigated using an immunohistochemical technique, and the intensity of immunostaining was quantitatively estimated using an image optical density (IOD) analyzer.

Figure (5):
A section of IDC grade II showing tumor cells with abundant eosinophilic cytoplasm and pleomorphic round-to-ovoid vesicular nuclei (thin arrow).The cells that are arranged in cords infiltrate the desmoplastic stroma (thick arrow) (H&E.Bar =50 µm).

Figure (6):
A section of IDC grade III (Atypical medullary variant) showing sheets of malignant ductal cells with wide areas of necrosis (thin arrow) and lymphocytes infiltration separated by fibrous tissue septa (thick arrow) (H&E.Bar=200 µm).

B-Immunohistochemical results:
I-GP88: a. Immunohistochemical reactivity of GP88:  Immunostaining reactivity of GP88 was detected as a diffuse, homogenous brown color in the cytoplasm and membrane of the ductal epithelial cells of the studied groups. GP88 immunostaining reactivity was negative (-ve) in 80% (8/10) of the control group and 57% (17/30) of the benign group, while it was moderate (2+) in 71% (32/45) of grade II IDC and strong (3+) in 77% (10/13) of grade III IDC as illustrated in figures (7).

Figure (7):
Immunostaining reactivity of GP88 in the different studied Groups.GP88 was overexpressed in the malignant group versus control and benign groups.

Figure (8):
Immunohistochemical staining of control breast tissue showing negative expression of GP88 in the cytoplasm of the ductal epithelial cell (Bar =50 µm).

Figure (9):
A benign breast tissue showing negative (-ve) expression of GP88 in the cytoplasm of the ductal epithelial cells (Bar =50 µm).

b. Integrated optical density (IOD) of GP88 in the different studied groups:
The mean values of GP88 IOD for control, benign, and IDC grade II and III were 35 ± 3, 41 ± 4, 140 ± 8 and 162 ± 7 respectively.A significant difference was noticed between GP88 IOD of grade II and III IDC and GP88 IOD of both control and benign groups (p < 0.00), but there was no statistically significant difference between GP88 IOD of control and benign groups (p = 0.1) as shown in table (2) and figure (12).

C. Correlation between GP88 IOD and histopathological parameters in breast cancer cases:
There was no statistically significant correlation between the GP88 IOD and patients' age (r= -.The mean values of Cath-D IOD for control, benign, and IDC grade II and III were 30 ± 3, 124 ± 3, 159 ± 9, and 168 ± 3 respectively.A statistically significant difference (p < 0.00) was noticed between the studied groups as shown in table (5) and figure (21).

DISCUSSION
Breast cancer remains a major scientific, clinical, and societal challenge.It is the most common malignancy and the second leading cause of cancer death in females worldwide following lung cancer (6,7) .In many developing countries, the incidence of breast cancer is now rising sharply due to changes in reproductive factors, lifestyle, and increased life expectancy (8) .
The results of the present study showed that 98% of the breast cancer cases were invasive ductal carcinoma (IDC), most of which were allocated to the age range of (>35-55) years.These results are consistent with several previous studies that reported that IDC is the most common histological type of invasive breast cancer, and in the developing world, it is characterized by an early peak age of onset (9,10) .
The present study was undertaken to assess the immunohistochemical expression of GP88 and Cath-D in human breast invasive carcinoma versus normal control and benign breast tumors, as well as to investigate the correlation of their immunohistochemical expression with clinicopathological parameters.
The ability of cancer cells to produce and respond to their own (autocrine) growth factors is important in the proliferation and progression of cancer cells (11) .
Progranulin or GP88 is an autocrine growth factor and pleiotropic regulatory protein that has been shown to play a role in tumorigenesis, including proliferation, survival, migration, angiogenesis invasion, and matrix metalloprotease activity, in addition to its role in wound healing and inflammation in normal tissues (12) .
The results of the present study showed a statistically significant increase in the immunohistochemical expression of GP88 in IDC, versus, normal tissues and benign tumors (p < .001).This result is in alignment with previous findings that reported a high level of GP88 expression in breast cancer biopsies versus benign lesions and normal mammary epithelial tissues (13) .
In addition, pathological studies with 203 formalinfixed paraffin-embedded human breast cancer tissue biopsies indicated that GP88 was preferentially expressed in ductal carcinoma with little expression in lobular carcinoma while benign lesions and normal mammary epithelial tissues were negative (14) .
Cath-D is a lysosomal aspartic protease that is overexpressed by epithelial and stromal breast cancer cells.This protease is an independent marker of poor prognosis in breast cancer as it is correlated with the incidence of clinical metastasis (15) .
The results of the current study showed that Cath-D expression was increased in breast cancer cases than in normal and benign cases.A Previous study reported that normal lobular or ductal epithelia both from non-tumoral and tumoral lesions showed no Cath-D specific Staining (16) .
Interestingly, the present results showed a statistically significant difference between the expression of Cath-D in normal and benign cases.
This finding agreed with Brujan, et al., (2009) (17) , who noticed that the expression of Cath-D in benign breast tumors was higher than in normal breast tissues, but still lesser than in malignant breast tumors.
Several approaches, such as immunohistochemistry, in situ hybridization, cytosolic immunoassay, and Northern and Western blot analyses have indicated that in most breast cancer tumors Cath-D is overexpressed 2-to 50fold compared to its concentration in other cells such as fibroblasts or normal mammary glands (18) .
The current results showed no statistically significant correlation between the immunohistochemical expression of Cath-D and patients' age (r = .22,P = .09).This lack of correlation between the expression of Cath-D and patients' age was also reported by several previous studies (19) .
The results of the current study showed no statistically significant correlation between the immunohistochemical expression of Cath-D and tumor size (r = .04,P = .77).This result is consistent with Gion, et al., (1995) (20) and Brujan, et al. (2009) (21) , but contrasted with Ruibal, et al., (2012) (22) who found that cytosolic concentration of Cath-D was associated with large tumors.
Cath-D is involved in the pathogenesis of neurodegenerative, skin, cardiovascular, and tumoral diseases (27) .In these pathologies, Cath-D is aberrantly produced and processed in malignancy and over-secreted to the cell microenvironment where it acts as tumor and stromal cells mitogen, also its hyper secretion leads to excessive degradation of the extracellular matrix, which contributes to tumor progression and metastases (28) .
A lack of correlation between ER and Cath-D in breast tumors was previously reported and the number of tumors with high concentrations of cathepsin D was not significantly different in ERpositive and ER-negative samples (29) .In contrast, NIKOLIAE-VUKOSAVLJEVIAE et al., (2002) (30) found a direct association between cathepsin D and ER and PR status.
, this may interpret their association in IDC in the present study.

Figure ( 2 )
Figure (2): A view of the acini present in a normal lobule.The acini are lined by cuboidal epithelium (thick arrow) with underlying myoepithelial cells having clear cytoplasm (thin arrow) (H & E. Bar =50 µm).

Figure ( 12 ):
Figure (12): The mean and SD of GP88 IOD in the different studied groups

Figure ( 13 ):
Figure (13): Correlation between GP88 IOD and tumor size/cm of the breast cancer cases.

Figure ( 17 )
Figure (17): A benign breast tissue showing moderate (2+) expression of Cath-D enzyme in the cytoplasm of the ductal epithelial cells and surrounding extracellular matrix (Bar=50 µm).

Figure ( 20
Figure (20): An IDC grade III breast tissue showing strong (3+) expression of Cath-D in the cytoplasm of the ductal epithelial cells (Bar=50 µm).
Figure (21): The mean and SD of Cath-D IOD in the different studied groups

Figure ( 22 ):
Figure (22): Correlation between Cath-D IOD and tumor grade of the breast cancer cases.
GP88 and Cath-D are representatives of cytokines, growth factors, and proteases.Stromal and tumor cells exchange enzymes, growth factors, and cytokines that modify the local extracellular matrix, stimulate migration and invasion, and promote the proliferation and survival of stromal and tumor cells

Table ( 2): The mean and SD of GP88 IOD in the different studied groups
p2: p-value for Post Hoc test (Scheffe) comparing between benign and each other group.p3: p-value for Post Hoc test (Scheffe) for comparing grade II with grade III.*: Statistically significant at p < 0.05
II-Cath-D: a. Immunohistochemical reactivity of Cath-D:   Table