AUTOPHAGY BIOMARKERS SQSTM1/P62 AND BECLIN-1 EXPRESSION IN BREAST CARCINOMA AND THEIR CLINICOPATHOLOGICAL SIGNIFICANCE.

Background: Clarifying the different mechanisms of molecular carcinogenesis of breast carcinoma could enable its better management, improving its prognosis and decreasing patient’s mortality. Autophagy is the process of lysosomal degradation which could remove the damaged components so as to preserve cells homeostasis. Autophagy had a complicated role in cancer as it might inhibit or stimulate cancer progression which depend on the type of cancer. Several proteins which control autophagy had been discovered in human cells such as Beclin-1and SQSTM1/p62 that played important roles in autophagy. aimed to assess expressions of autophagy markers SQSTM1/p62 and Beclin-1 in breast carcinoma comparing expression of both markers with clinicopathological parameters of that type of cancer. For advanced stage of breast carcinoma was 85.5 and the specificity was 97.9. We found an inverse relationship between p62 and Beclin-1 (Spearman’s r= – 0.806), Conclusion: SQSTM1-p62 over expression is a marker of poor prognosis, but Beclin-1 overexpression was a marker of good prognosis in breast carcinoma.


ISSN: 2320-5407
Int. J. Adv. Res. 6(2), 1846-1856 1847 For advanced stage of breast carcinoma was 85.5 and the specificity was 97.9. We found an inverse relationship between p62 and Beclin-1 (Spearman's r= -0.806), Conclusion: SQSTM1-p62 over expression is a marker of poor prognosis, but Beclin-1 overexpression was a marker of good prognosis in breast carcinoma. Aim of the work this study aimed to assess expressions of autophagy markers SQSTM1/p62 and Beclin-1 in breast carcinoma comparing expression of both markers with clinicopathological parameters of that type of cancer.

Patients and methods:-
We have included sixty formalin fixed paraffin-embedded blocks of breast carcinoma of different histological subtypes were collected from the Pathology Department, Faculty of Medicine, Beni-Suef University and Pathology Department, Kasr Al Aini Hospital.. We used the American Joint Committee on Cancer staging system (AJCC) classification 7 th edition carcinoma staging (Edge and Compton, 2010) and Nottingham (Elston-Ellis) modified Scarff-Bloom-Richardson grading system for carcinoma grading (Elston, 2002). We detected age of the patient, cancer size, histolopathological subtype, grade, stage, lymph node, and distant metastasis by examination of the patient's and the slide files in Pathology Department.

Evaluation of immunohistochemical expressions of both Beclin-1 and SQSTM1/p62 proteins:-
We have evaluated both extent and the intensity of stain for both markers. The extent had been graded as zero (negative), one (<30% positive) and two (>30% positive) and we have graded the intensity was as zero (negative), one (weak), two (moderate) and three (strong). Then we have multiplied both scores to have a total final score: negative (from zero to one), low (from two to four) and high (five and six) (Won et al., 2009), score less than four was considered as low expression and scores more than four as high expression.
Statistical analysis:-Continuous variables were expressed as the mean±SD and median (range) and the categorical variables were expressed as a number (%).A P value less than 0.05 was considered statistically significant. Percent of categorical variables were compared using Pearson's w2-test or Fisher's exact test when appropriate. The strength of the relationship between SQSTM1-p62, Beclin-1 and clinicopathological features was determined by computing Spearman's correlation coefficient. 1848

Results:-
Sixty females' patients were included in our study, with age ranged from 39-77 years (Mean ± SD: 56.35±10.99). Demographic data of all patients were detailed in table (1). SQSTM1/p62 expression in relation to clinicopathological features:-SQSTM1/p62 overexpression in breast carcinoma was significantly correlated with older age of the patients, higher grade (=0.002) and AJCC stage of the tumor, aggressive molecular type (=0.009), presence of lymph node metastases (=0.041), high KI67 index (p<0.001), negative ER& PR hormonal receptors (=0.01),, high Her2 neu expression (=0.03), and presence of distant metastasis (p=0.011). But it had no significant correlation with histopathological subtype of breast cancer. Tables 2& 4; fig 1 The sensitivity of high p62 expression as a predictor for advanced stage of infiltrating duct carcinoma (IDC) was 72.4 and the specificity was 97.9 Beclin-1 immunoexpression and its correlation with clinicopathological features:-Beclin-1 was cytoplasmic and its low expression was significantly correlated with older age of the patients, aggressive molecular type (=0.004), higher grade (p<0.001) and AJCC stage of the tumor (p=0.002), presence of lymph node metastases (=0.041), high KI67 index (p<0.001), negative ER& PR hormonal receptors (=0.003), high Her2 neu expression (=0.006), presence of distant metastasis (p=0.011). But it had no significant correlation with histopathological subtype of breast cancer. Tables 3& 4; fig 2 The sensitivity of low Beclin-1 expression as a predictor for advanced stage of IDC was 85.5 and the specificity was 97.9 Table 5 Correlation between immunohistochemical expression of SQSTM1/P62, Beclin-1, in IDC:-We found an inverse relationship between SQSTM1/P62 and Beclin-1 expression (Spearman's r= -0.806).

Discussion:-
Clinicopathological role of autophagy in cancer is still a point of research that could be due to variability in its role in carcinogenesis (White, 2012). In this study, we found that SQSTM1-p62 overexpression was correlated positively with a poor clinical behavior of cancer such as large size, higher incidence of positive lymph nodes, higher grade, and advanced stage and high incidence of distant metastases occurrence in patients with breast carcinoma (P<0.001). explained such conflicting results to differences in evaluation of positive areas in carcinoma tissues in between the peripheral and the central area, that were different in composition, while most researches had not analyze which positive part that they have found in cancer tissue. Many conflicting results had been discovered regarding clinicopathological role of Beclin-1, that is important the autophagy-related protein, expression in breast carcinoma. We declared that Beclin-1 expression in breast carcinoma was strongly negatively correlated to size, grade and stage of the tumor, the presence of nodal and/or distant metastasis. Beclin-1 overexpression was strongly positively correlated with good clinicopathological criteria in breast carcinoma. analysis had showed that Beclin-1 increased expression could be able to be a protective factor in stomach cancer and lymphoma that was similar to our results in cancer breast, but it had no clinicopathological relation to colon or lung cancers. On the contrary to our results, Han et al. 2014 had found that Beclin-1 over expression in cancer colon was associated with increased incidence of L.N, blood metastasis and associated with poor prognosis. So it had been found that both Beclin-1 increased or decreased expression were detected in human malignancies; as it may had a cancer suppressor role, by interacting with bcl-2 protein members (Cao and Klionsky, 2007). Otherwise the tumor stimulatory role of Beclin-1 overexpression in which it was associated with cancers aggressive behavior was done by anti-apoptotic machinery potentiation (Koukourakis et al. 2010), and another mechanism Beclin-1 overexpression during adverse cancer environmental conditions like hypoxia and increased acidity, so as to allow cancer cells to overcome such conditions by increasing autophagic cancer cells activity to recycle un needed proteins and damaged organelles to increased their survival (Samokhvalov et al.,  2008). However Beclin-1 over expression could be able to delay cancer progression by decreasing chromosomal instability and the occurrence of more mutation (Mathew et al., 2007). That may be due to Beclin-1-dependent autophagy which induced immunological response (Xu et al., 2008).
In summary, in our study we proved that SQSTM1/p62 expressions were strongly positively related to bad clinicopathological parameters of cancer breast patients and its expression was negatively correlated to Beclin-1 expression, which was proved to be a marker of favorable prognosis for cancer breast patients. There are conflicting results on the prognostic value of both markers in cancer breast. We recommended to do more studies confirm their role as clinical predictors of poor or good clinicopathological criteria and different outcome for breast cancer patients that may help to detect novel therapeutic targets to for them improving their prognosis.

Conclusion:-
SQSTM1/p62 is markers of a poor prognosis, while Beclin-1 is a marker of a good prognosis in in breast cancer.

Conflicts of interest
None declared.