ROLE OF S100P AS A NEW PROGNOSTIC MARKER IN WOMEN WITH METASTATIC BREAST CANCER AND ITS CORRELATION WITH BCL2 AND BAX EXPRESSION

* Lobna A. Abdelaziz 1 , Hoda F. Ebian 2 ,Shereen El Shorbagy 3 , Rham Z.Ahmed 3 , Ola A. Harb 4 , Mariem A. Elfeky 4 , Mohammed E.Eraki 5 and Loay M. Gertallah 5 . 1. Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Zagazig University, 2. Clinical Pathology Department, Faculty of Medicine, Zagazig University. 3. Medical Oncology Department, Faculty of Medicine, Zagazig University. 4. Pathology Department, Faculty of Medicine, Zagazig University. 5. General surgery Department, Faculty of Medicine, Zagazig University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Tissue specimens: Formalin fixed paraffin embedded blocks of PBC and MBC patients were collected. The seventh edition of the American Joint Committee on Cancer staging system (AJCC-7) classification was used for pathologic staging (13) and the Nottingham (Elston-Ellis) modification of the Scarff -Bloom-Richardson grading system was used for pathologic grading (14).
Immunohistochemical staining: Immunohistochemical staining was carried out using the streptavidin-biotin immunoperoxidase technique (15), the slides were incubated with mouse monoclonal Anti-Bcl-2 antibody [Bcl2/100] ab117115 was used at a dilution of 1:100 and primary rabbit polyclonal Anti-Bax antibody ab10813 diluted 1:1000 (Abcam, Cambridge, MA, USA) at 4°C overnight. Sections from normal tonsils were used as positive control for bcl2, sections from normal colon for bax and the negative control is by adding non-immune serum instead of the primary antibodies.

Evaluation of immunohistochemical expressions of Bcl2 proteins:
Cytoplasmic staining of bcl-2was scored as followed; bcl2 negative-no tumor cells stain or weak heterogeneous positive stain in less than 10 % of tumor cells and bcl-2 positive-more than 10 % of tumor cells stained (16).

Evaluation of immunohistochemical expression of bax:
Bax cytoplasmic expression was scored as positive if at least 20% of tumor cells showed clear cytoplasmic immunostaining (17).
Statistical Analysis:-Continuous variables were expressed as the mean ± SD & median (range), and the categorical variables were expressed as a number(percentage). Continuous variables were checked for normality by using Shapiro-Wilk test. Percent of categorical variables were compared using Pearson's Chi-square test or Fisher's exact test when was appropriate. Receiver operating characteristic (ROC) curve analysis was used to identify optimal cut-off value of S100 level with maximum sensitivity and specificity for discrimination between breast cancer and control. Overall Survival (OS) was calculated as the time from diagnosis to death or the most recent follow-up contact (censored). Progression Free Survival (PFS) was calculated as the time from start of treatment to date of progression or the most recent follow-up contact that patient was known as progression free. Stratification of OS and PFS was done according markers. These time-to-event distributions were estimated using the method of Kaplan-Meier plot, and compared using two-sided exact log-rank test. All tests were two sided. A p-value <0.05 was considered significant. All statistics were performed using SPSS 22.0 for windows (SPSS Inc., Chicago, IL, USA) and MedCalc windows (MedCalc Software bvba 13, Ostend, Belgium).
Comparison between the studied groups as regard the 3 studied markers •The median of plasma S100 P level was nearly the same for PBC patients and healthy control, in addition its level in MBC patients was higher than both PBC patients and healthy control (near the double) (p1 <0.001 between MBC vs Non-MBC; p2 <0.001 between MBC vs control) both of them are significant while p3 =0 .834 between Non-MBC vs control (non significant). •Regarding Bcl2 immunoexpression positive expression was found in 23 (32.9%) of MBC , 11 (91.7%) of Non-MBC and in 8 (100%) of the normal breast tissue (p1 <0.001 between MBC vs Non-MBC; p2 <0.001 between MBC vs control) both of them are significant while p3 =0.402 between Non-MBC vs control (non significant).

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•Regarding Bax immunoexpression; positive expression was found in 47 (67.1%) of cases of MBC but no positive expression was detected in either Non-MBC or in the normal breast tissue (p3 <0.001). (p1 <0.001 between MBC vs Non-MBC; p2 <0.001 between MBC vs control; p3 <0.001 between Non-MBC vs control all of them are significant (Table 1).  Effect of clinicopathological parameters on plasma S100p level in breast cancer patients plasma level of S100 in all breast cancer patients showed statistical correlation with capsular invasion (p =0.018), T (p=0.031), stage , bcl2 and bax expression (P <0.001 for each of them ),while S100 level in metastatic breast cancer patients showed significant correlation with capsular invasion and bcl2(P =0.002&<0.001 respectively). Furthermore, there is a significant association between elevation of S100P level and both the number and site of metastasis (P<0.001 for both); {presence of liver metastasis P=0.002, brain metastasis P=0.009 and bone metastasis P<0.001} (Table 2, 3 ) .

Correlation between clinicopathological parameters and Bcl2 expression in breast cancer patients
Bcl2 was cytoplasmic and its protein expressions in breast cancer tissues were lower than those in the relatively healthy and adjacent breast tissues .Bcl2 was positively expressed in 41.5% of all studied breast cancer patients, 32.9%of MBC, and 91.7% of PBC . Moreover, its low expression in all breast cancer patients was significantly negatively correlated with grade(P 0.001) ,ki67 (P 0.021), molecular subtype (P 0.050) , and each one of T ,N , stage ,BAX( P <0.001 for each of them ) ,S100p (P<0.05 ) . But its expression in MBC patients was statistically significant with, ki67 (P 0.044), molecular subtype (P 0.050) , and each one of grade, T ,N ,BAX( P <0.001 for each of them), number of metastasis (P=0.014) and presence of bone metastasis (P=0.013) ( Table 2,3 ).

Correlation between clinicopathological parameters and bax expression
Bax was cytoplasmic and its protein expression in breast cancer tissues was significantly higher than that in the relatively healthy, adjacent breast tissues. . Bax was positively expressed in 57.3% of all studied breast cancer patients, 67%of MBC, while all PBC didn't express bax.. Furthermore ,its high expression is statistically significant with grade (P=0.003) ,every one of molecular subtype ,T ,N and stage (P<0.001 for each ) ,ki67 (P<0.004) ,ER (P<0.010), PR(P<0.023) ,Her-2 neu (P<0.002) ,both bcl2 and BAX (P<0.001 for both) in all breast cancer patients . However in MBC patients it is statistically significant with grade (P=0.001), every one of molecular subtype, T, N (P<0.001 for each), ki67 (P<0.002) , site of metastasis (P=0.003), presence of brain metastasis (P=0.004) and lung metastasis (P=0.032)}( Table 2,3).

Effect of plasma S100p level, (bax and Bcl2) expression on treatment outcome in metastatic breast cancer patients:
The implication of plasma S100P levels on the prognosis of our 70 MBC patients was studied in our trial. Our patients were undergone follow up for 3 years (the median follow up period was 19 month) . The cut off points of plasma S100P was 7.6 ng / ml, which indicates that the values above it are high but the values below it are low. Evaluation of the effect of the markers on treatment outcome (response to treatment , occurance of progression and mortality ) was done , which reveals significant correlation between plasma S100P level with each one of the following; response , progression, and mortality ( P<0.001 for each ) (Table 4) . Similarly ,Bcl2 expression is significantly correlated with response ( P <0.001) , progression ( P 0.004) and mortality ( P 0.01) ( Table 4 ) . However, bax expression bax was not significantly correlated with response (P =0.78), progression ( P =0.80) or mortality ( P= 0.80) .

Effect of plasma S100p level, (bax and Bcl2) expression on Survival in metastatic breast cancer patients:
Mean PFS was 24.14 months, 3 y PFS was 57.9%. Mean OS was 30.75 month, 3y OS was 70.9%. The association between plasma S100P levels and PFS was assessed in our MBC patients. As illustrated in Kaplan-Meier curve, MBC patients with lower plasma S100P level had significantlylonger PFS in comparison to those who had higher plasma S100P levels (the mean PFS 35.36 months VS 9.94 months respectively, P=0.000, Fig.1B Fig.1D).
Overall survival (OS) in the studied MBC patients (N=70) showed that , patients who had low plasma S100P levels had significantly better OS than those who had high plasma S100P levels (the mean OS was 36.3 months VS 16.56 months respectively, P=0.000, Fig.2B Fig.2D). In our study ; we followed the patients for 3 years (the median follow up period was 19 month). The cut off points of plasma S100P was 7.6 ng / ml, which indicates that the values above it are highbut the values below it are low . S100P level in metastatic breast cancer patients are higher than PBC and healthy tissue. Elevation of Serum S100P level in metastatic breast cancer patients showed significant correlation with capsular invasion, bcl2(P =0.002 and <0.001 respectively), increased number of metastasis, metastatic site (P<0.001 for both ); { presence of liver metastasis P=0.002, brain metastasis P=0.009 and bone metastasis P<0.001}. There is a significant correlation between plasma S100P level with response, progression, and mortality (P<0.001 for each). MBC patients with lower plasma S100P level had longer but non-significant PFS time in comparison to those who had higher plasma S100P levels (the mean PFS time 35.36 months VS 9.94 months respectively, log-rank test P=0.000) Patients who had low plasma S100P levels had better but non-significant OS than those who had high plasma S100P levels (the mean OS time was 36.3 months VS 16.56 months respectively, log-rank test P=0.000)

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The small, calcium-binding protein S100P has gained the attention of researchers from different scientific fields due to its potential roles in both healthy and neoplastic tissues (22). S100P is a member of the large family of S100 calcium-binding proteins that mediate Ca 2+ dependent signal transduction pathways (23). Its expression has been found frequently and at high levels, in a variety of different tumor types in addition to its role in chemoresistance (3, 4). S100P could potentially serve as diagnostic marker, prognostic ,predictive indicator and therapy target for different carcinomas through its inhibition or inhibition of its targets, or its interactions which result in a decrease of cellular motility and metastatic potential (7,24). S100P plays an important role in oncogenesis (tumor cell proliferation, differentiation, apoptosis, weakening of cell: cell adhesion contacts, stimulation of cell motility and invasion and metastasis (25, 26). S100 P is expressed in tumor tissue and absent in most healthy tissues, so it was evaluated as a novel biomarker for detection of several cancers by using immunohistochemistry approaches (24). But in our study we used ELISA as a new modality for measurement of S100 P.
Indeed, anti-S100P antibodies have shown promising results (in vitro and in vivo) as single agents and in combination with other chemotherapeutic agents, such as gemcitabine in pancreatic cancer (3). So blocking S100P function is expected to improve responses to chemotherapeutic agents. However, this needs further investigations because there are other reports showed that overexpression of S100P (in vitro) led to sensitization of cancer cells to carboplatin, paclitaxel (27) and oxaliplatin (28) in ovarian and gastric cancer cells respectively. Despite this, S100P still represents a potentially very effective anti-cancer target, at least for in some cancer types, and further development of anti-S100P specific therapies will likely prove to be a fruitful and productive field of investigation (29).
In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumor behavior, indicating that this protein may have adverse prognostic value and poor survival in breast cancer patients (30).
Univariate and multivariate analyses in early breast cancer patients (stage II) showed that higher expression of nuclear S100P (S100Pn) was observed in cases of a shorter overall survival and disease-free time. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. The preliminary data indicated that, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value. (30).
Plasma S100P levels were measured in 381 women, including 60 healthy controls, 48 primary breast cancer patients (PBC) patients, and 273 metastatic breast cancer (MBC) with correlation between increased its level and MBC . In addition, assessment of prognostic value of S100P with enumeration of CTC and clinicopathological factors were done .The follow up period was 3.5 years. They found that the plasma S100P cut off point was 7 ng / ml and also there is association between high plasma S100P level (>7 ng/mL) and poor prognosis of MBC patients (median progression-free survival time: 5.0 vs. 8.7 months, log-rank test p < 0.001; median overall survival time: 22.5 vs. 31.6 months, log-rank test 1865 p < 0.001). The plasma S100P level added additional prognostic relevance to the prognostication model with clinicopathological factors and CTC enumeration. Furthermore, The examination of the value of plasma S100P levels as treatment monitoring marker was done and revealed, its significant reduction after treatment, This indicates its value in evaluation of treatment outcome. They concluded that plasma S100P level is a simple and cost-effective marker for the prognosis of metastatic breast cancer (4). The results of this trial are consistent with our result where, the cut off points of plasma S100P was 7.6 ng / ml, which indicate that the values above it are highbut the values below it are low. S100P level in metastatic breast cancer patients are higher than PBC and healthy tissue. There is significant correlation between plasma S100P level with response, progression, and mortality (P<0.001 for each). MBC patients with lower plasma S100P level had significant longer PFS time in comparison to those who had higher plasma S100P levels (the mean PFS time 35.36 months VS 9.94 months respectively, log-rank test P=0.000 ) Patients who had low plasma S100P levels had significant better OS than those who had high plasma S100P levels (the mean OS time was 36.3 months VS 16.56 months respectively, log-rank test P=0.000) .
There are many theories about the lack of activity of anti-cancer drugs in breast cancer. The disruption of the apoptotic pathways may be one of reasons. We therefore decided to assess the expression of those factors involved in apoptosis in the normal mammary gland, benign mammary dysplasia and primary cancer. The most significant findings of our study are that, Bcl2 positive protein expressions in breast cancer tissues were lower than those in the healthy and adjacent breast tissues. Moreover, its expression in all breast cancer patients was significantly correlated with good clinic pathological parameters like low grade and stage (P <0.001) and low ki67 level (P 0.021). Also its positive expression in MBC patients was statistically significant with low ki67 level (P 0.044), molecular subtype (P 0.050), and low grade, T, N, Bax (P <0.001 for each of them) decreased number of metastasis (P=0.014) and presence of bone metastasis (P=0.013).
Bax positive protein expressions in breast cancer tissues were higher than that in the relatively healthy, adjacent breast tissues. Furthermore, its positive expression was significantly correlated with poor clinic pathological parameters like high grade (P=0.003) and stage (P<0.001), high ki67 level (P<0.004), positive ER (P<0.010), PR (P<0.023) , Her-2 neu (P<0.002) and negative bcl2 level (P<0.001) in all breast cancer patients. However in MBC patients its positive expression is statistically significant with high grade (P=0.001), every one of molecular subtype, T, N (P<0.001 for each), high ki67 level(P<0.002), and site of metastasis (P=0.003) {brain (P=0.004), lung (P=0.032). However, bax expression is not significant with either response (P =0.78), progression (P =0.80) nor mortality (P= 0.80). Similarly, Bcl2 expression is significantly correlated with response (P <0.001), progression ( P 0.004) and mortality ( P 0.01) Mean PFS was 24.14 months, 3 (36) suggested that Bcl-2 and Bax expression were associated with a regulation of apoptosis in breast cancer.They found that Bcl-2 expression in tumours was associated with a better differentiation of the cancers (G1 -100% of Bcl-2-positive tumours, G2 -81%, G3 -60%), but there was no relationship between Bax and tumor grade . Dawson et al 2010 (39)revealed that BCL2 continues to be associated with favorable outcome. BCL2 belongs to a group of related proteins that are key regulators of apoptosis or programmed cell death (Cory et al, 2003) (40).
BCL2 protein expression in breast cancer is associated with an indolent phenotype of low-grade, slowly proliferating, ERþ breast tumours (Silvestrini et al, 1994; Lipponen et al, 1995)(41,42) . This 'paradoxical' favourable prognostic effect of BCL2 in breast cancer could be related to its non-apoptotic functions (43). Increased expression of BCL2 protein may also disrupt the balance with other members of the BCL2 family, including the expression of pro-apoptotic proteins (40) .The exact mechanism of differential BCL2 protein expression in breast cancer is complex. BCL2 is expressed in normal breast glandular epithelium and is known to be upregulated by oestrogen, possibly as a direct result of transcriptional induction (44) Also no differences in treatment response were found in patients with early breast cancer related to Bax expression in the tumor cells (45). While Krajewski et al. detected that, MBC patients with low Bax expression had poorer response to treatment and shorter OS (46). Pluta P, et al studied 62 breast cancer patients and control group of 11 breast fibroadenoma patients, bax expression was assessed by flow cytometer, bax expression was found in 82% of patients. Bax expression was lower in breast cancer patients than in controls, and this could be one of the mechanisms of apoptosis escaping by tumor cells (47).
Novel markers that could be used to save women from unnecessary cytotoxic adjuvant therapy are urgently needed and BCL2 provides valuable additional prognostic information to guide clinical decision making in this setting. In summaryour results proved that bcl2 and bax are independent and powerful prognostic protein marker in breast cancer patients more than other prognostic factors.
Our results indicate that overexpression of pro-apoptotic proteins could contribute to an increase in cell turnover and breast cancer development and progression, but we suggest that further studies should be carried out with increased sample size to fully assess Bak expression in breast cancer progression.
Conclusion: S100P, BCL2 and bax are promising prognostic markers in breast cancer patients but we recommend further studies with large sample size to be done to increase statistical power of the results.