Predictive and prognostic value of SALL4 and Tau protein in serous ovarian cancer patients treated with chemotherapy

Ola A. Harb 1 , Rasha Haggag 2 , Amira Amin Salem 1 , Walid Abdalla Abdesalam 3 and Reham Amin Salim 4 . 1. Department of pathology, Faculty of Medicine, Zagazig University, Sharkia, Egypt. 2. Department of Medical Oncology, Faculty of Medicine, Zagazig University, Sharkia, Egypt. 3. Department of Gynecology and Obstetrics, Faculty of Medicine, Zagazig University, Sharkia, Egypt. 4. Department of Clinical Oncology and Nuclear medicine, Faculty of Medicine, Zagazig University, Sharkia, Egypt.


Introduction:
Epithelial ovarian cancer (EOC) is the 4 th commonest malignancy in female [1], and it is one of the most common causes of postmenopausal women cancer mortality worldwide, serous ovarian carcinoma (SOC) forms high percentage of its types [1]. Despite the advancement in surgical intervention for management of SOC, its overall survival and progression-free survival rates are still poor as it is discovered at advanced stage especially in developing countries [3]. The primary goal of surgery is debulking due to the diffuse nature of SOC and the invasion of adjacent organs or peritoneal cavity via peritoneal fluid [4] followed by chemotherapy and molecular targeted therapies that play an important role in the postoperative treatment of SOC. The currently used chemotherapeutic agents are platinum plus taxane therapy [2]; they appeared with more resistance and less efficacy dealing with cancer patients. So a novel therapeutic agent that decrease the resistance to the current chemotherapeutic agents and that also specifically target the serous ovarian cancer is the main target for clinical cancer research [5][6]. Spalt-like transcription factor (SALL4) is a zinc finger transcriptional factor that regulates multiple downstream targeted genes involved in normal development, important in the control of pluripotency and self-renewal of embryonic stem cells (escs) [7], also recent research demonstrated that SALL4 is plays an important role in hematopoiesis and leukemogenesis, the abnormal expression of SALL4 was found in acute myeloid leukemia [8], and it functions as an oncogene in many cancers [9]. Tau protein (50-64 kd), a product of gene located in chromosome 17 (17q21) shows the ability of combining to beta-tubulin. It may bind to the exterior and to the interior microtubules surface [10].Tau was first isolated from brain and its function has been associated with neurodegenerative diseases [11].There is a real need to adequately understand the mechanisms of SOC invasion, progression and development of more effective therapeutic molecular targets and prognostic biomarkers. The predictive or prognostic value of Tau protein and SALL4 in SOC is still points under research, and no previous studies had explored the relation between immunohistochemical expression of SALL4 and its relation to chemotherapy resistance in SOC patients or detect the relation between both SALL4 and Tau protein immunoexpression.
To explore and assess the predictive and prognostic role of SALL4 and Tau protein immunohistochemical expressions in serous ovarian carcinoma patients treated with paclitaxel/platinum first-line chemotherapy.

Patients and Methods:-
This retrospective study was carried out at departments of Medical Oncology, Pathology, Clinical Oncology and nuclear medicine, and Gynecology and Obstetrics, Faculty of Medicine, Zagazig University in the period from March 2013 to March 2016.
We included 50 histologically confirmed serous ovarian cancer (SOC) International Federation of Gynaecology and Obstetrics (FIGO) stage IC-IV. All patients had history of debulking surgery followed by first-line Chemotherapy regimen: paclitaxel (175 mg/m2) with carboplatin (AUC 6), administered every 3 weeks for 6 cycles. We used the tumor-node-metastasis (TNM) and International Federation of Gynecology and Obstetrics (FIGO) classifications for staging of SOC [12] and the WHO grading system, for pathologic grading [13]. We identified sex, age, tumor size, grade, stage, lymph node, distant metastasis, response to first-line chemotherapy according to RECIST criteria of the patients by retrospective examination of the patients' records at the involved departments. Local Research Ethics Committee approval of the study was obtained.
Immunohistochemical staining:-Immunohistochemical staining was carried out using using streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China)5μm thick sections cut from paraffin blocks, put on positively charged slides, then de-paraffinized in xylene and rehydrated in rising grades of ethyl alcohol. Antigen retrieval was done by boiling sections in citrate buffer (ph6.0) for 20 min and then after washing with phosphate buffered saline (PBS), the slides were incubated with rabbit monoclonal anti-Tau antibody [E178] (ab32057) (Abcam, Cambridge, MA, USA) was used at a dilution of 1:500 and Mouse monoclonal anti-Sall4 antibody (ab57577) (Abcam,Cambridge, MA, USA)diluted 1/100 at 4°C overnight. The sections were then washed in PBS and incubated with Poly-peroxidaseanti-mouse/rabbit igg (Zymed Laboratories, San Francisco, CA, USA) for 20 min. 3; 30-Diaminobenzidine was used as the chromogen. At the end, the sections were counterstained with hematoxylin. Normal ovarian epithelium taken from areas near benign ovarian cyst was used as an external positive control for Tau protein [14], human yolk sac tumor tissue as a positive control for SALL4 [15] and the negative controls by replacing the primary antibodies by the non-immune serum.

Evaluation of immunohistochemical expression of Tau protein:-
Specimens were assessed as follows: IHC score 0 -no staining; 1+ − poor focal staining or very poor diffuse staining (less intense than normal ovarian epithelium); 2+ average diffuse staining (similar to normal ovarian epithelium) or strong staining (more intense than normal ovarian epithelium) in less than 25% cells; 3 + strong staining in 25% of tumors cells or more. Tau expression was interpreted as negative (0 and 1+) or positive (2+ and 3+). The final staining results were determined by using staining intensity of normal epithelial cells as a reference. We consider results as; low (0-1, 5) and high (2)(3)

Results:-
Patients:-Fifty patients of serous carcinoma of the ovary were included in our study with age ranged from (25-75) years (Mean: 55.48 ± 10.97 years), 18 cases were of low grade and 32 cases were of high grade type.

Correlation between SALL4 and Tau expressions with clinicopath-ological variables of the 50 SOC patients:-
The expression of SALL4 in serous ovarian carcinoma was significantly positively correlated with grade, stage, lymph node metastasis (p<0.001 for all), ECOG performance status (p= 0.01), local recurrence of the tumor (p=0.002) and distant metastasis (p=0.005; Table 1; Figures 1, 2).
As well as, the expression of Tau protein was significantly positively correlated with grade, stage, lymph node, distant metastasis, ECOG performance status (p<0.001 for all), and local recurrence of the tumor (p=0.002; Table 1; Figs 3,4).  Table 2). Combined High Tau protein and SALL4 immunoexpression were significantly positively correlated with lower response to treatment (p=0.02; Table 2).
Overall survival (OS) associations were identified in univariate analysis and presented in Table 4. Statistical significance was achieved in following factors: FIGO stage at diagnosis (p<0.001), age (p=< 0.001), tumor grade (p< 0.001) tau expression status (p< 0.001) and SALL4 expression level (p=0.008). The results are presented in tables 3&4 and figure 5.
In multivariate analysis, the Tau protein and SALL4 immunoexpressions were statistically independent parameter associated with OS (HR-3.4, 8.4, and P=0.001, 0.01, respectively).    Ovarian cancer is the leading cause of morbidity among malignant gynecologic diseases. The most frequent subtype among ovarian cancer is SOC [17]. It can directly broadly invade adjacent organs or peritoneal cavity via peritoneal fluid. Because of the diffuse nature of SOC, the objective of surgery is often cytoreductive, but not radical [18].
However, disease that initially responds well to this treatment frequently relapses, indicating that the existing therapeutic molecular targets and prognosis markers are not sensitive and efficacious enough. Therefore, it is necessary to make further research on new therapies and treatment targets of SOC. In this study, we investigated the prognostic role of SALL4 and Tau protein in SOC.
We found that SALL4 immunohistochemical expression was significantly positively correlated with grade, stage, lymph node metastasis (p<0.001), local recurrence of the tumor (p=0.002) and distant metastasis (p=0.005). Our follow up data showed that high expression of SALL4 expression was significantly positively correlated with response to treatment, performance status worse 3-year overall survival (OS) and local recurrence free survival rate (P =0.018, 0.01, 0.008 and <0.001 respectively).
Yang et al. [19], proved the same results in SOC that were near that of the previous studies on the function of SALL4 in solid tumors [20][21][22][23].
Our results can be explained by, Zeng et al. [24] that explained how SALL4 regulate stemness in embryonic and hematopoietic stem cells and activated in a hepatocellular carcinoma subtype with stem cell features, deletion of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity, also the function of SALL4 in promoting lymph node metastasis and advanced stage might be due to the interaction with b-catenin and subsequent aberrant activation of Wnt/b-catenin signaling pathway [15,25,26].
The aim of adjuvant chemotherapy is prolongation of OS rate that effect is possible to achieve if the cancer is chemo-sensitive. Thus, chemosensitivity is a good prognostic factor and drug resistance is a significant obstacle that affects the overall survival rate for advanced serous ovarian cancer patients.
We tried to find relation between the immunohistochemical expression of our markers and response to chemotherapeutic agents we found that SALL4 can participate in mediating cellular resistance to chemotherapeutic drugs and that low immunohistochemical expression of SALL4 in SOC is associated with better efficacy of chemotherapy (p= 0.626).
There are no many previous studies studied the relation between SALL4 and drug resistance in SOC, but our results are the same like that of Liu et al., [30] who proved that SALL4 expression is positively correlated with increased resistant to chemotherapeutic agents in endometrial carcinoma, and results of other studies that detected SALL4induced chemotherapeutic resistance in a variety of cancers [31][32] and our results can be explained by that the majority of drug resistance in human cancer are associated with ATP-binding cassette (ABC) multidrug transporters. SALL4 induces chemotherapeutic drug resistance through the regulation of ABCB1 in endometrial cancer cells. ABC transporters were thought to be closely related to drug resistance ABCB1 is the prototype of this gene superfamily, and its deregulation has been associated with drug resistance in several types of cancers [30].
C-Myc plays an important roles in many oncogenic processes, including tumor drug resistance and metastasis. SALL4 in endometrial cancer cells promoted c-Myc transcriptional activity [27].
In our study Tau protein-immunoexpression in SOC was significantly positively correlated with grade, stage, lymph node, distant metastasis (p<0.001) and local recurrence of the tumor (p=0.002) and we analyzed the role of its expression as a predictive factor for paclitaxel-containing chemotherapy. High Tau protein expression were significantly positively correlated wit-h response to treatment, performance status, worse 3-year OS rate (p<0.001) and resistance to the paclitaxel/ carboplatin first-line chemotherapy (p= 0.021).
Smoter et al. [14] found similar results that the negative status of Tau protein in SOC is associated with better response to chemotherapy.
Our results were explained by understanding paclitaxel's action which is competitive to Tau protein. Paclitaxel binds beta-tubulin on microtubule's inner surface, in the same point as Tau protein [33], and the presence of Tau protein on the microtubules 'surface creates difficulties in paclitaxel attachment to these structures. Low Tau expression may result in better paclitaxel connection with microtubules and more effective chemotherapy action, expressed in higher objective responses rate and better PFS [13].
Low Tau protein expression was associated with statistically significant more frequent achievement of complete response (CR) to paclitaxel in breast cancer and inhibition of Tau protein may enhance paclitaxel activity [10].
Tau protein -negative patients with breast cancer were sensitive to paclitaxel therapy, compared with Tau protein positive patients [34].
The predictive value of low Tau expression for paclitaxel therapy was confirmed in gastric cancer [35].
Paclitaxel has a more obvious function to cells with low expression of Tau protein; the apoptosis rate is also higher. Paclitaxel may be easily combined with tubulin under low concentrations of Tau protein. The high concentration has a stabilizing effect to microtubules, reducing the harmful effects of paclitaxel, which leads to drug resistance. However, resistance mechanisms to paclitaxel and their associated prognostic value needs further study [36].
Abd elaziz et al. [37] proved results that are similar to ours. Steffensen et al. [38], results were different from ours they reported that Tau protein was not associated with OS rate or progression free survival.
Our results differ from those obtained in the studies on breast cancer, where co-expression of Tau protein and estrogen receptor was considered as good prognostic factor [39].This divergence might be caused by Tau significance evaluation in different cancer sites.
Our study was the first to correlate the immunohistochemical expression of SALL4 and Tau protein immunohistochemical expression, prognosis and response to chemotherapy and we found a positive correlation between the expression of both markers, poor prognosis and resistant to chemotherapy (Spearman's r= +0.198; p=0.161).
Therefore the targeted therapy against these markers can decrease the resistance to the currently used chemotherapeutic agents and anti SALL4 could be a novel therapeutic drug that specifically target SOC cells decreasing its spread and improving its prognosis.

Conclusion:-
SALL4 and Tau protein may be useful prognostic and predictive markers in ovarian cancer patients receiving chemotherapy. More prospective studies with larger group of patients, may confirm their predictive and prognostic value.