Study on the small breast epithelial mucin tumor tissue expression difference and its relationship with survival in triple-negative breast cancer patients in Han, Miao and Buyi ethnic

Objective: To investigate Small breast epithelial mucin(SBEM) expression difference in mammary tissues and relationship with survival in thetriple-negative breast cancer(TNBC) patients in Han, Buyi and Miao ethnic. Methods: In our study, SBEM protein expressions were detected by means of immunohistochemistry in 297 patients diagnosed from 2014 to 2015,including 99 normal breast tissue specimens, 99 cases of breast benign tumor tissue specimens and 99 tissues specimens from TNBC patients.Each set of tissue specimens contains 33 samples from Han,Miao and Buyi ethnics, respectively.We analyze the expression of SBEM in different mammary tissues in different ethnics and the association of different SBEM expression levels in tissue of TNBC patients with clinical- pathological features, DFS and OS in Han,Miao and Buyi ethnics,respectively. Results: SBEM expression in breast cancer tumor cells were related to the ki67 in the Han, Miao and Buyi ethnic (P=0.034 (cid:0) 0.027 (cid:0) 0.047), respectively. There was a marked associations between the SBEM expression level and lymphatic metastasis (P = 0.042 (cid:0) 0.039) in the Han and Miao ethnic, while the same results were not found in the Buyi people (P=0.072).There was signicant difference in DFS ( P =0.028 (cid:0) 0.013)and OS ( P =0.09 (cid:0) 0.037) between the high expression group and low expression group in the Han and Miao ethnic.But there was no signicant difference in DFS (P = 0.053 ) and OS (P = 0.088 )in the Buyi people. Conclusion: The SBEM positive detection rate was no signicant difference in the han, miao and buyi ethnic groups. SBEM was associated with DFS and OS in the Han and Miao ethnic, while no correlation in the Buyi ethnic.

Study on the small breast epithelial mucin tumor tissue expression difference and its relationship with survival in triple-negative breast cancer patients in Han, Miao

Introduction
Breast cancer is the most common malignancy and is second only to lung cancer in mortality among women worldwide [1,2] .While the incidence rate of breast cancer continues to rise over the last few decades, its mortality rate,on the other other hand, declines every year thanks for the development of diagnosis and targeting medication [3,4] .Triple-negative breast cancer (TNBC), one of ve molecular subtypes recognized in 2000 [5,6] , lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression.TNBC is characterized by high incidence in young women, early recurrence and shows a relative sensitivity of chemotherapy. Most studies [7][8][9] showed that the prognosis of TNBC was less favorable than that of non-TNBC.
Micro metastasis is considered to be a key factor for poor prognosis of breast cancer patients [10] .Despite the improvement in detection of breast cancer,about 30% of patients are still detected with micro metastasis at their rst visit.The micro metastasis of breast cancer before primary treatment is considered to be one of the recurrence reasons that will directly impact the survival of patients.The development of distant metastasis is the major cause of their death [11] .So,it is crucial to nd speci c markers to detected micro metastasis and provide useful information to guide early therapeutic methods of breast cancer patients,especially TNBC.
Small breast epithelial mucin (SBEM) was a tissue speci c protein and only expresses in mammary and salivary glands [12] .High SBEM expression was found to be strongly associated with the histopathological detection of lymph node metastasis [12] . SBEM could serve as a useful marker for breast micro metastasis,also as a speci c targets for the treatment of breast cancer [12] . O 'brien [13] con rmed that rates of detection of SBEM in protein levels were 51% (52/103) and 4% (1/26) in breast cancer and non-breast cancer tissue, respectively, which further con rmed the SBEM expression was signi cantly higher in breast cancer tissue than non-breast tissue.Skliris GP [14] study showed that SBEM expression rate was 18% in 300 patients with breast cancer using the method of immunohistochemical analysis and the SBEM expression rate was signi cantly higher in ER negative(22%) than in ER positive (13%) breast cancer tissue.Several laboratories showed that SBEM expression correlated with higher tumor grade,TNM staging and lymph node metastasis at both mRNA and protein levels.
SBEM has signi cant guidance for clinical activities. Liu ZZ [15] reported that SBEM has the potential to be a speci c marker for predicting hematogenous micro metastasis and response to neo-adjuvant chemotherapy in breast cancer.Valladares-Ayerbes M et al. [16] studied the expression pro les of SBEM gene in silico and in vitro, and demonstrated that SBEM-mRNA could serve as a marker for bone marrow micro metastasis in breast cancer patients and SBEM-mRNA could serve as a marker for bone marrow micro metastasis in breast cancer patients [15] .Research of Liang Liu et al [17] suggested SBEM 3 + score was cut-off value of prognosis and signi cantly correlated with decreased DFS and OS in TNBC patients.SBEM is an independent risk predictor and may offer utility as a prognostic marker in TNBC patients [17] .
Based on the resules above,SBEM might play an important role in progression and metastasis of breast cancer indifferent races, especially in TNBC. But humans have genetic diversity and every race and nationality's genome has its own characteristics. Only to gure out what each nation's genetic structure characteristics and the change rule, we can analyze their origins and the genetic relationship with other ethnic groups.This kind of population genetics research is a di cult project.The genetics investigation need to rely on all sorts of genetic markers, especially those with racial speci city and individual speci c genetic markers,which can provide enough valuable genetic information. or if the score was 2 + but non-ampli cation by uorescence in situ hybridization (FISH), and positive if the score was 3+. Based on the past research results, SBEM expression with SBEM 3 + score is the SBEM cut-off value of prognosis. We divided the cases into two groups, one is the SBEM < 3 + group de ned as a high expression group,the other is SBEM = 3 + group de ned as a high expression group. or if the score was 2 + but non-ampli cation by uorescence in situ hybridization (FISH), and positive if the score was 3+. Based on the past research results, SBEM expression with SBEM 3 + score is the SBEM cut-off value of prognosis. We divided the cases into two groups, one is the SBEM < 3 + group de ned as a high expression group,the other is SBEM = 3 + group de ned as a high expression group.

Sbem Expression And Evaluation Of Ihc
All tissues were collected surgically under the supervision of an experienced pathologist. SBEM expression was measured by IHC. Streptavidinperosidase (S-P) IHC staining was performed using SBEM antibody of mouse monoclonal (diluted 1/800, Abcam plc. Cambridge, UK). The detailed procedures were done as described by Jennbacken [18] . PBS was used to replace the primary antibody in negative controls. SBEM was a secreted protein and it was mainly located in cell membrane, secondly in cytoplasm. Normal breast tissues were in general weakly or negative. So it was evaluated and scored if cell membrane and/or cytoplasm reactivity were observed [8] . According to our data and TMA IHC grading method by Serrero G [19] Pan [20] and Liu Liang [17] , our scoring was semiquantitatively categorized as: ≤5% of tumor cells staining with/without weakly stained was negative (0), followed by a score of 1 (> 5% of tumor cells and with weak/focal positive staining or ≤ 5% of tumor cells with strongly stained), 2 (> 5% of tumor cells and with moderate/focal positive staining), 3 (> 5% of tumor cells and with strong/diffuse positive staining).

Statistical analysis
The correlation between SBEM, Clinicopathological characteristics and survival outcomes was compared by Pearson's Χ 2 test. Survival analyses, including DFS and OS, were performed with the log rank test and all results were displayed in Kaplan-Meier. DFS was de ned as the time interval from date of diagnosis to the time of last disease free follow-up or at death for those patients who died without a previous recurrence [21] . OS was de ned as the time interval from date of diagnosis to time of last follow-up or death [21] . Statistical signi cance was de ned as P value < 0.05. SPSS17.0 software package was used for all statistical analyses.   The clinicopathological characteristics of patients were described in Table 2

Discussion
Breast cancer is pushed into rst place in the United States and many other parts of world.Breast cancer alone is expected to account for 29% (226,870) of all new cancer cases among women [22] .Although incidence rate of breast cancer remains relatively stable in recent 5 years,its death rate declines by 34% because of the development of diagnosis and targeting medication [4,10] .The micro metastasis of breast cancer before primary treatment is considered to be one of the recurrence reasons that will directly impact the survival of patients.So,it is crucial to nd speci c markers to detected micro metastasis and provide useful information to guide early therapeutic methods of TNBC patients.
The de nition of tumor markers is broad.Tumor cells may express certain molecules at a different rate from that of normal cells,and these substances are released in the bloodstream or in other biologic uids.Although various biological markers had been proposed for the detection of breast cancer cells,they were often affected by tumor differentiation,lower speci city and detection rate. But almost all the current clinical application of tumor markers can not reach ideal level in the identi cation of the tumor speci city and sensitivity, especially in TNBC at present.
SBEM was a tissue speci c protein, a member of the MUC family, and its expression is highly speci c to mammary gland tissue.High SBEM expression was found to be strongly associated with the histopathological detection of lymph node metastasis [12] .SBEM was a secreted protein and it was mainly located in cell membrane.Normal breast tissues were in general weakly or negative.SBEM could serve as a useful marker for breast micro metastasis,also as a speci c targets for the treatment of breast cancer [12] .
However,as the genomes of different races and nationalities have their own characteristics,it is of guiding signi cance to explore the genetic Researchers have conducted many studies on SBEM as a tumor marker of breast cancer micro metastasis worldwide.RT-PCR and immunohistochemistry are used to detect the expression of gene and protein nowadays [15,23] .The research of O'Brien et al [13] has shown that SBEM are expressed relatively exclusively in breast tissue than non-breast tissue and are potential new markers for breast cancer [14] .Skliris GP [14] study showed that the SBEM expression rate was signi cantly higher in ER negative(22%) than in ER positive (13%) breast cancer tissue.Zhong Lei et al [24] study showed that SBEM expression rate was 53.3% in 60 patients with breast cancer the negative SBEM-mRNA resules were found in peripheral blood in 20 patients with mammary gland broma and 10 cases of healthy people. SBEM expression in TNBC tumor cells were related to TNM staging and axillary lymph node metastasis.
The similar results were found by Yang Hua-wei et al [25] .This research shows that SBEM is mainly expressed in the cell membrane, the second expression in the cytoplasm, few expression in the nucleus. SBEM positive detection rate of breast cancer tissue is signi cantly higher than the breast benign tumor tissue and normal breast tissue. But there was no signi cant difference in the han, miao, buyi ethnic in breast cancer tissue, breast benign tumor tissue and normal breast tissue. and P53, which can evaluate DFS and OS [26][27] . Ki67 is also a important marker of cell proliferation, and it has important signi cance for prognosis judgment in breast cance [28] . In our study, SBEM expression in TNBC tumor cells were related to the ki67 in the Han, Miao and Buyi ethnic (P = 0.034 0.027 0.047), respectively. There was a marked associations between the low expression of SBEM group and high expression group in lymphatic metastasis (P = 0.042 0.039) in the Han and Miao ethnic, while the same results were not found in the Buyi people (P = 0.072)).Thus it can be seen that there is difference in different ethnic groups between SBEM different expression levels and metastatic tumor recurrence and the prognosis.
The past studies have shown that SBEM correlated with the prognosis of patients.
Breast cancer stem cell scan obtain higher resistance and high invasive due to their high plasticity [29] . The previous studies have not been reported whether there is the same prediction results or the clinical meaning for the same tumor marker in different ethnics.The experiment has carried on the preliminary exploration to this. In our study, we found that DFS and OS function curves showed the large separation between the high expression group and low expression group. There was signi cant difference in DFS ( P = 0.028 0.013)and OS ( P = 0.09 0.037)between the high expression group and low expression group in the Han and Miao ethnic.But there was no signi cant difference in DFS (P = 0.053 ) and OS (P = 0.088 )in the Buyi people.Thus it can be seen there is different prognostic signi cance in different ethnic groups for SBEM different expression levels.
In conclusion, this research shows that SBEM positive detection rate was no signi cant difference in the han, miao and buyi ethnic in breast cancer tissue, breast benign tumor tissue and normal breast tissue.The SBEM expression level is related to the prognosis of TNBC patients and its clinical signi cance is not exactly the same in different ethnics ,which can provide a more reasonable choice for clinical individualized treatment and prognostic judgement and reduce a certain amount of medical costs. However, it needs more centers to participate in further study because this is just a retrospective single-center study and the small sample size limits to some extent the generalization of the ndings made in the study.

Conclusion
We have demonstrated that the SBEM positive detection rate was no signi cant difference in the han, miao and buyi ethnic groups in breast cancer tissue, breast benign tumor tissue and normal breast tissue.The expression of SBEM does signi cantly correlate with a DFS and an OS of TNBC patients in the han, miao and buyi ethnic. But it is not obvious for prognostic signi cance in Buyi ethnic.The distinction in different ethnic groups shall certainly provide a more reasonable choice for clinical individualized treatment and prognostic judgement in TNBC patients. Kaplan-Meier estimates for OS by SBEM scores in Han, Miao and Buyi ethnic