Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia

Background: Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Methods: Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Results: Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC ( p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64. Conclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC.

We revised the introduction focusing on TNBC epidemiology. We emphasised the introduction related to TNBC classification, characteristics and behaviour. We also updated the role of p63 in cancer within the introduction.
In the conclusion, we included more information related to the frequency of Basal and Non-basal type of TNBC and this finding related to the expression of SOX2 and P63 both in Basal and Nonbasal type of TNBC. We also included the acknowledgement section in the current version. We also revised the author list; Harapan Harapan, MD was requested to be mentioned in the acknowledgement section rather than as co-author due to current changing of his research field of interest.

Introduction
Breast cancer, accounting for 25% of all cancer cases and 15% of all cancer deaths among females, is the most frequently diagnosed cancer among female worldwide 1,2 . The incidence of breast cancer increased significantly, approximately by 30% in developed countries 3 and currently it has been also rising in many developing countries 2 . In Asia, 639,824 breast cancer cases and 228,926 deaths were recorded in 2012, from which 48,998 cases and 19,750 deaths occurred in Indonesia 4 . Triple-negative breast cancer (TNBC), a group of breast cancers with the absence of oestrogen receptor and progesterone receptor and no overexpression of human epidermal growth factor receptor 2 (HER2), represents 10%-20% of invasive breast cancer. A global data base, National Cancer Data Base (NCDB), reveals that TNBC was present in 13% of breast cancer patients, ranged from 23.7% in African-Americans to 8.9% in Filipino patients 5 . In Southeast Asia, a study found that TNBC presented in 10.5% among 1227 breast cancer patients 6 . In Indonesia, a study that was conducted between 2010 and 2011 in Bandung found that 11.9% of breast cancer patients were TNBC 7 .
Immunohistochemically, TNBC could be divided into two subtypes: Basal-like (positive for the expression of highmolecular-weight/basal cytokeratins 5/6 (CK5/6) and epidermal growth factor receptor (EGFR)) and Non basal-like (negative for the expression of CK5/6 and EGFR). Basal-like TNBC usually has p53 mutation, EGFR overexpression, loss of function of BRCA1, c-MYC amplification, and high histological grade indicating more aggressive characteristics and aggressive behavior 8,9 . In addition, majority of Basal-like cancer cannot be managed effectively with trastuzumab and hormonal treatments 10 .
Advanced screening and diagnosis methods for breast cancer such as mammograms, ultrasound, magnetic resonance imaging and fine-needle aspiration, have allowed for detection of small lesions at the early stage. Identifying breast cancer at the early stage will increase the potential for curative treatment and therefore increases the survival rate 11-14 . However, smaller lesions are more challenging to diagnose. Therefore, it is essential to use an advanced immunohistochemical approach for evaluation of smaller tissue specimens that target more specific markers 15 .
A previous study using a MCF7 breast cancer cell line to produce MCF7-derived tumour xenografts found that P63 and SOX2 immunostainings were two potential markers for breast cancer 16 . P63, involved in cellular differentiation, is a homolog of tumour protein P53 and in normal breast ducts and lobules it is expressed frequently in the nuclei of myoepithelial cells 17 . Mutation of the p53 gene results in a very high risk of breast cancer 18 . The roles of p63 in tumorigenesis, cancer progression, and metastasis are still being discovered. However, in animal model found that p63 deficiency may be a causative factor for metastatic spread 19,20 . In addition, clinical evidence suggests that a robust correlation between reduced p63 expression and cancer progression 21 .
A study revealed that the total percentage of P63-positive cells was related to marked nuclear pleomorphism and the intensity of P63 staining was associated with syncytial growth pattern in TNBC 22 . In addition, data also reveals that p63 gene expression in breast cancer could be used as a specific marker of metaplastic carcinoma 17 , and P63 immunohistochemical staining could improve diagnostic accuracy of breast cancer even in small tissue specimens 23 .
SOX2 is a transcription factor belonging to the SOX family and functions as an activator or suppressor of gene transcription 24,25 . Data shows that SOX2 promotes cellular proliferation of breast tissue 26 and regulates self-renewal in cancer stem cells 27 . The scientific evidence reveals that SOX2 acts as an oncogene in epithelial cancers 25 and in the breast, a study found that silencing of sox2 gene was associated with reduction of the size of the cancer stem cells and restoration of tamoxifen sensitivity 28 . All together, these data indicate that P63 and SOX2 have pivotal role in breast cancer and therefore are potential to be used as specific biomarkers. This study was conducted to assess the immunoexpression of P63 and SOX2 in TNBC cases in order to provide insight regarding their potential diagnostic value (single or in combination) to differentiate TNBC types.

Study setting and histological slides
A cross-sectional study to assess the immunoexpression of P63 and SOX2 in TNBC cases (negative expression of estrogen and progesterone receptors and c-erbB2) was conducted.

Immunohistochemistry
Forty archival paraffin blocks from TNBC cases were subjected to immunohistochemical staining to assess the immunoexpression of CK 5/6, P63 and SOX2. Briefly, 4 μm sections of each paraffin block were prepared using standard procedure 29 . Immunohistochemical staining was conducted using primary antibodies as follows: anti-CK5/6 monoclonal antibody (Biocare Medical, Concord, CA, USA), anti-P63 monoclonal antibody (Biocare Medical, Concord, CA, USA) and anti-SOX2 monoclonal antibody (Abcam, Cambridge, UK). Starr Trek Universal HRP Detection (Biocare Medical, Concord, CA, USA) was used as second antibody. A chromogen 3,3'-diaminobenzidine (DAB) (Biocare Medical, Concord, CA, USA) was used to develop the colour. For each experiment, appropriate controls were used.
Immunoreactivity score was calculated by multiplying staining intensity and the percentage of positivity, and the score therefore ranged from 1 to 16. The immunoreactive score was then divided into low (≤ 5), moderate (≥ 6 -10) and high (≥11 -16). Immunoexpression of P63 was measured both in cytoplasm and nucleus while SOX2 immunoexpression was measured in nucleus only.

Statistical analysis
Normality of the data was assessed using the Shapiro-Wilk test and therefore the analysis tests chosen based on the normality of the data. The correlations between immunoexpression of P63 (cytoplasm and nucleus) and SOX2 were assessed using Pearson correlation and Spearman correlation, respectively. The associations of P63 and SOX2 immunoexpression and type of TNBC were assessed using Mann Whitney test. The predictive diagnostic values of P63 cytoplasm for diagnosing Basal-like type TNBC were estimated using several immunoreactivity score cut-off points. Receiver operating characteristic curve (ROC) was plotted and area under the ROC curves (AUC) was estimated. For all analyses, estimates were considered statistically significant for two-tailed values of p<0.05. All analyses were conducted using Statistical Package for the Social Sciences software (SPSS for Windows, Version 16, Chicago, IL).

Clinicopathology and classification of TNBC
The histopathology of the TNBC samples used in this study is described in Table 1. Approximately 45% of the samples were classified as metaplastic carcinomas. In addition, immunohistochemical staining for CK 5/6 revealed that 23 (57.5%) of samples were Basal-like type TNBC and while 17 (42.5%) samples were Non basal-like type TNBC.
Immunoreactivity score of P63 and SOX2 Immunoexpression of P63 and SOX2 in samples, categorized by immunoreactivity score, are presented in Table 2. For both types of TNBC (basal and non basal-like type), all immunoreactivity scores for P63 in the nucleus were classified as low grade, while 11 (27.5%) and 7 (17.5%) samples were classified as moderate and high grade, respectively for the P63 in the cytoplasm. The immunoreactivity grade for SOX2 was similar to P63 in the cytoplasm, and therefore correlation analyses were conducted.

Correlation between immunoexpression of P63 and SOX2
There was a strong negative correlation between immunoexpression of P63 in the cytoplasm and immunoexpression of SOX2 in the nucleus in metaplastic carcinoma (a sub-type of TNBC basal-like type) (r=-0.73, p=0.013) ( Table 3). In addition, linear regression showed a relatively strong correlation between P63 cytoplasm and SOX2 immunoexpression in metaplastic carcinoma (r=0.49, p=0.012). There was no significant correlation between P63 cytoplasm and SOX2 immunoexpression in Non basal-like type TNBC, and no significant correlation between P63 nucleus and SOX2 immunoexpression either in Basal-like type or Non basal-like type of TNBC.

SOX2, n (%)
Low (   Predictor diagnostic value of P63 in the cytoplasm for diagnosing Basal like type TNBC As mentioned above, immunoexpression of P63 in the cytoplasm was the only marker that was significantly different between TNBC types. Therefore, immunoreactivity score of P63 cytoplasm was further analysed to determine its ability to predict Basallike type TNBC. Table 5 shows the predictive values of P63 in the cytoplasm for determining Basal-like type TNBC, using seven immunoreactivity score cut-off values from 3 to 9. It shows that P63 has relatively weak diagnostic value in diagnosing Basal-like type TNBC. The highest sensitivity was achieved at immunoreactivity score 3, while specificity was increasing with a higher immunoreactivity score.
Using the average score of P63 cytoplasm immunoexpression for Basal-like type TNBC in this study, 5.6 or 6, the sensitivity and specificity of P63 cytoplasm immunoreactivity score to predict Basal-like type TNBC was 56.5% and 72.6%, respectively with area under curve 0.64. The receiver operating curve of predictive diagnostic value of P63 cytoplasm for determining Basal-like type TNBC is plotted in Figure 1.

Discussion
To the best of our knowledge, this is the first study conducted to assess the immunoexpression of P63 and SOX2 in TNBC cases in Indonesia. Some studies have been conducted to assess the predictive values of P63 as specific marker for breast cancer 17,30 . In addition, the idea of utilization of a cocktail of specific markers has been proposed previously to provide higher sensitivity and specificity for diagnosing specific breast cancers 15,22,31 . However, none of the previous studies had been conducted to assess the diagnostic value of immunoexpression of P63 and SOX2 in combination. This study, at the beginning, sought to assess predictive value of combination both of those markers for specific type of TNBC. However, we found that there was no difference in immunoexpression of SOX2 between Basal-like type TNBC and Non basal-like type TNBC. Nevertheless, we found that immunoexpression of P63 cytoplasm, but not P63 nucleus, was higher in Basal-like type TNBC compared to Non basal-like type TNBC.
P63 has been proposed as a breast cancer marker for a long time, but with conflicting results. A study demonstrated that immunoexpression of P63 was associated with breast cancers, for example References the metaplastic carcinoma type of breast cancer 17 , but there was no difference in immunoexpression of P63 between medullary breast carcinomas and atypical medullary breast carcinomas of TNBC 30 . In our study, we found that the sensitivity and specificity of P63 cytoplasm immunoexpression to diagnose Basal-like type TNBC was 56.5% and 72.6%, respectively, with area under curve of 0.64. This sensitivity and specificity seems higher compared to a previous study, with 14% and 94%, respectively in determining a Basal-like type in infiltrative ductal carcinomas (TNBC) 22 . All together, these data indicate a weak predictive value of P63 immunoexpression as marker for Basal-like type TNBC. However, a study found that P63 is a specific marker for metaplastic carcinomas of the breast (a sub-type of Basal-like type TNBC) 17 . In our study, we could not assess the predictive value of P63 cytoplasm immunoexpression for determining metaplastic carcinomas, due to the small sample size (see Table 3).
We found that SOX2 immunoexpression grade was classified as moderate and high grade in 55% of TNBC cases (Table 2,) and it has been indicated previously that SOX2 has strong roles in promoting breast cancers [26][27][28]32 . However, there was no different in immunoexpression between Basal-like type TNBC and Non basal-like type TNBC. This result indicates that SOX2 expression is not different amongst TNBC types. This finding was in line with a previous study that indicated that SOX2 was expressed across different breast cancer subtypes 33 . A study found that SOX2 antibody in the sera was is higher in patients with breast cancer compared to healthy women and therefore it could be used to discriminate between breast cancer patients and healthy controls 34 . In addition, a meta-analysis found that SOX2 expression was associated with tumor size, histological grade, the aggressiveness and lymph node metastasis in TNBC patients 35 . All together, these results indicate that there was a possibility SOX2 expression could be used for diagnosing breast cancers, but there was no difference in expression amongst breast cancer types, and therefore it could not be used as specific marker for differentiating TNBC types.
There are some limitations to this study. The sample size was relatively small, and therefore some analyses could not be conducted. In addition, the diagnostic specimens were collected from different procedures such as from biopsy, mastectomy or lumpectomy, and this might influence the immunoexpression of the markers.

Conclusions
Immunoexpression of P63 cytoplasm is higher among Basal-like type TNBC compared to Non basal-like type TNBC. However, the predictive diagnostic value of P63 immunoexpression in the cytoplasm for Basal-like type TNBC is relatively low, with 56.5% sensitivity and 72.6% specificity.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.
I. -In introduction there is only little information regarding of nuclear expression of p63.
-It doesn't mention about cytoplasmic p63 expression. What is significance function of p63 in tumor progression or disease progression? It written in result and analysis. -In introduction you haven't discussed yet about the incidence of triple negative breast cancer in the worldwide, Asia or Indonesia ?
II. In the methods, to classify TNBC morphology into Basal like and non Basal like, based on only, antibody (CK 5/6) but you didn't perform EGFR staining. Which is one of the marker of TNBC.
-Is there any reference about the scoring of intensity and the percentage of positivity of expression of p63?
-Is there any reference about imunoreactive score?
III. In discussion, there is also no information about cytoplasmic p63 and its role in disease progression.
-Researcher only stated that cytoplasmic p63 expression has predictive value to classify breast cancer into TNBC and there is no comparison of p63 expression sensitivity and specificity with other TNBC marker as like CK 5/6 and EGFR which has been routinely used.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Referee Expertise: Pathologist with major interest in colorectal cancer I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Harapan Harapan
Thank you for comments and suggestion. We have deleted some sentences related to the general Thank you for comments and suggestion. We have deleted some sentences related to the general introduction of breast cancer and added specific information related to TNBC. We would like to confirm that predictive diagnostic analysis in this study was conducted using qualified statistician. There is no evidence to support the importance of the negative correlation between immunoexpression of P63 cytoplasm and immunoexpression SOX2 nucleus in metaplastic carcinoma, and therefore we are unable to discuss this finding in depth. In conclusion section, we have added some additional principal finding as suggested by the reviewer.
There is no competing interest in this study.

Harapan Harapan
Thank you for comments and suggestion. We have deleted some sentences related to the general introduction of breast cancer and added specific information related to TNBC. In the revised manuscript, the data of the incidence from global, Southeast Asian countries and Indonesia are included.
In our study, we used the histological paraffin blocks that have been confirmed as TNBC previously by testing ER, PR and HER2. To differentiate Basal like and non Basal like, either CK 5/6 or EGFR staining could be used with no significant different sensitivity and specificity (Livasy , 2006;et al. Nielsen , 2004). In this study, CK 5/6 was employed to differentiate TNB into Basal like and et al. non Basal like and this procedure was conducted during the study. This method is established method to define basal phenotype (Sasa , 2008;Rakha , 2007). et al.
et al.
In our study, we used the histological paraffin blocks that have been confirmed as TNBC previously by testing ER, PR and HER2. To differentiate Basal like and non Basal like, either CK 5/6 or EGFR staining could be used with no significant different sensitivity and specificity (Livasy , 2006;et al. Nielsen , 2004). In this study, CK 5/6 was employed to differentiate TNB into Basal like and et al. non Basal like and this procedure was conducted during the study. This method is established method to define basal phenotype (Sasa , 2008;Rakha , 2007). CK 5/6 and EGFR. et al. et al.
As mentioned Immunohistochemistry Section, the principle of the scoring system for immunoexpression of P63 and SOX2 used in this study was have been elsewhere with modification (Thike , 2010). In detailed: Staining intensity was scored as follows: 1 (no et al. staining), 2 (weak staining), 3 (moderate staining) and 4 (strong staining). The percentage of positively stained tumour cells was assessed as a proportion of the total number of tumour cells present in the section as follows: 1 (<20%), 2 (≥20-50%), 3 (>50-80%) and 4 (>80%). Then from staining intensity and percentage of positively stained cells, we created immunoreactivity score by multiplying staining intensity and the percentage of positivity. The score, therefore, ranged from 1 to 16 (divided into low (≤ 5), moderate (≥ 6 -10) and high (≥11 -16). However, specifications for staining intensity and the percentage of positivity used in this study are the standard system used by Pathology Anatomy Laboratory of Dr. Hasan Sadikin Hospital Bandung since 1990. These score systems are also adopted in some breast cancer diagnostic centres in Indonesia.
Immunoreactive score system used in this study have been published elsewhere (Thike , et al. 2010). We mentioned this in Immunohistochemistry Section of our Methods. In this study produce all raw data related to: type of TNBC (Basal like and Non Basal-like), subtype of cancer, expression of SOX2, P63 and CK 5/6 including staining intensity, distribution of the positively cells and immunoreactive score. of cancer, expression of SOX2, P63 and CK 5/6 including staining intensity, distribution of the positively cells and immunoreactive score.
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