Nuclear Expression Loss of SSBP2 Is Associated with Poor Prognostic Factors in Colorectal Adenocarcinoma

Single-stranded DNA binding protein 2 (SSBP2) is involved in DNA damage response and may induce growth arrest in cancer cells, having a potent tumor suppressor role. SSBP2 is ubiquitously expressed and the loss of its expression has been reported in various tumor types. However, the correlation between SSBP2 expression and colorectal cancer (CRC) prognosis remains unclear. SSBP2 nuclear expression was evaluated immunohistochemically in 48 normal colonic mucosae, 47 adenomas, 391 primary adenocarcinomas, and 131 metastatic carcinoma tissue samples. The clinicopathological factors, overall survival (OS), and recurrence-free survival were evaluated, and associations with the clinicopathological parameters were analyzed in 391 colorectal adenocarcinoma patients. A diffuse nuclear SSBP2 expression was detected in all normal colonic mucosa and adenoma samples. SSBP2 expression loss was observed in 131 (34.3%) primary adenocarcinoma and 100 (76.3%) metastatic carcinoma samples. SSBP2 expression was significantly associated with poor prognostic factors, such as vascular invasion (p = 0.005), high pT category (p = 0.045), and shorter OS (p = 0.038), using univariate survival analysis. Nuclear SSBP2 expression loss was significantly observed in colorectal carcinoma and metastatic carcinoma tissues, being associated with poor prognostic factors. SSBP2 acts as a tumor suppressor and may be used as a CRC prognostic biomarker.


Introduction
Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality worldwide, accounting for approximately about 700,000 deaths per year. Based on the GLOBOCAN series of the International Agency for Research on Cancer (IARC), CRC is the third most common cancer (10% of the total number) in men and the second most common (9.2% of the total number) in women [1].
CRC is a heterogeneous disease that exhibits variable underlying molecular changes, with genetic instability. Two major mechanisms of genetic instability include chromosomal instability (CIN, the most common type) and microsatellite instability (MSI) [2]. CIN includes changes in the chromosome number and structure, such as deletions, gains, translocations, and other chromosomal rearrangements [3]. MSI occurs due to a defective DNA mismatch repair. For example, Lynch syndrome or hereditary nonpolyposis colorectal cancer syndrome is caused by inherited mutations in one of the mismatch performed on patients with colorectal peritoneal metastasis (CPM) that underwent CRS (cytoreductive surgery)-HIPEC (hyperthermic intraperitoneal chemotherapy) (n = 62), using IHC. They described that patients exhibiting a lower expression of SSBP2 potentially had a poorer overall survival (OS) and a shorter disease-free survival (DFS), compared to those with a higher expression, although they were not statistically significant [27]. Third, Perilli et al. found that an increased level of miR-182-5p (miR-182), one of the most upregulated oncogenic microRNAs (miRNAs) in CRC, was associated with a significant decrease in SSBP2 mRNA levels in the tumor tissues, compared to matched normal mucosa [28].
In this study, we examined the expression of SSBP2, its prognostic significance, and its association with the clinicopathological features in CRC patients.

Patients
We retrospectively collected data from patients with colorectal adenocarcinoma who underwent curative surgery at the Hanyang University Hospital, Seoul, between January 2005 and December 2010. A total of 391 patients were included after excluding patients who received neoadjuvant chemotherapy and/or radiation therapy, the ones who died within 30 days of surgery, or had insufficient tissue material for analysis. Primary colorectal adenocarcinoma tissues (n = 391), matched normal colonic mucosa tissues (n = 48; randomly selected from 391 cases), and matched metastatic carcinoma tissues (n = 131; 92 lymph node metastasis and 39 distant organ metastasis) were obtained from the colorectal adenocarcinoma patients. In addition, 40 patients diagnosed with adenoma with low-grade dysplasia who underwent biopsy or polypectomy at the Hanyang University Hospital, Seoul, between January 2013 and December 2014, were randomly selected. Four patients diagnosed with adenoma exhibited multiple adenomatous polyps, and 47 adenoma tissue samples were obtained from them.
A total of 617 formalin-fixed, paraffin-embedded tissue samples were collected and 2.0-mm-core tissue microarray (TMA) blocks were constructed with one representative core for each case. The percentage of tumors in each cancer tissue core was greater than 70%. Clinical data, including age, sex, tumor location, tumor size, histologic grade, lymphovascular invasion, perineural invasion, tumor deposit, tumor budding, and TNM (tumor (T), nodes (N), and metastases (M)) staging were obtained from the medical records. Staging was determined according to the American Joint Committee on Cancer (8th edition) [29]. This study was approved by the Hanyang University Hospital (No. 2016-12-030-001), and the requirement for informed consent was waived.

Immunohistochemical Stainings and Interpretation
SSBP2 expression was evaluated using immunohistochemical staining of 4-µm-thick sections from TMA blocks. Rabbit monoclonal anti-SSBP2 antibody (1:100, ab177944, Abcam, Cambridge, UK) was used. The sections were first deparaffinized in xylene and then rehydrated through a graded ethanol series. For antigen retrieval, we heated the samples to 100 • C for 30 min in sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked using a peroxidase blocking solution (S2023, DakoCytomation, Carpinteria, CA, USA). TMA slides were incubated with primary antibodies at 4 • C overnight and then incubated with the labeled polymer (DAKO REAL EnVision/HRP, K5007, DakoCytomation, Glostrup, Denmark) for 30 min at room temperature. Then, 3,3-diaminobenzidine was used as a chromogen for visualization, and Mayer's hematoxylin counterstain was applied.
The expression of SSBP2 was evaluated according to the extent of tumor cell nuclear staining, using a light microscope, by two pathologists (YM and SS) who were blinded to the clinical data. The patients were subsequently subdivided into negative (proportion of positive tumor cells ≤ 10% of the total tumor cells) and positive (proportion of positive tumor cells > 10% of the total tumor cells) subgroups ( Figure 1). The evaluation of tumor positivity for a given marker is frequently performed using predetermined standard cutoffs, such as 10% [30][31][32][33][34][35]. The adoption of a categorical scoring system for interpretation simplifies the division of positive and negative groups by pathologists and is Diagnostics 2020, 10, 1097 4 of 11 further supported by practical observer reproducibility. However, it is assumed that no additional relevant data from the detailed analysis of protein expression of 10-100% will be provided in the production of the results.
Diagnostics 2020, 10, x FOR PEER REVIEW 4 of 11 system for interpretation simplifies the division of positive and negative groups by pathologists and is further supported by practical observer reproducibility. However, it is assumed that no additional relevant data from the detailed analysis of protein expression of 10-100% will be provided in the production of the results.

Statistical Analyses
Pearson's chi-square test or Fisher's exact test were used to evaluate any potential association between SSBP2 expression and the clinicopathological parameters, including age, sex, tumor location, tumor size, histologic grade, lymphatic invasion, vascular invasion, perineural invasion, tumor deposit, tumor budding, and AJCC (American Joint Committee on Cancer) staging. The differences in SSBP2 expression between groups were compared using the Mann-Whitney U test and Wilcoxon signed-rank test. The overall survival (OS) was defined as the duration between the date of curative resection and the date of death or the last follow-up. Recurrence-free survival (RFS) was defined as the duration between curative resection and the date of the first recurrence. Kaplan-Meier survival curves, the log-rank test, and the Cox proportional hazard regression model were used for survival analysis. Two-sided p-values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 24.0 (SPSS Inc., Chicago, IL, USA).

Patient Characteristics and SSBP2 Expression
The median age of patients with colorectal adenocarcinoma and adenoma was 64 years (range, 27-89 years) and 60 years (range, 30-82 years), respectively, and the male-to-female ratios were 1.56:1 and 1:1, respectively. The median follow-up period for the patients in this study was 108 months (range, 1-166 months). Among the 391 colorectal adenocarcinoma patients, 25 (6.4%) patients exhibited metastatic disease at the time of initial diagnosis, 81 (20.7%) patients had recurrence at the time of analysis, and 154 (39.4%) patients had died at the time of analysis.
Overall, a loss of nuclear SSBP2 expression was observed in 134 (34.3%) primary colorectal adenocarcinoma and 100 (76.3%) metastatic carcinoma tissues, while all normal colonic mucosa and adenoma tissues showed a positive SSBP2 expression. The mean values (%) of SSBP2 expression were 99.17, 94.47, 25.38, and 9.92, respectively. SSBP2 expression was significantly decreased in primary adenocarcinoma and metastatic carcinoma tissues (all, p < 0.001; Table 1, Figure 2A). There was no significant difference between the lymph node metastasis and distant organ metastasis groups (p =

Statistical Analyses
Pearson's chi-square test or Fisher's exact test were used to evaluate any potential association between SSBP2 expression and the clinicopathological parameters, including age, sex, tumor location, tumor size, histologic grade, lymphatic invasion, vascular invasion, perineural invasion, tumor deposit, tumor budding, and AJCC (American Joint Committee on Cancer) staging. The differences in SSBP2 expression between groups were compared using the Mann-Whitney U test and Wilcoxon signed-rank test. The overall survival (OS) was defined as the duration between the date of curative resection and the date of death or the last follow-up. Recurrence-free survival (RFS) was defined as the duration between curative resection and the date of the first recurrence. Kaplan-Meier survival curves, the log-rank test, and the Cox proportional hazard regression model were used for survival analysis. Two-sided p-values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 24.0 (SPSS Inc., Chicago, IL, USA).

Patient Characteristics and SSBP2 Expression
The median age of patients with colorectal adenocarcinoma and adenoma was 64 years (range, 27-89 years) and 60 years (range, 30-82 years), respectively, and the male-to-female ratios were 1.56:1 and 1:1, respectively. The median follow-up period for the patients in this study was 108 months (range, 1-166 months). Among the 391 colorectal adenocarcinoma patients, 25 (6.4%) patients exhibited metastatic disease at the time of initial diagnosis, 81 (20.7%) patients had recurrence at the time of analysis, and 154 (39.4%) patients had died at the time of analysis.
Overall, a loss of nuclear SSBP2 expression was observed in 134 (34.3%) primary colorectal adenocarcinoma and 100 (76.3%) metastatic carcinoma tissues, while all normal colonic mucosa and adenoma tissues showed a positive SSBP2 expression. The mean values (%) of SSBP2 expression were 99.17, 94.47, 25.38, and 9.92, respectively. SSBP2 expression was significantly decreased in primary adenocarcinoma and metastatic carcinoma tissues (all, p < 0.001; Table 1, Figure 2A). There was no significant difference between the lymph node metastasis and distant organ metastasis groups (p = 0.389; Figure 2B). Among the 39 distant organ metastasis cases, 30 affected the liver, six affected the lungs, and three affected the ovaries; SSBP2 expression was significantly lower in the liver than in other organs (mean value = 7.3% vs. 32.2%, p = 0.011). A paired-samples Wilcoxon signed-rank test was conducted to compare SSBP2 expression in distant organ metastatic tissues and matched primary CRC tissues. There was a significant difference in the expression proportion (mean value = 32.7% vs. 16.8%, p = 0.008). 0.389; Figure 2B). Among the 39 distant organ metastasis cases, 30 affected the liver, six affected the lungs, and three affected the ovaries; SSBP2 expression was significantly lower in the liver than in other organs (mean value = 7.3% vs. 32.2%, p = 0.011). A paired-samples Wilcoxon signed-rank test was conducted to compare SSBP2 expression in distant organ metastatic tissues and matched primary CRC tissues. There was a significant difference in the expression proportion (mean value = 32.7% vs. 16.8%, p = 0.008).

Correlation between SSBP2 Expression and Clinicopathological Features
To assess the correlation between SSBP2 expression and the clinicopathological parameters, SSBP2 expression was evaluated in 391 primary colorectal adenocarcinomas. The loss of SSBP2 expression was more frequently observed in tumors with vascular invasion (p = 0.005) and a high pT category (p = 0.045). There was no significant correlation between SSBP2 expression and other clinicopathological parameters (Table 2).

Correlation between SSBP2 Expression and Clinicopathological Features
To assess the correlation between SSBP2 expression and the clinicopathological parameters, SSBP2 expression was evaluated in 391 primary colorectal adenocarcinomas. The loss of SSBP2 expression was more frequently observed in tumors with vascular invasion (p = 0.005) and a high pT category (p = 0.045). There was no significant correlation between SSBP2 expression and other clinicopathological parameters (Table 2).

Prognostic Significance of SSBP2 Expression
The OS of CRC patients with a loss of SSBP2 expression was significantly shorter (p = 0.038, Figure 3A). However, SSBP2 expression did not affect the RFS (p = 0.368, Figure 3B). The univariate survival analysis for OS showed that SSBP2 expression, age, sex, pT category, nodal status, stage, histologic grade, lymphatic invasion, vascular invasion, perineural invasion, and tumor budding were significantly associated with OS (p < 0.05 for all cases) ( Table 3). Multivariate Cox regression analysis, including SSBP2 expression, age, sex, pT category, nodal status, histologic grade, lymphatic invasion, vascular invasion, perineural invasion, and tumor budding, revealed that age (p < 0.001), sex (p = 0.004), and vascular invasion (p < 0.001) were independent prognostic factors for a poor OS, while SSBP2 expression was not statistically significant (Table 3).
Diagnostics 2020, 10, x FOR PEER REVIEW 7 of 11 sex (p = 0.004), and vascular invasion (p < 0.001) were independent prognostic factors for a poor OS, while SSBP2 expression was not statistically significant (Table 3).

Discussion
In this study, we showed that SSBP2 expression is significantly associated with OS and poor prognostic factors, such as vascular invasion and a high pT category.
SSBP2 is downregulated in several malignancies, such as esophageal squamous cell carcinoma, prostate cancer, and acute myeloid leukemia, and in previous studies, it has been speculated that promoter methylation may be the main mechanism of loss of SSBP2 expression. Recent studies have revealed that SSBP2 is one of the genes that are downregulated by the methylation pathway [18,20,21,24,36]. In 2011, Huang et al. compared SSBP2 methylation in normal and tumor tissues in 20 pairs of esophageal squamous cell carcinoma and matched normal esophageal tissues using TaqMan-MSP analysis, and a higher degree of SSBP2 methylation in paired tumors than in paired normal tissues was observed in 15 of 20 esophageal squamous cell carcinoma patients [18]. Jun-Wei et al. reported that the SSBP2 promoter region was hypermethylated in 61.4% (54 of 88) of prostate cancer cases, whereas none of the 23 benign prostatic hyperplasia cases showed hypermethylation [21]. They further examined the SSBP2 expression pattern using immunohistochemistry and showed

Discussion
In this study, we showed that SSBP2 expression is significantly associated with OS and poor prognostic factors, such as vascular invasion and a high pT category.
SSBP2 is downregulated in several malignancies, such as esophageal squamous cell carcinoma, prostate cancer, and acute myeloid leukemia, and in previous studies, it has been speculated that promoter methylation may be the main mechanism of loss of SSBP2 expression. Recent studies have revealed that SSBP2 is one of the genes that are downregulated by the methylation pathway [18,20,21,24,36]. In 2011, Huang et al. compared SSBP2 methylation in normal and tumor tissues in 20 pairs of esophageal squamous cell carcinoma and matched normal esophageal tissues using TaqMan-MSP analysis, and a higher degree of SSBP2 methylation in paired tumors than in paired normal tissues was observed in 15 of 20 esophageal squamous cell carcinoma patients [18]. Jun-Wei et al. reported that the SSBP2 promoter region was hypermethylated in 61.4% (54 of 88) of prostate cancer cases, whereas none of the 23 benign prostatic hyperplasia cases showed hypermethylation [21]. They further examined the SSBP2 expression pattern using immunohistochemistry and showed that SSBP2 was significantly downregulated in most of the primary prostate cancer tissues, compared with normal prostate tissues. In addition, another study was conducted to identify a panel of epigenetic biomarkers that can distinguish cholecystitis from gallbladder cancer patients. This study revealed that promoter methylation statuses of SSBP2 (p = 0.01) were significantly different in patients with gallbladder cancer when compared to those of patients with cholecystitis [20]. Furthermore, in ovarian cancer, SSBP2 methylation was found in 9% of tumor cases, whereas no cases showed methylation of the SSBP2 promoter in normal tissues [37].
The prognostic impact of SSBP2 expression in CRC has been mentioned in only a few studies. Shannon et al. reported that patients with a lower expression of SSBP2 were potentially correlated with a poorer OS and a shorter DFS, compared to those with a higher expression, using IHC. However, these results were not statistically significant. According to the article, the authors determined the staining results semi-quantitatively based on the staining intensity and percentage of stained tumor cells. Although patients with a lower expression of SSBP2 potentially have a poorer OS and DFS, they did not reach statistical significance (median OS, 29.8 months vs. 42.3 months for a low and high expression, respectively, HR 1.886, 95% CI 0.812-4.378, p = 0.140; median DFS, 14 months vs. 30 months for a low and high expression, respectively; HR 1.913; 95% CI, 0.825-4.437; p = 0.131) [27]. Perilli et al. studied the changes in expression of transcription factors, such as HIST1H2BH, NABP1 (also known as SSBP2), RND3, and TRIO genes after miR-182 silencing of oncogenic miR-182. As a result, all of them showed a significant upregulation, as confirmed using transcript-specific qRT-PCR assays. In particular, the SSBP2 gene showed a remarkably high expression in the anti-miR-182-treated tumorigenic cell line. Interestingly, they also reported that a significant decrease in the SSBP2 mRNA levels was identified in primary CRC samples compared to matched normal colon mucosa samples [28].
In this study, we found that the SSBP2 expression pattern is correlated with the OS and several poor clinicopathological factors of the patient. An SSBP2 expression loss was found in 34.3% of primary adenocarcinoma and 76.3% of metastatic adenocarcinoma tissues; however, no expression loss was found in matched normal colonic mucosa and adenoma cases. These results suggested that the loss of SSBP2 expression is possibly associated with aggressive clinical behavior of CRC.
In addition, it is meaningful to combine the results of the previous study [28]. Although Perilli et al. conducted a study with many other transcription factors besides SSBP2, only in the case of SSBP2 it was reported that the mRNA level was markedly reduced in colorectal cancer compared to normal tissues. This result is consistent with the present study, which performed immunohistochemical staining using SSBP2 antibody. This can be positively evaluated in that it suggests the possibility of performing a prognosis-related test for colorectal cancer by a simple method such as immunohistochemistry.
Further studies on SSBP2 promoter methylation and its association with SSBP2 expression in a large cohort of CRC patients using fresh tissues are necessary to understand the mechanism underlying SSBP2-related carcinogenesis. The findings of our study lay the foundation for designing future studies. If the same results are gathered in more groups in the future and target therapy studies for SSBP2 are progressed, it will be a cornerstone for the opening of a new treatment for colorectal cancer, which remains a challenge.

Conclusions
In conclusion, we showed that SSBP2 expression was significantly decreased in colorectal adenocarcinoma and metastatic carcinoma tissues and was associated with poor prognostic factors. SSBP2 acts as a tumor suppressor and may be used as a prognostic biomarker in colorectal cancer.

Conflicts of Interest:
The authors declare that they have no competing interests.
Availability of Data and Materials: All data generated or analyzed during this study are included in this published article.: