The association of clinicopathologic factors with the expressions of FAM83H and ZNF16 in gallbladder carcinomas
To verify the specificity of the antibodies for FAM83H and ZNF16, western blots for FAM83H and ZNF16 were performed on SNU-308 gallbladder carcinoma cells after inducing knock-down or overexpression of FAM83H. Knock-down of FAM83H decreased expression of FAM83H and ZNF16, and overexpression of FAM83H increased expression of FAM83H and ZNF16 (Fig 1a). Immunohistochemical expression of FAM83H and ZNF16 in gallbladder carcinomas are presented in Figure 1b. The expression of FAM83H and ZNF16 were detected in both cytoplasmic and nuclear areas of adenocarcinoma components (Fig 1b). In squamous cell carcinoma components, the expression of FAM83H and ZNF16 were detected in the cytoplasmic membrane, cytoplasm, and nuclei of tumor cells (Fig 1b). Immunohistochemical expression of FAM83H and ZNF16 were grouped into negative or positive groups by receiver operating characteristic curve analysis. The cut-off points for the expression of nuclear FAM83H, cytoplasmic FAM83H, nuclear ZNF16, and cytoplasmic ZNF16 were eleven, fourteen, eight, and fourteen, respectively (Fig 1c). With these cut-off values, nuclear FAM83H expression was significantly associated with histologic grade (p = 0.044) and the expression of cytoplasmic FAM83H (p < 0.001) and nuclear ZNF16 (p = 0.002) (Table 1). Cytoplasmic expression of FAM83H was significantly associated with distant metastasis (p = 0.022), tumor stage (p = 0.040), histologic grade (p = 0.003), and the expression of nuclear ZNF16 (p = 0.019) and cytoplasmic ZNF16 (p = 0.009) (Table 1). Positivity for nuclear ZNF16 was significantly associated with tumor stage (p =0.028), T category of the tumor stage (p =0.014), histologic grade (p < 0.001), and the cytoplasmic expression of ZNF16 (p < 0.001) (Table 1). Cytoplasmic expression of ZNF16 was clearly associated with the age of the patients (p = 0.046), tumor stage (p =0.003), and T category of the tumor stage (p < 0.001) (Table 1).
The expressions of FAM83H and ZNF16 are associated with shorter survival of gallbladder carcinoma patients
The factors clearly associated with OS or RFS of gallbladder carcinomas were age, preoperative serum level of CA19-9, tumor stage, T category of tumor stage, lymph node metastasis, distant metastasis, lymphovascular invasion, histologic type, histologic grade, and the expression of nuclear FAM83H (OS; p < 0.001, RFS; p < 0.001), cytoplasmic FAM83H (OS; p < 0.001, RFS; p = 0.001), nuclear ZNF16 (OS; p < 0.001, RFS; p < 0.001), and cytoplasmic ZNF16 (OS; p = 0.035, RFS; p = 0.068) (Table 2). Nuclear FAM83H positivity predicted a 2.823-fold [95% confidence interval (95% CI); 1.716-4.646] greater risk of death and a 2.685-fold (95% CI; 1.640-4.395) greater risk of relapse or death of gallbladder carcinoma patients. Cytoplasmic FAM83H positivity predicted a 2.292-fold (95% CI; 1.413-3.720) greater risk of death and a 2.201-fold (95% CI; 1.360-3.564) greater risk of relapse or death of gallbladder carcinoma patients. Patients with nuclear ZNF16 positive carcinomas had a 3.287-fold (95% CI; 1.888-5.722) greater risk of death and a 3.038-fold (95% CI; 1.765-5.229) greater risk of relapse or death from gallbladder carcinoma. Patients with cytoplasmic ZNF16 positive carcinomas had a 1.675-fold (95% CI; 1.038-2.703) greater risk of death from gallbladder carcinoma (Table 3). Figure 2 presents Kaplan-Meier survival curves for OS and RFS of gallbladder carcinoma patients according to the nuclear and cytoplasmic expression of FAM83H and ZNF16.
Multivariate analysis was performed with the factors clearly associated with OS or RFS; age, preoperative serum level of CA19-9, tumor stage, T category of tumor stage, lymph node metastasis, distant metastasis, lymphovascular invasion, histologic type, histologic grade, and the expression of nuclear FAM83H, cytoplasmic FAM83H, nuclear ZNF16, and cytoplasmic ZNF16. Multivariate analysis showed age (OS; p < 0.001, RFS; p < 0.001), tumor stage (OS; overall p < 0.001, RFS; overall p < 0.001), and nuclear FAM83H expression (OS; p = 0.005, RFS; p = 0.005) to be independent prognostic indicators of gallbladder carcinoma patients (Table 3). Nuclear FAM83H positivity predicted a 2.094-fold (95% CI; 1.243-3.525) greater risk of death and a 2.108-fold (95% CI; 1.260-3.527) greater risk of relapse or death of gallbladder carcinoma patients by the multivariate analysis (Table 3).
The co-expression pattern of nuclear FAM83H and nuclear ZNF16 was associated with shorter survival of gallbladder carcinoma patients
In our results, the expression of FAM83H was significantly associated with ZNF16 expression. Moreover, the expression of nuclear FAM83H, cytoplasmic FAM83H, nuclear ZNF16, and cytoplasmic ZNF16 were associated with the survival of gallbladder carcinoma patients. In addition, as shown in Table 2, the prognostic predictability of nuclear expressions of FAM83H and ZNF16 were stronger than cytoplasmic expressions of FAM83H and ZNF16 when incorporating their hazard ratios and P values. Therefore, based on the relationship between FAM83H and ZNF16 expression and the prognostic value of the nuclear expression of FAM83H and ZNF16, we further evaluated the clinical significance of the co-expression pattern of nuclear FAM83H and nuclear ZNF16 (nFAM83H/nZNF16). When we sub-grouped gallbladder carcinomas into nFAM83H-/nZNF16-, nFAM83H-/nZNF16+, nFAM83H+/nZNF16-, and nFAM83H+/nZNF16+ subgroups, the nFAM83H-/nZNF16- subgroup had the longest survival and the nFAM83H+/nZNF16+ subgroup had the shortest survival (Fig 3a) (Table 4). However, the difference in survival among nFAM83H-/nZNF16+, nFAM83H+/nZNF16-, and nFAM83H+/nZNF16+ subgroups were minimal (Fig 3a). Based on these survival analyses, we grouped gallbladder carcinomas into two subgroups: favorable (nFAM83H-/nZNF16-) and poor (nFAM83H-/nZNF16+, nFAM83H+/nZNF16-, or nFAM83H+/nZNF16+) subgroups (Fig 3b) (Table 4). These prognostic subgroups were significantly associated with tumor stage (p = 0.016), T category of tumor stage (p = 0.006), and histologic grade (p < 0.001) (Table 5). In univariate Cox regression analysis, the poor prognostic subgroup with co-expression of nFAM83H/nZNF16 predicted a 5.463-fold (95% CI; 2.598-11.487, p < 0.001) higher risk of death and a 4.796-fold (95% CI; 2.367-9.717, p < 0.001) higher risk of relapse or death of patients (Table 6). In multivariate analysis, the co-expression of nFAM83H/nZNF16 was also an independent indicator of poor prognosis of gallbladder carcinoma patients (Table 6). The poor prognostic subgroup co-expressing nFAM83H/nZNF16 had a 4.808-fold (95% CI; 2.143-10.791, p < 0.001) higher risk of death and a 4.204-fold (95% CI; 1.958-9.029, p < 0.001 higher risk of relapse or death of patients compared with the favorable prognostic subgroup (Table 6).
Furthermore, we evaluated the prognostic significance of the individual and co-expression patterns of nuclear FAM83H and nuclear ZNF16 in 23 gallbladder carcinoma patients who received adjuvant chemotherapy. As shown in Figure 4, individual expression of nuclear FAM83H and nuclear ZNF16, and two prognostic subgroups according to the co-expression patterns of nFAM83H/nZNF16 [(nFAM83H-/nZNF16-) versus (nFAM83H-/nZNF16+, nFAM83H+/nZNF16-, or nFAM83H+/nZNF16+) subgroups] were significantly associated with OS and RFS (Fig 4).