Prognostic significance of TMEM16A, PPFIA1, and FADD expression in invasive ductal carcinoma of the breast

We collected 98 cases of invasive ductal carcinoma surgically resected at Uijeongbu Mary’s Hospital from 2002 to 2004. Patients’ age was between 29 and 77 (mean, 49.1) years old. Forty-six cases were treated with adjuvant chemotherapy and 33 cases received hormone therapy. Adjuvant radiotherapy was given to 22 cases. Disease-free survival data (11.0 to 103.3 months; mean, 61.9 months) was available. The disease recurred in 21 cases and 7 cases died of the disease. We selected representative archival tissues resected from the cases and tissue microarray was constructed using manual tissue arrayer, MTA-1 (Estigen Tissue Science, Estonia). Human tissue acquisition and its use followed the Institutional Review Board-approved protocol (CUMC11U058) at the Catholic University of Korea, School of Medicine.

The immunohistochemical staining of breast cancer tissue followed the previously reported protocol [26]. Briefly, tissue sections were cut in 4-μm thicknesses and transferred to ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA, USA). To minimize tissue loss during boiling procedure and to get rid of excess paraffin on the slides, tissues were incubated for two hours in a 56°C dry chamber (Agilent Technologies, Santa Clara, CA, USA). The sections were deparaffinized in xylene three times and hydrated through 100%, 90%, 80%, 70% ethanol and Tris-buffered saline (TBS, pH 7.4). To make the epitopes more accessible to the primary antibodies used in the current study, the tissues were boiled in 10 mM sodium citrate buffer (pH 6.0) using a microwave for 20 minutes. To quench endogenous peroxidase, we treated the tissues with 3% hydrogen peroxide in PBS. The tissues were incubated with the respective primary antibodies at 4°C overnight (Table 1). After incubating the tissue with biotinylated secondary antibody, diluted (1:50) ExtrAvidin (Sigma-Aldrich, St. Louis, MO, USA) was used to amplify signal intensity. For visualization, liquid DAB + substrate chromogen system (Dako, Glostrup, Denmark) was used. Scoring of immunohistochemical staining was divided into three groups. Positive staining in less than 5% of tumor cells was considered negative. Cases showing a brown color in more than 50% of tumor cells were considered to be a strongly positive group. Cases showing light brown color in more than 5% and dark brown color in less than 50% of tumor cells were counted as a weakly positive group [14, 20].

Where appropriate, the Chi-square test or Fisher’s exact test was used to evaluate association of immunoreactivity with clinicopathologic parameters. For disease-free survival analysis, Kaplan-Meier method and the nonparametric log-rank test was used. We used R ver. 3.0.2 (R foundation, Vienna, Austria) for statistical tests and their graphic presentations.

Among 98 cases in total, 5 (5.1%) cases of grade I, 51 (52.0%) cases of grade II, and 40 (40.8%) cases of grade III were included in this study. Two (2.0%) cases did not have information on grade status. T1, T2, and T3 stages were 23 (23.5%), 64 (65.3%), and 11 (11.2%) cases, respectively. For nodal stages, N0 (41 cases, 41.8%) and N1 (34 cases, 34.7%) were most common. Estrogen receptor (ER) was positive in 63 cases and progesterone receptor (PR) was positive in 65 cases (Table 2). ER and PR positivities of nine cases were not known.

TMEM was positive in 86 cases (strongly positive 5 cases, weakly positive 81 cases). FADD was positive in 62 cases (strongly positive 3 cases, weakly positive 59 cases). PPFIA1 was positive in 88 cases (strongly positive 24 cases, weakly positive 64 cases) (Figure 1). Strongly positive and weakly positive groups were pooled in the positive group for association tests and survival analysis. In association analysis of immunoreactivity of each protein and pathological parameter, significant association was only found between FADD expression and T stage (P = 0.046). Neither TMEM16A nor PPFIA1 showed significant association with any pathological parameters studied (Table 3). To see if the expressions of the three genes are associated, we calculated correlation of coefficient of each expression. TMEM16A and PPFIA1 expression was marginally correlated (r = 0.6). However, FADD expression showed low correlation with PPFIA1 and TMEM16A expression (r = 0.35, each).

Survival difference was significant in grade (P = 0.01) and N stage (P = 0.00) but not in age (P = 0.85) and T stage (P = 0.189). Both PPFIA1 and TMEM16A expression showed tendency of association with poor survival group (P = 0.161 and 0.114, respectively). However, the survival differences were not strong enough to show statistical significance. For FADD expression, positive expression showed a tendency towards better survival, but this also failed to show statistical significance (P = 0.182).

To evaluate net effect of the closely located genes, the score representing combined effect of the three gene expressions was calculated and we named it ‘hazard score’. Because FADD expression showed a tendency towards better patient survival, we calculated a combined score using the following equation:

We found the hazard score was associated with perineural invasion status of the cases (Table 3). The cases with the hazard scores of 2 or more showed significant association with poor disease-free survival (P = 0.034, Figure 2).