We retrospectively reviewed 78 patients with ampullary carcinoma who underwent pancreatoduodenectomy at our institution between January 2000 and December 2021. The study protocol was approved by the Institutional ethics committee (approval no. 2110-003), and was conducted in accordance with the Declaration of Helsinki.

Clinicopathological data were extracted from our database: age, sex (male or female), body mass index, American Society of Anesthesiologists (ASA) physical status (grade 1, 2, or 3), hypertension, diabetes mellitus, preoperative biliary drainage, operative time, estimated blood loss, postoperative outcome [major complication defined as Clavien grade ≥3 (11) and mortality), pathological factors evaluated by the General Rules for Clinical and Pathological Studies on Cancer of the Biliary Tract of Japan (12) (T and N factors), histopathologic subtype evaluated by a pathologist (intestinal, pancreatobiliary, and mixed type) (13), recurrence (absence or presence), status at the last follow-up (survival or death), and cause of death (primary disease-related or others).

We employed a previously reported protocol for evaluating CAFs (14–16). First, the presence of tumor cells was confirmed by hematoxylin and eosin (H&E) staining. Sections on the microslides were deparaffinized with xylene, hydrated using a diluted alcohol series, and immersed in H2O2 with methanol to quench endogenous peroxidase activity. To reduce nonspecific staining, each section was blocked with a serum-free protein block (Dako, Agilent Technologies, Santa Clara, CA, USA) for 15 min. After heat-mediated antigen retrieval with Tris/EDTA buffer, the sections were incubated with anti-SMA antibody (1:1000 dilution; Sigma-Aldrich, St. Louis, MO, USA) or anti-FAP antibody (1:250 dilution; Abcam, Cambridge, UK) diluted in Dako REAL Antibody Diluent (Dako, Agilent Technologies, Santa Clara, CA, USA) and incubated overnight at 4°C. The sections were then incubated with Envision+ anti-mouse/rabbit antibodies (Dako, Agilent Technologies, Santa Clara, CA, USA) for 30 min at RT. The chromogen used was liquid DAB+ (Dako, Agilent Technologies, Santa Clara, CA, USA). The sections were visualized with a 3,3’-diaminobenzidine tetrahydrochloride solution, and nuclei were counterstained with Meyer’s hematoxylin. CAFs were defined as spindle-shaped cells expressing α-SMA or FAP, and evaluated using an area index calculated in low-magnification fields using ImageJ software (http://rsb.info.nih.gov/ij/). Three fields, including stromal cells per sample, were carefully selected to evaluate CAFs. The mean value obtained from each sectioned tissue sample was defined as the area index. The α-SMA and FAP positive rates were calculated as each area index. In this study, the median values of the area index for α-SMA and FAP were used as cut-off values to define the low and high groups.

The primary endpoint was the prognostic factors for survival after surgery. The secondary endpoint was survival after surgery, focusing on CAFs. The recurrence-free (RFS) and disease-specific (DSS) survival rates were analyzed.

RFS and DSS rates were investigated using the Kaplan–Meier method, and the log-rank test was used to evaluate differences between the groups. DSS was defined as the duration from surgery to the date of death as a result of the primary disease. Patients who died of causes unrelated to the primary disease were excluded.

The prognostic factors associated with RFS and DSS were investigated using a Cox proportional hazards model with hazard ratios (HR) and 95% confidence intervals (CI). As a significant correlation between α-SMA and FAP was found, the multivariable analyses were generated by including α-SMA and FAP separately. In Model 1, relevant factors, including α-SMA, were included in the multivariable analyses. In Model 2, the multivariable analyses included relevant factors, including FAP.

JMP version 11 software (SAS Institute, Cary, NC, USA) was used for statistical analyses.

Of the 78 patients, 67 were available for immunohistochemical analysis. The characteristics of 67 patients are shown in Table 1 . Pathological T factors included Tis (n = 10), T1 (n = 21), T2 (n = 22), and T3 (n = 17). Lymph node metastases were observed in 14 patients (21%). Histopathologic subtypes of ampullary carcinoma included the intestinal (n = 36, 54%), pancreatobiliary (n = 20, 30%), and mixed type (n = 11, 16%). Postoperative recurrence was observed in 17 patients (25%) during a mean follow-up period of 5.3 years.

CAFs were identified as stromal cells expressing α-SMA and FAP. The mean (standard deviation [SD]) area indices for α-SMA and FAP were 11.0 (10.5) and 6.9 (9.6), respectively ( Figure 1 ). Using the median area indices for α-SMA and FAP, the cut-off values were set at 7.1 for α-SMA and 1.2 for FAP. Microscopic images of H&E, anti-α-SMA, and FAP staining, as well as the images generated using Image J, are shown in Figure 2 . The area index of α-SMA significantly correlated with that of FAP (r² = 0.55; p < 0.001), as shown in Figure 3 .

Relationship between CAF expression and histopathologic subtype of ampullary carcinoma is depicted in Table 2 . A significant difference was found in the mean (SD) area index for α-SMA and FAP between three groups: 4.8 (5.9) and 2.2 (5.8) in the intestinal type; 19.1 (9.9) and 12.8 (10.1) in the pancreatobiliary type; and 16.0 (10.8) and 11.1 (11.0) in the mixed type. The high-α-SMA and high-FAP was 25% and 19% in the intestinal type, 90% and 80% in the pancreatobiliary type, and 73% and 91% in the mixed type, respectively.

The 5-year RFS and DSS rates were 63.9% and 78.5%, respectively. RFS and DSS curves stratified by α-SMA and FAP are shown in Figure 4 . Patients with high α-SMA expression had a significantly worse RFS than those with low α-SMA expression (5-year RFS, 47.6% vs. 82.2%; p = 0.003; Figure 4A ). Furthermore, the 5-year DSS rates in the low- and high-α-SMA groups were 93.3% and 67.5%, respectively, with a significant difference between the groups (p = 0.01; Figure 4B ). Regarding the effect of FAP on survival, the high-FAP group had significantly worse RFS (5-year RFS, 53.5% vs. 73.7%; p = 0.04; Figure 4C ) and DSS (5-year DSS, 65.7% vs. 94.4%; p = 0.02; Figure 4D ) than the low-FAP group.

The results of the univariate and multivariable analyses for investigating the prognostic factors of RFS are shown in Table 3 . Univariate analyses revealed that α-SMA, FAP, and lymph node metastasis were significant factors, but histopathologic subtypes were not an independent factor. In model 1, multivariable analysis revealed that high α-SMA expression was an independent predictor (HR: 3.68; 95% CI: 1.21–12.4; p = 0.02). In contrast, high FAP was not an independent index for RFS in model 2.

Table 4 shows the results of the univariate and multivariable analyses for DSS. Univariate analyses identified high α-SMA (HR: 9.48; p = 0.005) and FAP (HR: 4.50; p = 0.03) as independent predictors of DSS. However, only high α-SMA level (HR: 8.54; 95% CI: 1.21–170; p = 0.03) was a significant factor associated with DSS in the multivariable analyses.

The relationship between α-SMA expression and clinicopathological parameters is shown in Table 5 . No significant differences were found between the low- and high-α-SMA groups in terms of the demographic variables. High α-SMA expression was significantly associated with advanced T stage as well as higher incidences of lymph node metastases and recurrence. In fact, the low α-SMA group had lymph node metastasis in only one patient (3%) and no recurrence after surgery.