Survival Outcomes According to Adjuvant Treatment and Prognostic Factors Including Host Immune Markers in Patients with Curatively Resected Ampulla of Vater Cancer

Background Ampulla of Vater cancer (AoV Ca) is a rare tumor, and its adjuvant treatment has not been established. The purpose of this study was to find out prognostic factors including host immunity and role of adjuvant treatment in AoV Ca. Methods and Findings We reviewed 227 AoV Ca patients with curative resection. Clinical characteristics, adjuvant treatment, disease-free survival (DFS) and overall survival (OS) were analyzed. Among all patients, 63.9, 36.1 and 33.9% had T1/T2, T3/T4 stage and lymph node-positive disease (LN+), respectively. OS of all patients was 90.9 months (95% CI: 52.9–129.0). OS was different according to neutrophil-to-lymphocyte ratio (HR 1.651, 95% CI: 1.11–2.47), platelet-to-lymphocyte ratio (HR 1.488, 95% CI: 1.00–2.21) and systemic inflammatory index (HR 1.669, 95% CI: 1.13–2.47). In multivariate analysis, adverse prognostic factors for OS included vascular invasion (HR 2.571, 95% CI: 1.20–5.53) and elevated CA 19–9 (HR 1.794, 95% CI: 1.07–3.05). A total of 104 patients (46.3%) received adjuvant treatment (25 out of 111of T1/T2 & LN (-), 79 out of 116 of T3/T4 or LN (+)). In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the longest OS (5-year OS rate: 47.0 vs. 41.4%). Conclusions Vascular invasion and elevated CA 19–9 were adverse prognostic factors in resected AoV Ca. In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the best survival outcome. Adjuvant treatment should be further defined in AoV Ca, especially with poor prognostic factors.


Introduction
The annual incidence of biliary tract cancer (BTC) in the Western world is about 5-6 per 100,000, while the annual incidence in Korea is 10 per 100,000. [1,2] BTC has a worse prognosis than other malignancies. [2] Surgical resection is the only treatment modality which offers a chance of cure. [3] Approximately 40~50% of cholangiocarcinoma and 30% of gallbladder cancer patients undergo surgery; however, even in those resected cases, many patients experience cancer recurrence. [4,5] In 1999, there was a randomized controlled study to evaluate the role of adjuvant concurrent chemoradiotherapy (CCRT) in pancreatic and biliary cancers by The European Organization for Research and Treatment of Cancer, which failed to show survival gain. [6] Other retrospective studies of the role of radiotherapy after surgical resection showed better 5-year loco-regional disease-free survival (DFS) and overall survival (OS) rates, and several retrospective analyses also showed significantly better survival outcomes in lymph node-positive patients on adjuvant CCRT. [7][8][9] Ampulla of Vater cancer (AoV Ca) accounts for 10-15% of BTC in Korea, which arises from distal to the confluence of the common bile duct with the main pancreatic duct. [10] Initial presentations of AoV Ca are usually related to biliary obstruction such as jaundice, red urine and pruritus, potentially resulting in early detection. [11] Approximately 80% of AoV Ca patients were detected at a potentially resectable stage at the time of diagnosis. [12] Prognosis of AoV Ca has been favorable compared with other biliary tract cancers originating from the intrahepatic or extrahepatic bile duct or gallbladder. However, resected patients relapse in many cases, which leads to an eventual 5-year survival rate of 20~50%. [7,13] The identification of patients with poor prognosis after curative resection is important to improve survival outcomes. In parallel, the role of adjuvant treatment should be accurately defined in patients with poor prognosis. Because of the relatively low incidence of AoV Ca, a prospective study design is extremely difficult to answer those questions.
Several studies have reported on the prognostic factors of AoV Ca. Nowadays, host immunity and peritumoral inflammation are considered important factors in the carcinogenesis and prognosis of solid tumors. [14][15][16][17] However, in BTC, including AoV Ca, the prognostic role of host immunity and peritumoral inflammation has not been well documented.
In this study, we evaluated the prognostic factors to define the AoV Ca patients with poor prognosis after curative resection. In this analysis, we included immunity/inflammation markers. The other important purpose of this study was to determine the role of adjuvant treatment in AoV Ca.

Patients and data collection
This study was a retrospective analysis of de-identified patient-level data from medical charts. Patients who were diagnosed with AoV Ca and who underwent curative resection at the Seoul National University Hospital between 1997 and 2012 were enrolled. Diagnosis was confirmed by tissue pathology. Data of baseline demographics were collected, including gender, ECOG, stage, laboratory tests (total bilirubin, albumin, carcinoembryonic antigen (CEA), carbohydrate antigen , and neutrophil, platelet and lymphocyte counts). The data of adjuvant treatment patterns were also collected, including chemotherapy, radiotherapy and CCRT. Survival outcomes including disease-free survival (DFS) and overall survival (OS) were obtained as well.

Statistical analysis
Statistical analysis of categorical variables was performed using Pearson's chi-square test or Fisher's exact test, as appropriate. A t-test was used for comparison of means. Median DFS and OS for all patients were calculated using the Kaplan-Meier method and comparisons between groups were made using log-rank tests.
Neutrophil, lymphocyte and platelet count were obtained from preoperative laboratory tests. We calculated neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) as neutrophil and platelet counts divided by lymphocyte counts, respectively. We also used the systemic inflammatory index (SII) which was determined as neutrophil x platelet/ lymphocyte. [18] The cut-off values for NLR, PLR and SII were obtained using receiver operating characteristic (ROC) curve analysis for predicting OS.
The impact of continuous numerical variables on clinical outcomes was evaluated using Cox regression. Multivariate analysis for DFS and OS was also performed using Cox regression models. Factors with p<0.05 in univariate analysis were examined in multivariate regression models. All statistical tests were two-sided, with significance defined as p<0.05.

Ethics
The study protocol was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (H-1306-109-500). All studies were conducted according to guidelines for biomedical research (Declaration of Helsinki). Written informed consent was not given by participants but patients' record and information was anonymized and de-identified prior to analysis.

Patient characteristics
A total of 227 patients were included in this analysis (  (Fig 1).

Prognostic factor and clinical outcomes
In univariate analysis, aged <60, CEA, CA-19-9, total bilirubin, NLR, PLR, SII and T/N stage were significant prognostic factors for 5-year OS ( Table 2). Patients with lower NLR showed longer survival than patients with higher NLR (not achieved vs. 58.2 months, HR 1.651 (95% CI: 1.11-2.47), p = 0.012) (Fig 2A). In a similar way, lower PLR was associated with better  (Table 3). In the higher NLR group, a higher proportion of T3/4 stage, stage II/ III, lymphatic/perineural invasion, high PLR, high SII was observed. Regarding pathologic findings, degree of differentiation and lymphatic/vascular/perineural invasion were also significant prognostic factors for OS. On multivariate analysis, vascular invasion and elevated CA 19-9 were significant poor prognostic factors for 5-year OS ( Table 2).
Adverse prognostic factors for 5-year DFS were differentiation, lymphatic/vascular/perineural invasion, CEA, CA 19-9, total bilirubin and T/N stage on univariate analysis. Differentiation and T/N stage showed significant differences for DFS on multivariate analysis (S1 Table).

The patterns of adjuvant treatment
After curative resection of tumor, 104 patients (45.8%) received adjuvant treatment. Adjuvant treatment modalities according to tumor stage are shown in S2 Table. A total 59 patients received adjuvant CCRT with maintenance chemotherapy, and 32 patients received adjuvant CCRT. Eight and five patients received adjuvant chemotherapy only and adjuvant radiotherapy only, respectively. The most commonly used chemotherapy was 5-FU based one. During CCRT, the regimen 5-FU 500 mg/m 2 , D1,2,3 q 4 weeks was most commonly used, followed by 5-FU/leucovorin (375 mg/m 2 , 20 mg/m 2 , respectively, D1-5, q 4 weeks). During maintenance chemotherapy or adjuvant chemotherapy alone, 5-FU 500 mg/ m 2 , D1-5 q 4 weeks was most commonly used for 6 months. Radiotherapy was administered at a dose of 45 Gy in 25 fractions. When we analyzed survival outcomes according to adjuvant treatment, there was no significant difference in stage 1A and 1B. However, in T3/T4 or LN (+) stage, the patients who received adjuvant CCRT with maintenance chemotherapy had better 5-year OS, even though the finding was not statistically significant (Table 4, Fig 3).
In patients who received adjuvant treatment, NLR, PLR and SII were all important factors for OS. However, this was not the case in patients without adjuvant treatment (Table A and B in S1 File).

Discussion
In this study, we found that in curatively resected AoV Ca, vascular invasion in pathologic examination and elevated CA 19-9 were poor prognostic factors. Patients who had T3/T4 or LN (+) tumors showed good survival when they received adjuvant CCRT with maintenance chemotherapy.
Tumor stage, lymph node involvement and vascular/perineural invasion were well-known prognostic factors in biliary tract cancer. [19] In our study, T/N stage, presence of lymphatic/  vascular/perineural invasion, histologic differentiation and elevated total bilirubin/CEA/CA 19-9 were adverse prognostic factors.
In cancer development and progression, the role of inflammation has been highlighted. [15][16][17]20] As systemic inflammatory response is activated, neutrophils increase, and in parallel, lymphocytes decrease in peripheral blood. For several years, the index representing the systemic inflammatory state has been developed and several markers such as NLR, PLR and SII have been analyzed in various tumor conditions except AoV Ca. [21,22] Tumor antigens elicit an adaptive immune response by inflammatory cells, macrophages and lymphocytes. CD4+ T cells and CD8+ T cells have important roles in this process, and especially tumor-infiltrating CD8+ T lymphocytes improve prognosis in several cancers. [23,24] NLR, PLR and SII may represent these immune response processes and be of prognostic significance. [25,26] We analyzed the association of OS and host immunity and inflammation status such as NLR, PLR and SII. The patients with NLR 1.78 or PLR 192.0 or SII 780.0 showed significantly prolonged OS. We selected the cut-off values of NLR, PLR and SII using ROC analysis for OS. NLR and PLR showed a linear relationship (r 2 = 0.82) and NLR and SII also showed a linear relationship (r 2 = 0.88). Patients with higher NLR included a higher proportion of T3/4 stage, stage II/III and lymphatic/perineural invasion compared with patients with lower NLR. To the best of our knowledge, this is the most extensive analysis in AoV Ca patients focused on host immunity and inflammation status.
The role of adjuvant treatment in BTC patient has not been established. An earlier retrospective analysis of survival outcomes in patients with adjuvant therapy showed that OS was improved insignificantly. [27] Recently, another study also reported that neoadjuvant and adjuvant chemotherapy did not provide survival benefit. [28] However, a meta-analysis reported survival benefit in patients with LN (+) or R1 resection by adjuvant therapy. [29] The study was reported that the patients with KRAS G12D mutation show poor prognoses and high risk of early recurrence, and adjuvant therapy will be effective in the high risk patients. [30] In these circumstances, according to National Comprehensive Cancer Network (NCCN) guidelines, adjuvant therapy is recommended for R1 or R2 resected or LN (+) patients. In case of R0 resection with no LN involvement or carcinoma in situ at resection margins, four options are all recommended, that is, observation or fluoropyrimidine-based chemoradiation or fluoropyrimidine-based or gemcitabine-based chemotherapy or clinical trial.
In our study, 25% of patients received adjuvant treatment in T1/T2 & LN (-) stage. The percentage of delivered adjuvant treatment was increased with stage, where nearly 70% of patients One of the limitations of our study was the design, i.e., retrospective, single center study. The adjuvant treatment was not applied based on a consistent principle of guidelines, and  Nonetheless, our study has a value of providing information on adverse prognostic factors including host immunity and inflammation status and clinical outcomes of adjuvant treatment modalities in a relatively large AoV Ca cohort.
In conclusion, the AoV Ca patients with vascular invasion and elevated CA 19-9 showed poor prognosis after curative resection. Host immunity and inflammation status represented