Impact of Positive Lymph Nodes and Resection Margin Status on the Overall Survival of Patients with Resected Perihilar Cholangiocarcinoma: The ENSCCA Registry

Citation: Nooijen, L.E.; Banales, J.M.; de Boer, M.T.; Braconi, C.; Folseraas, T.; Forner, A.; Holowko, W.; Hoogwater, F.J.H.; Klümpen, H.-J.; Groot Koerkamp, B.; et al. Impact of Positive Lymph Nodes and Resection Margin Status on the Overall Survival of Patients with Resected Perihilar Cholangiocarcinoma: The ENSCCA Registry. Cancers 2022,14, 2389. https://doi.org/10.3390/ cancers14102389 Academic Editor: David Wong Received: 25 March 2022 Accepted: 10 May 2022 Published: 12 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article Impact of Positive Lymph Nodes and Resection Margin Status on the Overall Survival of Patients with Resected Perihilar Cholangiocarcinoma: The ENSCCA Registry Lynn E. Nooijen 1,2 , Jesus M. Banales 3,4,5,6, Marieke T. de Boer 7, Chiara Braconi 8, Trine Folseraas 9, Alejandro Forner 10 , Waclaw Holowko 11 , Frederik J. H. Hoogwater 7, Heinz-Josef Klümpen 2,12 , Bas Groot Koerkamp 13, Angela Lamarca 14 , Adelaida La Casta 15, Flora López-López 16, Laura Izquierdo-Sánchez 3, Alexander Scheiter 17 , Kirsten Utpatel 17, Rutger-Jan Swijnenburg 2,18, Geert Kazemier 1,2 and Joris I. Erdmann 2,18,* on behalf of the ENSCCA Group 1Department of Surgery, Amsterdam UMC—Location Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands; [email protected] (L.E.N.); [email protected] (G.K.) 2Cancer Center Amsterdam, Cancer Treatment and Quality of Life, 1081 HV Amsterdam, The Netherlands; [email protected] (H.-J.K.); [email protected] (R.-J.S.) 3Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), 20014 San Sebastián, Spain; [email protected] (J.M.B.); [email protected]g (L.I.-S.) 4 National Institute for the Study of Liver and Gastrointestinal Diseases, CIBERehd, “Instituto de Salud Carlos III” (ISCIII), 28029 Madrid, Spain 5 Department of Biochemistry and Genetics, School of Sciences, University of Navarra, 31080 Pamplona, Spain 6Ikerbasque, Basque Foundation for Science, 48009 Bilbao, Spain 7 Section of HPB Surgery & Liver Transplantation, Department of Surgery, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; [email protected] (M.T.d.B.); [email protected] (F.J.H.H.) 8The Royal Marsden NHS Trust, London SW3 6JJ, UK; [email protected] 9Norwegian PSC Research Center and Section of Gastroenterology, Department of Transplantation Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital Rikshospitalet, 0372 Oslo, Norway; [email protected] 10 BCLC Group, Liver Unit, Hospital Clinic Barcelona, IDIBAPS, CIBEREHD, University Barcelona, 08036 Barcelona, Spain; [email protected] 11 Department of General, Transplant and Liver Surgery, Medical University of Warsaw, 02-091 Warsaw, Poland; [email protected] 12 Department of Medical Oncology, Amsterdam UMC—Location University of Amsterdam, 1105 AZ Amsterdam, The Netherlands 13 Department of Surgery, Erasmus MC Cancer Institute, 3015 GD Rotterdam, The Netherlands; [email protected] 14 Department of Medical Oncology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, UK; [email protected] 15 Department of Medical Oncology, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), 20014 San Sebastián, Spain; [email protected] 16 Department of Medical Oncology, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain; [email protected] 17 Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany; alexander[email protected]egensburg.de (A.S.); [email protected]egensburg.de (K.U.) 18 Department of Surgery, Amsterdam UMC—Location University of Amsterdam, 1105 AZ Amsterdam, The Netherlands *Correspondence: [email protected]; Tel.: +31-(0)-20-5627624 Simple Summary: Lymph node metastasis and positive resection margins have been reported to be major determinants of overall survival (OS) and poor recurrence-free survival (RFS) for patients after resection for perihilar cholangiocarcinoma (pCCA). The aim of current study was to assess the prognostic value of positive lymph nodes and resection margin status on OS. We found a major negative effect of positive lymph nodes on survival and recurrence, which, surprisingly, was Cancers 2022,14, 2389. https://doi.org/10.3390/cancers14102389 https://www.mdpi.com/journal/cancers Cancers 2022,14, 2389 2 of 13 independent of margin status. ECOG performance status, positive lymph nodes, and the grade of tumor differentiation were found as the main independent indicators of oncological outcome. This suggests that tumor biology and performance status have a higher impact on survival than extended or radical surgery. Abstract: Background: Lymph node metastasis and positive resection margins have been reported to be major determinants of overall survival (OS) and poor recurrence-free survival (RFS) for patients who underwent resection for perihilar cholangiocarcinoma (pCCA). However, the prognostic value of positive lymph nodes independently from resection margin status on OS has not been evaluated. Methods: From the European Cholangiocarcinoma (ENSCCA) registry, patients who underwent resection for pCCA between 1994 and 2021 were included in this retrospective cohort study. The primary outcome was OS stratified for resection margin and lymph node status. The secondary outcome was recurrence-free survival. Results: A total of 325 patients from 11 different centers and six European countries were included. Of these, 194 (59.7%) patients had negative resection margins. In 113 (34.8%) patients, positive lymph nodes were found. Lymph node status, histological grade, and ECOG performance status were independent prognostic factors for survival. The median OS for N0R0, N0R1, N+R0, and N+R1 was 38, 30, 18, and 12 months, respectively (p< 0.001). Conclusion: These data indicate that in the presence of positive regional lymph nodes, resection margin status does not determine OS or RFS in patients with pCCA. Achieving negative margins in patients with positive nodes should not come at the expense of more extensive surgery and associated higher mortality. Keywords: perihilar cholangiocarcinoma; lymph nodes; resection margin; overall survival; recurrence-free survival 1. Introduction Radical surgical resection is still the only treatment with a chance of long-term survival for patients with perihilar cholangiocarcinoma (pCCA). Radical (R0) resection rates range from 60 to 90%, which is associated with a 5-year overall survival (OS) up to 60%, while a margin positive (R1) resection is associated with a 5-year OS of less than 20% [ 1 – 5 ]. On the other hand, the prognosis of patients with resected pCCA is also significantly determined by the presence of lymph node metastases [1,6,7]. At exploration, about 35% of patients have loco-regional positive lymph nodes, which is associated with a 5-year OS of less than 20% versus 55% in case of negative lymph nodes [ 1 , 6 , 7 ]. During resection, the retrieval of a low number of less than four lymph nodes has been reported to negatively influence survival, possibly due to the understaging of unresected positive nodes [ 8 ]. In addition, a recent systematic review found that an extended lymphadenectomy does not lead to a survival benefit [9]. Nevertheless, a high number of involved lymph nodes has been reported to be a major determinant of OS and an indicator of poor recurrence-free survival (RFS) [ 8 , 10 , 11 ]. Buettner et al. concluded that the actual survival benefit of resection in patients with positive lymph nodes is shorter than seven months and should be weighed against considerable postoperative morbidity and mortality [12]. The updated AJCC guidelines (8th edition, 2017) distinguish between the number ( n≤3 or n> 3) instead of the location of regional positive lymph nodes [ 13 , 14 ]. Several studies investigated the value of this new staging system and found a slightly improved prognostic predictability, implying that the amount of involved lymph nodes is more predictive for survival outcome than their location [15–17]. In clinical practice, surgeons are often confronted with one or more positive lymph nodes at frozen section analysis in a patient undergoing surgical exploration with the intention to undergo an oftentimes major hepatobiliary resection. The technical feasibility of a potential margin-free resection and concomitant morbidity and mortality should be evaluated in the light of potential oncological outcomes. Until now, the relationship Cancers 2022,14, 2389 3 of 13 between positive lymph nodes and resection margins and their independent influence on OS remained unclear, making the question of whether patients with positive lymph nodes benefit from extended resections to obtain negative margins hard to answer. Therefore, we performed a retrospective cohort study, in cooperation with the ENSCCA Registry, to determine the impact of positive lymph nodes and resection margin status on the OS of patients after resected pCCA. 2. Materials and Methods 2.1. ENSCCA Registry The ENSCCA registry, endorsed by the European Network for the Study of Cholangiocarcinoma (ENSCCA), is an international, multicenter, and observational retrospective and prospective study including patients with histologically or radiologically proven cholangiocarcinoma (intrahepatic, perihilar, and distal cholangiocarcinoma). This registry includes several different variables regarding surgical and oncological outcomes. The ENSCCA registry protocol was approved by the Ethic Committee of Euskadi, Spain (Code: PI2016137) and the Ethic Committee of the Amsterdam UMC (location AMC), the Netherlands (W20_050), as coordinating Centers. Additionally, each participating center obtained local ethical approval (or equivalent). 2.2. Included Patients From the ENSCCA registry, patients with pCCA who underwent surgery including diagnostic laparoscopy, explorative laparotomy, liver transplantation, or liver resection were identified. Only patients from centers that had registered a minimum of 10 patients undergoing resection for pCCA were included. This was in order to secure centers with expertise in treating patients with pCCA for inclusion. Eligible centers were asked to complete potentially missing key variables (e.g., resection margin, lymph node status, and last date of follow-up) or to check discrepant variables. For the analysis, only patients who received curative-intent resection were included. 2.3. Outcomes The primary outcome was to assess the impact of the resection margin and lymph node status on OS. The secondary outcome was the impact of resection margin and lymph node status on recurrence-free survival (RFS). 2.4. Statistical Analysis Descriptive statistics were used to summarize the data. For continuous variables with normal distribution, the data were presented as mean ± standard deviation (SD) and for non-normal distributions, as median and interquartile range (IQR). Comparisons between the groups (N0 and N+) were analyzed using chi-square tests for proportions, Mann–Whitney U for medians, and unpaired t-tests for means. The OS was defined as the time (in months) after surgery to death or last follow-up, and the median OS was estimated using the Kaplan–Meier method. The RFS was defined as the time (in months) after surgery to recurrence or last medical visit. Survival curves were compared using the log-rank test. The reversed Kaplan–Meier-based method was used to calculate median follow-up. Univariable and multivariable logistic regression were used to examine the relationship between the presence of positive lymph nodes as a dependent variable and the possible predictors as independent variables. Therefore, the following factors were included: age, ECOG, and tumor size > 2.5 cm. Factors with p-values < 0.2 at univariable analysis were included in the multivariable analysis. The independent predictors of survival determined prior to conducting the study (i.e., age, ECOG, tumor size > 2.5 cm, carbohydrate antigen 19.9 (CA19.9), lymph node status (AJCC 8th), margin status, and tumor differentiation [8,11]) were analyzed using uniand multivariable Cox regression analysis. In addition, the independent predictors of recurrence, also determined prior to conducting the study (tumor size > 2.5 cm, T status (AJCC 8th), lymph node status Cancers 2022,14, 2389 4 of 13 (AJCC 8th), resection margin, and tumor differentiation) were analyzed using uniand multivariable Cox regression analysis [ 10 , 18 ]. Factors with p-values < 0.1 at univariable analysis were included in the multivariable analysis. A two-sided p-value of < 0.05 was considered to indicate statistical significance. The data were analyzed using IBM SPSS statistics, version 25.0 (IBM Corp, Armonk, NY, USA). Survival curves were displayed using GraphPad Prism 8. 3. Results 3.1. Baseline Characteristics A total of 1126 patients with pCCA were extracted from the registry; from these, 618 (55%) patients underwent any type of surgery. Forty-one patients were excluded as these were from centers that registered fewer than ten patients. Thirty-nine patients were excluded because of missing information. From this dataset, patients undergoing palliative resection (n= 103), undergoing liver transplantation (n= 15), who had R2 residual disease (n= 12), unknown residual disease (n= 7), unknown lymph node status (n= 4), who had no resection (n= 60), other diagnosis (n= 3), or were double in the registry (n= 9) were excluded, leaving a total of 325 patients from 11 different centers in six countries. A flowchart is presented in Figure 1. Cancers 2022, 14, x FOR PEER REVIEW 4 of 14 carbohydrate antigen 19.9 (CA19.9), lymph node status (AJCC 8th), margin status, and tumor differentiation [8,11]) were analyzed using uniand multivariable Cox regression analysis. In addition, the independent predictors of recurrence, also determined prior to conducting the study (tumor size > 2.5 cm, T status (AJCC 8th), lymph node status (AJCC 8th), resection margin, and tumor differentiation) were analyzed using uniand multivariable Cox regression analysis [10,18]. Factors with p-values < 0.1 at univariable analysis were included in the multivariable analysis. A two-sided p-value of < 0.05 was considered to indicate statistical significance. The data were analyzed using IBM SPSS statistics, version 25.0 (IBM Corp, Armonk, NY, USA). Survival curves were displayed using GraphPad Prism 8. 3. Results 3.1. Baseline Characteristics A total of 1126 patients with pCCA were extracted from the registry; from these, 618 (55%) patients underwent any type of surgery. Forty-one patients were excluded as these were from centers that registered fewer than ten patients. Thirty-nine patients were excluded because of missing information. From this dataset, patients undergoing palliative resection (n = 103), undergoing liver transplantation (n = 15), who had R2 residual disease (n = 12), unknown residual disease (n = 7), unknown lymph node status (n = 4), who had no resection (n = 60), other diagnosis (n = 3), or were double in the registry (n = 9) were excluded, leaving a total of 325 patients from 11 different centers in six countries. A flowchart is presented in Figure 1. Figure 1. Flow diagram of included patients. Figure 1. Flow diagram of included patients. A total of 206 out of the 325 patients (63.4%) were male and the median age at surgery was 64 (58–71) years. The median Bismuth–Corlette classification was IIIb (20.6%). The median preoperative tumor size was 2.5 (1.9–3.7) cm. Neoadjuvant therapy, consisting of radiotherapy 5 ×5 Gy, was administered to 26 (8.0%) patients. Cancers 2022,14, 2389 5 of 13 Out of the 325 patients, 194 (59.7%) had undergone an R0 resection. In 113 (34.8%) patients, positive lymph nodes were found. In the latter group, the median number of retrieved lymph nodes was 5 (IQR: 3–7) and the median number of positive lymph nodes was 2 (IQR: 1–3), as shown in Table 1. When selected for resection margin and lymph node status, the following groups were formed: 138 patients with N0R0, 74 patients with N0R1, 56 patients with N+R0, and 57 patients with N+R1. Following the AJCC 8th edition, N2 disease was found in 20 (17.7%) patients and N1 in 93 (82.3%) patients. The location of the lymph nodes was only mentioned in 14 patients (12.4%); therefore, we were not able to include the location or specific lymph node stations in the analysis. Table 1. Baseline and postoperative characteristics according to resection margin and lymph node status. Total N0 N+ p-Value Patients n= 325 n= 212 n= 113 Age at surgery (median) 64 (58–71) 67 (58.3–72.0) 63 (57–69) 0.028 Male (%) 206 (63.4%) 135 (63.7%) 71 (62.8%) 0.880 ECOG performance status 0.901 0 143 (44.0%) 96 (45.3%) 47 (41.6%) 1 81 (24.9%) 51 (24.1%) 30 (26.5%) 2 17 (5.2%) 12 (5.7%) 5 (4.4%) 3 4 (1.2%) 3 (1.4%) 1 (0.9%) unknown 80 (24.6%) 50 (23.6%) 30 (26.5%) Bismuth–Corlette 0.931 I 18 (5.5%) 11 (5.2%) 7 (6.2%) II 20 (6.2%) 14 (6.6%) 6 (5.3%) IIIa 40 (12.3%) 26 (12.3%) 14 (12.4%) IIIb 67 (20.6%) 47 (22.2%) 20 (17.7%) IV 49 (15.1%) 31 (14.6%) 18 (15.9%) Unknown 131 (40.3%) 83 (39.2%) 48 (42.5%) CA19.9 67 (11–313) 57 (10–252) 174 (13–604) 0.070 Bilirubin 12 (2–116) 12 (2–131) 13 (3–106) 0.706 Preoperative tumor size (cm) (median) (N = 221) 2.5 (1.9–3.7) 2.3 (1.7–3.6) 2.8 (2.1–4.0) 0.022 Neoadjuvant therapy ±26 (8.0%) 17 (14.8%) 9 (16.4%) 0.789 Size and extent (T) 0.011 T1 20 (6.2%) 18 (8.5%) 2 (1.8%) T2a 80 (24.8%) 59 (28.0%) 21 (18.8%) T2b 93 (28.8%) 60 (28.4%) 33 (29.5%) T3 92 (28.5%) 50 (23.7%) 42 (37.5%) T4 31 (9.6%) 18 (8.5%) 13 (11.6%) Tx 7 (2.2%) 6 (2.8%) 1 (0.9%) Regional lymph nodes (N #) - N0 212 (65.2%) 212 (100%) 0 N1 93 (28.6%) 0 93 (82.3%) N2 20 (6.2%) 0 20 (17.7%) Total number of resected lymph nodes (median) 4 (2–7) 4 (2–6) 5 (3–7) 0.024 Number of positive lymph nodes (median) (n= 103) 2 (1–3) - 2 (1–3) - Resection margin 0.007 R0 194 (59.7%) 138 (65.1%) 56 (49.6%) R1 131 (40.3%) 74 (34.9%) 57 (50.4%) Differentiation grade 0.550 Grade 1: well differentiated 40 (12.3%) 29 (13.7%) 11 (9.7%) Grade 2: moderately differentiated 150 (46.2%) 93 (43.9%) 57 (50.4%) Grade 3: poorly differentiated 62 (19.1%) 43 (20.3%) 19 (16.8%) Not available 73 (22.5%) 47 (22.2%) 26 (23.0%) Adjuvant therapy 59 (18.2%) 34 (17.3%) 23 (23.1%) 0.213 Recurrence * 151 (46.5%) 89 (42.0%) 62 (54.9%) 0.003 Location of recurrence 0.990 Cancers 2022,14, 2389 6 of 13 Table 1. Cont. Total N0 N+ p-Value Local recurrence 54 (35.8%) 31 (34.8%) 23 (37.1%) Liver 31 (20.5%) 18 (20.2%) 13 (21.0%) Distant 63 (41.7%) 38 (42.7%) 25 (40.3%) Unknown 3 (2.0%) 2 (2.2%) 1 (1.6%) 90-day mortality 70 (21.5%) 44 (20.8%) 26 (23.0%) 0.638 p-values based on complete case analysis unless unknown is displayed. * Censored at 5-year follow-up. # According to AJCC 8th edition. ±Radiotherapy 5 ×5 Gy. 3.2. N+ versus N0 When comparing the included patients for lymph node status, the age at surgery, size and extent of the tumor, number of resected lymph nodes, resection margin, and recurrence were significantly different (Table 1). Patients with positive lymph nodes were approximately four years younger and presented with a higher tumor (T) stage. In these patients, a higher median number of lymph nodes were dissected in contrast to lymph node-negative patients (N+: 5 vs. N0: 4). The R1 resection margin (N+: 50.4% vs. N0: 34.9% ) and higher tumor recurrence rate were associated with N+ status ( N+: 54.9% vs. N0: 42.0% ). On univariable logistic regression analyses, age (OR: 0.98 (95% CI 0.96–1.00 )) and tumor size > 2.5 cm (OR: 1.62 (95% CI 0.92–2.85)) were independently associated with positive lymph nodes. However, on multivariable logistic regression analysis, these factors were not independently associated with positive lymph nodes (Table S1). 3.3. Overall Survival Out of the 325 patients, 114 patients were alive and 211 had died at final follow-up. The median follow-up of patients alive at last medical visit was 59 months (95% CI 57.6–60.4). Out of the 325 patients, 59 (18%) patients received adjuvant (chemo)therapy. This concerned chemotherapy in 52 (16%) patients, chemo–radiotherapy in four (1%), and radiotherapy in one (0.3%) patient. For two patients, this was unknown. The median and 5-year OS for the whole group was 26 months (95% CI 21.0–31.0) and 29%, respectively. The median and 5-year OS divided for lymph node status were 34 months (95% CI 27.9–40.1) and 36% for N0 patients, and 15 months (95% CI 10.6–19.4) and 18% for N+ patients (p< 0.001; Figure 2a). The median and 5-year OS divided for resection margin was 31 months (95% CI 23.0–39.0) and 34% for R0 disease, and 21 months (95% CI 14.9–27.2) and 23% for R1 disease (p< 0.05; Figure 2b). Including both resection margin and lymph node status (N0R0, N0R1, N+R0 and N+R1), the median and 5-year OS were 38 months (95% CI 28.0–48.0) and 36%, 30 months (95% CI 19.8–40.2) and 36%, 18 months (95% CI 10.7–25.3) and 30%, and 12 months (95% CI 7.7–16.3) and 8%, respectively ( p< 0.001 , Figure 2c). The comparison per status was as follows: N0R0 vs. N+R0: p= 0.064, N0R1 vs. N+R1: p< 0.001, N+R0 vs. N+R1: p= 0.061, N0R0 vs. N0R1: p= 0.61. The prognostic factors for OS were analyzed using a uniand multivariable Cox regression analysis. On univariable analysis, ECOG performance status, lymph node status, resection margin, and tumor differentiation grade were negatively associated with OS ( p< 0.1 ). After multivariable Cox regression analysis, ECOG performance status (ECOG 2: HR 2.95 (95% CI 1.55–5.63)), positive lymph nodes (N1: HR 1.92 (95% CI 1.29–2.85)), and tumor differentiation (poorly differentiated: HR 1.80 (95% CI 1.14–2.84)) were identified as independent prognostic factors for OS (p< 0.05; Table 2). Cancers 2022,14, 2389 7 of 13 Cancers 2022, 14, x FOR PEER REVIEW 7 of 14 Figure 2. ( a ): Kaplan–Meier curve for overall survival and lymph node-negative (N0) and lymph node-positive (N+) patients. Only the first 5 years are displayed. Median OS was 34 months (95% CI 27.9–40.1) for the N0 versus 15 months (95% CI 10.6–19.4) for the N1 patients (p< 0.001). (b): Kaplan–Meier curve for overall survival of resection margin-negative (R0) and resection marginpositive (R1) patients. Only the first 5 years are displayed. Median OS was 31 months (95% CI 23.0–39.0) for R0 versus 21 months (95% CI 14.9–27.2) for R1 patients (p= 0.037). ( c ): Kaplan–Meier curve for overall survival of N0R0, N0R1, N+R0, and N+R1 patients. Only the first 5 years are displayed. Median OS was 38 months (95% CI 28.0–48.0) for N0R0, 30 months (95% CI 19.8–40.2) for N0R1, 18 months (95% CI 10.7–25.3) for N+R0, and 12 months (95% CI 7.7–16.3) for the N+R1 patients (p< 0.001). N0R0 vs. N+R0: p= 0.064, N0R1 vs. N+R1: p< 0.001, N+R0 vs. N+R1: p= 0.061, N0R0 vs. N0R1: p= 0.61. Cancers 2022,14, 2389 8 of 13 Table 2. Uniand multivariable Cox regression analysis for overall survival. Univariable Analysis Multivariable Analysis HR (95% CI) p-Value $HR (95% CI) p-Value # Age at surgery 1.01 (0.99–1.02) 0.287 ECOG performance status ECOG 0 Reference Reference ECOG 1 1.21 (0.84–1.74) 0.297 1.41 (0.92–2.15) 0.114 ECOG 2 1.80 (1.01–3.20) 0.045 2.95 (1.55–5.63) 0.001 ECOG 3 3.39 (1.23–9.36) 0.018 2.73 (0.84–8.87) 0.096 Tumor size > 2.5 cm 1.07 (0.75–1.52) 0.705 CA19.9 > 37 1.02 (0.62–1.69) 0.932 Lymph node status (N) * N0 Reference Reference N1 1.72 (1.27–2.33) 0.001 1.92 (1.29–2.85) 0.001 N2 1.97 (1.15–3.39) 0.014 1.21 (0.55–2.63) 0.640 Resection margin (R) R0 Reference Reference R1 1.34 (1.01–1.78) 0.042 1.14 (0.78–1.67) 0.501 Tumor differentiation Well differentiated (G1) 0.67 (0.41–1.07) 0.094 0.69 (0.41–1.17) 0.168 Moderately differentiated (G2) Reference Reference Poorly differentiated (G3) 2.39 (1.41–4.04) 0.013 1.80 (1.14–2.84) 0.011 * According to AJCC 8th edition, $p< 0.100, #p< 0.05, p-values based on complete case analysis. 3.4. Recurrence During follow-up, 156 (48%) patients developed recurrence. The recurrence status at final follow-up was unknown for 52 (16%) patients. Recurrence occurred most often distant from the primary tumor site (n= 63, 41.7%), being mostly found in the peritoneum ( n= 28, 49.1% ; Table 1). From the patients who had recurrence, 32 (21%) patients received and 120 (77%) patients did not receive adjuvant (chemo)therapy. For four patients, this was unknown. The overall median recurrence-free survival was 23 months (95% CI 19.2–26.8). The recurrence-free survival divided for resection margin and lymph node status (N0R0, N0R1, N+R0, and N+R1) was 27 months (95% CI 20.4–33.6), 33 months (95% CI 19.5–46.5), 14 months (95% CI 7.4–20.6), and 11 months (95% CI 5.7–16.3), respectively (p< 0.001; Figure 3). The comparison per status was: N0R0 vs. N+R0: p= 0.151, N0R1 vs. N+R1: p< 0.001, N+R0 vs. N+R1: p= 0.109, N0R0 vs. N0R1: p= 0.331. The prognostic factors for recurrence-free survival were also analyzed using a uniand multivariable Cox regression analysis. On univariable analysis, lymph node status, T stage (AJCC 8th), and tumor differentiation were negatively associated with OS (p< 0.1). After multivariable Cox regression analysis, positive lymph nodes (N1: HR 2.62 (95% CI 1.68–4.08)) and tumor differentiation (poorly differentiated: HR 1.65 (95% CI 1.05–2.58)) were identified as independent prognostic factors for recurrence (p< 0.05; Table 3). Table 3. Uniand multivariable Cox regression analysis for recurrence-free survival. Univariable Analysis Multivariable Analysis HR (95% CI) p-Value $HR (95% CI) p-Value # Tumor size > 2.5 cm 1.25 (0.82–1.90) 0.303 T status (T) T1 1.12 (0.56–2.23) 0.754 1.60 (0.61–4.20) 0.336 T2a 0.53 (0.34–0.84) 0.007 0.60 (0.36–1.01) 0.056 T2b 1.12 (0.73–1.73) 0.594 1.27 (0.76–2.12) 0.367 T3 Reference Reference T4 1.01 (0.54–1.86) 0.988 1.04 (0.52–2.11) 0.908 Cancers 2022,14, 2389 9 of 13 Table 3. Cont. Univariable Analysis Multivariable Analysis HR (95% CI) p-Value $HR (95% CI) p-Value # Lymph node status (N) * N0 Reference Reference N1 1.72 (1.20–2.47) 0.003 2.62 (1.68–4.08) <0.001 N2 1.86 (0.99–3.50) 0.055 1.249 (0.70–3.17) 0.303 Resection margin (R) R0 Reference R1 0.99 (0.71–1.38) 0.940 Tumor differentiation Well differentiated (G1) 0.98 (0.57–1.70) 0.946 0.98 (0.57–1.70) 0.416 Moderately differentiated (G2) Reference Reference Poorly differentiated (G3) 1.65 (1.05–2.58) 0.029 1.65 (1.05–2.58) 0.001 * According to AJCC 8th, $p< 0.100, #p< 0.05, p-values based on complete case analysis. Cancers 2022, 14, x FOR PEER REVIEW 9 of 14 N0R1, N+R0, and N+R1) was 27 months (95% CI 20.4–33.6), 33 months (95% CI 19.5–46.5), 14 months (95% CI 7.4–20.6), and 11 months (95% CI 5.7–16.3), respectively (p < 0.001; Figure 3). The comparison per status was: N0R0 vs. N+R0: p = 0.151, N0R1 vs. N+R1: p < 0.001, N+R0 vs. N+R1: p = 0.109, N0R0 vs. N0R1: p = 0.331. Figure 3. Estimated cumulative recurrence probability of N0R0, N0R1, N+R0, and N+R1 patients. Only the first 5 years are displayed. The median recurrence-free survival was 27 months (95% CI 20.4–33.6) for N0R0, 33 months (95% CI 19.5–46.5) for N0R1, 14 months (95% CI 7.4–20.6) for N+R0, and 11 months (95% CI 5.7–16.3) for the N+R1 patients (p < 0.001). N0R0 vs. N+R0: p = 0.151, N0R1 vs. N+R1: p < 0.001, N+R0 vs. N+R1: p = 0.109., N0R0 vs. N0R1: p = 0.331. The prognostic factors for recurrence-free survival were also analyzed using a uniand multivariable Cox regression analysis. On univariable analysis, lymph node status, T stage (AJCC 8th), and tumor differentiation were negatively associated with OS (p < 0.1). After multivariable Cox regression analysis, positive lymph nodes (N1: HR 2.62 (95% CI 1.68–4.08)) and tumor differentiation (poorly differentiated: HR 1.65 (95% CI 1.05–2.58)) were identified as independent prognostic factors for recurrence (p < 0.05; Table 3. Table 3. Uniand multivariable Cox regression analysis for recurrence-free survival. Univariable Analysis Multivariable Analysis HR (95% CI) p -Value $ HR (95% CI) p -Value # Tumor size > 2.5 cm 1.25 (0.82–1.90) 0.303 T status (T) T1 1.12 (0.56–2.23) 0.754 1.60 (0.61–4.20) 0.336 T2a 0.53 (0.34–0.84) 0.007 0.60 (0.36–1.01) 0.056 T2b 1.12 (0.73–1.73) 0.594 1.27 (0.76–2.12) 0.367 T3 Reference Reference T4 1.01 (0.54–1.86) 0.988 1.04 (0.52–2.11) 0.908 Lymph node status (N) * N0 Reference Reference N1 1.72 (1.20–2.47) 0.003 2.62 (1.68–4.08) <0.001 N2 1.86 (0.99–3.50) 0.055 1.249 (0.70–3.17) 0.303 Resection margin (R) R0 Reference R1 0.99 (0.71–1.38) 0.940 Tumor differentiation Figure 3. Estimated cumulative recurrence probability of N0R0, N0R1, N+R0, and N+R1 patients. Only the first 5 years are displayed. The median recurrence-free survival was 27 months (95% CI 20.4–33.6) for N0R0, 33 months (95% CI 19.5–46.5) for N0R1, 14 months (95% CI 7.4–20.6) for N+R0, and 11 months (95% CI 5.7–16.3) for the N+R1 patients (p< 0.001). N0R0 vs. N+R0: p= 0.151, N0R1 vs. N+R1: p< 0.001, N+R0 vs. N+R1: p= 0.109, N0R0 vs. N0R1: p= 0.331. 4. Discussion This study shows that for patients who undergo a resection for pCCA, positive lymph nodes have a major negative effect on recurrence and survival. Surprisingly, this was independent of resection margin status. In addition, ECOG performance status, positive lymph nodes, and the grade of tumor differentiation were found as the main independent indicators of oncological outcome. These findings suggest that tumor biology and performance status have a higher impact on survival than extended or radical surgery. Several studies describe the outcomes of patients with resected pCCA. However, most of these studies are single-center or single-country studies and often only include small numbers of patients. The present analysis is the first study from the ENSCCA registry addressing resected pCCA. The registry represents a large cohort, with patients from multiple expert centers across Europe. Previous studies reported comparable differences between the survival rates for patients with and without lymph node involvement [ 4 , 6 , 19 ]. For patients with locally advanced disease who do not undergo resection, median OS rates of 12 months are reported. With the OS for N+ between 12 and 18 months and the mortality rate of 23% found in this