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Impact of the number of examined lymph nodes on outcomes in patients with lymph node-negative gallbladder carcinoma

AIM To determine whether the number of examined lymph nodes (LNs) is correlated with the overall survival of gallbladder carcinoma (GBC) patients. METHODS Patients were collected from the Surveillance Epidemiology and End Results database (2004-2013) and categorized by the number of LNs into six groups: 1 LN, 2 LNs, 3 LNs, 4 LNs, 5 LNs, and ≥ 6 LNs. Survival curves for overall survival were plotted with a Kaplan-Meier analysis. The log-rank test was used for univariate comparisons. RESULTS In a cohort of 893 patients, the median number of examined LNs was two for the entire cohort. The survival for the 1 LN group was significantly poorer than those of the stage I and II disease groups and for the entire cohort. By dichotomizing the number of LNs from 1 to 6, we found that the minimum number of LNs that should be examined was four for stage I, four or five for stage II, and six for stage IIIA disease. Therefore, for the entire cohort, the number of examined LNs should be at least six, which is exactly consistent with the American Joint Committee on Cancer criteria. CONCLUSION The examination of higher numbers of LNs is associated with improved survival after resection surgery for N0 GBC. The guidelines for GBC surgery, which recommend that six LNs be examined at least, are statistically valid and should be applied in clinical practice widely.


INTRODUCTION
Gallbladder carcinoma (GBC) is one of the most lethal carcinomas and has a poor prognosis [1][2][3] . To date, surgery remains the only radical treatment strategy for patients, translating into 5-year survival rates of approximately 5% [4][5][6][7] . Lymph node (LN) status is an important prognostic factor for GBC patients [8] . Unfortunately, LN metastases occur in more than 50% of patients, and LN-positive patients are widely known to have very poor survival [4] . The role of regional and extended lymphadenectomy for GBC has been previously investigated [9][10][11][12] , but there is not a general consensus about the number of LNs that should be examined. In the 8 th edition of the tumornode-metastasis (TNM) staging system for GBC from the American Joint Committee on Cancer (AJCC), the N category was defined by the number of metastatic LNs instead of the location of the metastatic LNs, as used in the previous edition, and was correlated with prognosis. These guidelines recommend examining a minimum of six LNs to accurately classify patients with GBC [13] . Thus, this study aimed to assess patients with LN-negative (N0) GBC to determine whether the number of examined LNs was correlated with overall survival of GBC patients. We used the Surveillance, Epidemiology, and End Result (SEER) database to determine the influence of the number of examined LNs on prognosis in patients with N0 GBC.

Patients
The SEER database (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013) was used to identify patients with GBC. Patients who met the following criteria were included: (1)  The clinical characteristics of the entire cohort and patients with stage Ⅰ, Ⅱ, and ⅢA disease are listed in Table 1. There was no difference among patients with stage Ⅰ, Ⅱ, or ⅢA disease in terms of age, sex, race, and number of LNs examined. In addition, compared with patients with stage Ⅰ and Ⅱ disease, a larger proportion of patients with stage ⅢA disease had poor/ undifferentiated tumors and received radiation therapy.
The median number of examined LNs was 2 for the entire cohort, 1 LN for the stage Ⅰ group, 2 LNs for the stage Ⅱ group, and 2 LNs for the stage ⅢA group. More than 40% of the patients had only 1 LN examined, and a lower proportion of patients had more LNs examined ( Figure 1). The number of examined LNs did not differ by stage (P = 0.59).
Patients were categorized by the number of examined LNs into the following 6 groups: 1 LN, 2 LNs, 3 LNs, 4 LNs, 5 LNs and ≥ 6 LNs. Survival in relation to the number of examined LNs was assessed separately for the entire cohort and patients with stage Ⅰ, Ⅱ and ⅢA disease ( Table 2). For the entire cohort, a median survival of 18 mo and a 5-year survival rate of 0.393 were noted for patients with one LN examined (n = 398). The survival for the 1 LN group was significantly poorer than that of the other groups (P < 0.001, Figure 2). However, there was no difference in survival among the other five groups (P > 0.05). For patients with stage Ⅰ disease, the median survival for the 1 LN, 2 LNs, 3 LNs, 4 LNs, 5 LNs, and ≥ 6 LNs groups was 24, 43, 30, 22, 38, and 26 mo, respectively. Similar survival results according to the LN groups were demonstrated for patients with stage Ⅰ and Ⅱ disease but not for patients with stage ⅢA disease (Table 2). However, compared with patients with stage Ⅰ and Ⅱ disease, the median survival and 5-year survival rate of patients with stage ⅢA disease was obviously decreased in all the LN groups. For example, the 5-year survival rate in the 1 LN group was 0.473 for stage Ⅰ, 0.445 for stage Ⅱ, and 0.177 for stage ⅢA disease. As shown in Table 3, there was no difference in  LN numbers varied among the three stages. The largest survival difference was observed at 4 LNs for stage Ⅰ disease (P = 0.004; Figure 3A), at 4 or 5 LNs for stage Ⅱ disease (P < 0.001 for both; Figure 3B and C), and at 6 LNs for stage ⅢA disease (P = 0.019; Figure 3D). For the entire cohort, the optimal number of examined LNs was 4, 5, or 6 (P < 0.001 for all; Figure 3E-G).
A stepwise Cox regression identified race and sex as significant prognostic factors for the entire cohort (Table  3); however, race was not a significant factor for patients with stage Ⅱ and ⅢA disease. Grade was a significant prognostic factor for patients with stage Ⅰ, Ⅱ, and ⅢA disease but not for the entire cohort; and radiation therapy was a significant prognostic factor only for patients with stage ⅢA disease.

DISCUSSION
GBC is associated with a high incidence of invasion through the layers of the gallbladder wall into adjacent structures and LNs. The influence of LN metastases on primary GBC is supported by one series of reports, which showed that the 5-year survival rate of T1N0 patients was 33% compared with a 3% survival rate in T1N1 patients [14] . As a consequence, several large-scale studies were conducted to examine the role of extended LN dissection to determine whether the removal of survival between the stage Ⅰ and Ⅱ groups (HR: 1.089, 95%CI: 0.793-1.497, P = 0.598 for stage Ⅱ, referred to stage Ⅰ), but the survival of patients with stage ⅢA disease was significantly lower than that of patients with stage Ⅰ disease (HR: 3.730, 95%CI: 2.635-5.280, P < 0.0001).
To identify the cutoff point for the optimal number of examined LNs, we compared the survival of the entire cohort with stage Ⅰ, Ⅱ and ⅢA groups with X-tile software. The ranges for the significant dichotomization   additional LN basins would influence the survival of GBC patients [9,15] . Early retrospective reports suggested improved survival for late-stage GBC patients treated with extended regional lymphadenectomy compared with standard regional lymphadenectomy [9] . However, the minimum clearance and/or number of LNs that should be examined have yet to be established. In the study, we evaluated the impact of the number of examined LNs on survival in N0 GBC. Using the SEER database, we discovered that the median number of examined LNs was two for the entire cohort, one for stage Ⅰ, two for stage Ⅱ, and two for stage ⅢA disease. We used the smallest median value, 1 LN, as the basis for our categorization of the SEER patient cohort into six groups that reflected the extent of lymphadenectomy: 1 LN, 2 LNs, 3 LNs, 4 LNs, 5 LNs, and ≥ 6 LNs. There was a significant difference in survival among the six groups for the entire cohort and for the stage Ⅰ and Ⅱ groups but not for the stage ⅢA group. With X-tile software, we found that the minimum number of LNs that should be examined was four for stage Ⅰ, four or five for stage Ⅱ, and six for stage Ⅲ A disease. Therefore, for the entire cohort, the number of examined LNs should be at least six, which is exactly consistent with the AJCC guidelines.
The general phenomenon that the more LNs are examined, the better the survival for N0 disease, has some potential explanations. For example, the final LN count may be a proxy for surgeon experience and surgical technique and may be reflective of more thorough pathological assessment and identification of nodes from the surgical specimen [16] . It is also related to the concept of stage migration, where inadequate removal of LNs may result in the misclassification of LN-positive patients as N0 [17] . However, the removal of too many LNs may result in side effects such as lymphatic leakage.
Our results were consistent with those of previous studies that investigated the relationship between LN count and survival in GBC patients and demonstrated that at least six LNs should be examined to improve survival after resection surgery [18] . Notably, except for LN dissection, nerve dissection may be required, especially for T3 or T4 disease [19] .
In conclusion, the analysis suggests that examining higher numbers of LNs is associated with improved survival after resection surgery in N0 GBC. As recommended in the AJCC guidelines, at least six LNs should be examined for patients with N0 GBC.

Research background
The American Joint Committee on Cancer (AJCC) tumor-node-metastasis staging system for gallbladder carcinoma (GBC) has been updated recently to the 8 th edition. The N category is re-defined by the number of metastatic lymph nodes (LNs) instead of the location of the metastatic LNs, as defined in the 7 th edition.

Research motivation
The new staging system for GBC has not been validated yet. Thus, we used Surveillance, Epidemiology, and End Result (SEER) database to evaluate its impact on clinical practice.

Research objectives
The primary purpose of this study was to evaluate the impact of the number    of examined LNs on the prognosis of N0 GBC. The secondary purpose was to verify the rationality of the guideline recommendation that at least six LNs should be harvested and evaluated.

Research results
The survival for the 1 LN group was significantly lower than that of the stage Ⅰ and Ⅱ disease groups and for the entire cohort. By dichotomizing the number of LNs from one to six, we found that the minimum number of LNs that should be examined was four for stage Ⅰ, four or five for stage Ⅱ, and six for stage ⅢA disease. Thus, at least six LNs should be examined for the entire cohort, which was exactly consistent with the AJCC criteria.

Research conclusions
The examination of higher numbers of LNs is associated with improved survival after resection surgery for N0 GBC. As recommended in the guidelines, at least six LNs should be examined for patients with N0 GBC.

Research perspectives
The results validated the new recommendation in the AJCC guidelines, which can be applied widely in clinical practice.