The Impact of Metastatic Lymph Node Size on Long-Term Outcomes Following Curative Colectomy for Pathological Stage III Colon Cancer

Background: In node-positive colon cancer, the impact of MLN size on prognosis is controversial. The aim of this study was to clarify the impact of metastatic lymph node (MLN) size on long-term outcomes in patients undergoing curative resection for pStage III colon cancer. Methods: This study enrolled patients who underwent curative colectomy for pStage III colon cancer between January 2013 and December 2015. All eligible patients were divided into four groups based on the short-axis diameter of the largest MLN: Group A, < 5 mm; Group B, ≥ 5 mm and < 10 mm; Group C, ≥ 10 mm and < 15 mm; and Group D, ≥ 15 mm. We performed univariate and multivariate analysis using Cox proportional hazard regression models to identify clinicopathological factors affecting recurrence-free survival (RFS). Results: A total of 209 patients were analyzed. We evaluated 7305 LNs, of which 644 were metastatic. The 5-year RFS rates of Groups A, B, C, and D were 82.3%, 74.6%, 74.5%, and 60.7%, respectively. In univariate analysis, age older than 70 years, Group D (largest MLN ≥ 15 mm), and the absence of adjuvant chemotherapy were signicantly associated with RFS. In multivariate analysis, Group D (hazard ratio [HR], 3.95; 95% condence interval [CI], 1.34–11.65; p=0.01) and the absence of adjuvant chemotherapy (HR, 2.44; 95% CI, 1.26-4.72; p<0.01) were independently associated with worse RFS. Conclusion: A maximum MLN ≥ 15 mm was signicantly associated with worse RFS in stage III colon cancer. Bulky MLNs might be a poor prognostic factor in node-positive colon cancer.


Introduction
Lymph node (LN) metastasis is one of the most important prognostic factors in patients undergoing curative resection for colon cancer [1]. To assess the degree of LN metastasis, it is essential to determine the number of metastatic LNs (MLNs); this number was incorporated into the TNM staging system both in the Union for International Cancer Control and in the American Joint Committee on Cancer [2,3].
Furthermore, multiple studies demonstrated that the number of LNs examined was associated with longterm outcomes in colon cancer because stage migration was avoided by harvesting a large number of LNs [4][5][6]. Little is known about how LN size in uences prognosis. In node-negative colon cancer, large non-MLNs were reported to be associated with better prognosis due to a strong immune response [7][8][9][10]. In node-positive colon cancer, the impact of MLN size on prognosis is controversial because few studies have examined this question, and their results differed in terms of whether MLN size affected long-term outcomes [11,12]. This study was conducted to clarify the impact of MLN size on long-term outcomes in patients undergoing curative resection for pathological stage III colon cancer.

Patient selection
Patients who underwent curative colectomy for primary pathological stage III colon cancer at Shizuoka Cancer Center in Japan between January 2013 and December 2015 were included. The exclusion criteria were synchronous or metachronous colorectal cancer and concomitant surgical procedures for other cancers. Patient characteristics and pathological and surgical ndings were recorded in a prospective database. Data collection and analysis were approved by the institutional review board of Shizuoka Cancer Center Hospital (institutional code: J2019-135). The cecum, ascending colon, and transverse colon were classi ed as the right-sided colon, and the descending colon and sigmoid colon were classi ed as the left-sided colon. Pathological T or N stage was classi ed according to the tumor node metastasis (TNM) classi cation system [2].

Measurement of lymph node size
After removal of fresh specimens, colorectal surgeons manually identi ed all LNs. The short-axis diameter of each dissected LN was measured, and the LN was xed in 10% buffered formalin for 24 hours; the pathological diagnosis was recorded for each dissected LN, as previously reported [13][14][15]. All eligible patients were divided into four groups based on the short-axis diameter of the largest MLN: Group A, < 5 mm; Group B, ≥ 5 mm and < 10 mm; Group C, ≥ 10 mm and < 15 mm; and Group D, ≥ 15 mm. Clinicopathological characteristics and long-term outcomes were compared between groups. surgery was performed only when laparoscopic surgery was judged unsuitable for ensuring oncological safety or when patients refused laparoscopic surgery. In principle, 5-uorouracil-based adjuvant chemotherapy was administered to patients younger than 75 years who did not have any severe comorbidity.

Surveillance protocol
Surveillance was performed for 5 years after surgery. The surveillance protocol at our institution was as follows: an interview, physical examination, and blood tests, including carcinoembryonic antigen and cancer antigen 19 − 9, were performed every 3 months for the rst 3 years after surgery and then every 6 months thereafter. Chest, abdominal, and pelvic CTs were performed every 6 months. Colonoscopy was performed 1, 3, and 5 years after surgery. Recurrence was con rmed by pathological assessment or by progressively increasing tumor size in imaging studies.

Statistical analysis
Categorical variables are presented as numbers (percentages). Continuous variables are presented as medians (range). Categorical variables were compared using the chi-squared test and continuous variables were compared using the Mann-Whitney U test. Overall survival (OS) and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method and compared using the log-rank test. We performed univariate and multivariate analyses using the Cox proportional hazards regression model to identify clinicopathological factors affecting RFS. All statistical analyses were performed with the statistical program R version 1.40 (http://www.r-project.org/). A p value less than 0.05 was considered statistically signi cant.

Patient characteristics
A total of 216 patients underwent curative colectomy for primary pathological stage III colon cancer between January 2013 and December 2015. Patients who had synchronous or metachronous colorectal cancer (n = 5) and who underwent concomitant surgical procedures for other cancers (n = 2) were excluded. The remaining 209 patients were analyzed. We evaluated a total of 7305 LNs, of which 644 were metastatic. The median short-axis diameter of MLNs was 5 mm (range 1-40 mm) and the median short-axis diameter of the largest MLN in each patient was 8 mm (range, 1-40 mm). Among the eligible patients, 40 (19%), 79 (38%), 51 (24%), and 39 (19%) were categorized into Groups A, B, C, and D, respectively. The patient characteristics are presented in Table 1. There were no signi cant differences between Group A and the other Groups in terms of age, sex, operative approach, histology, or number of dissected LNs. The proportion of patients with right-sided colon cancer was higher in Group D than in Group A, the median tumor size was signi cantly larger in Groups C and D than in Group A, the proportion of patients undergoing D3 dissection was higher in Groups B, C, and D than in Group A, and the proportion of patients with T3 cancers was higher in Group D than in Group A. The proportion of patients with N2 cancers and the number of MLNs were signi cantly higher in Groups B, C, and D than in Group A. The proportion of patients who underwent adjuvant chemotherapy was higher in Group C than in Group A.

Long-term outcomes
The median follow-up time was 54 months. Figures 1 and 2 show the OS and RFS curves of each Group, respectively. Table 2 shows the 5-year OS and RFS rates of each Group. There were no signi cant differences in OS rates between Groups. The RFS rate in Group D was signi cantly lower than that in Group A. The RFS rates in Groups B and C were also lower than that in Group A, although the differences were not signi cant. A larger maximum MLN diameter was associated with a lower RFS.   [11,12]. One, which used a cutoff value of 5 mm to de ne a large MLN, reported that there were no signi cant differences in OS or disease-free survival (DFS) between patients with large and small MLNs [11]. The other, which used a cutoff value of 10 mm, demonstrated that patients with large MLNs had worse OS and DFS than those with small MLNs, and large MLN diameter was identi ed as an independent poor prognostic factor in a multivariate analysis of DFS [12]. Based on these results, patients in our study were classi ed into four groups to more precisely evaluate the relationship between MLN diameter and prognosis. Schrembs et al. analyzed the association between OS and the LN metastasis to LN size ratio (MSR) and demonstrated that smaller MSR values correlated with longer OS [17]; that is, a high proportion of tumor components in MLNs was associated with worse prognosis. In our study, a maximum MLN diameter ≥ 15 mm was signi cantly associated with worse prognosis; therefore, most of the normal lymphatic tissue might be replaced by tumor cells in bulky MLNs. Furthermore, in gastric cancer and esophageal cancer, MLN size was also reported to be a poor prognostic factor [18][19][20]. MLN size might re ect malignant potential in gastrointestinal cancers.
The absence of adjuvant chemotherapy was also associated with worse RFS in the multivariate analysis in this study. In pathological stage III colon cancer, adjuvant chemotherapy for 6 months is recommended by the Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines [16]. Recent large studies reported that adjuvant chemotherapy for 3 months was noninferior to 6 months in terms of disease-free survival in low-risk stage III colon cancer, including T1, T2, T3, and N1 disease [21,22].
However, even for this type of cancer, adjuvant chemotherapy for 6 months might be optimal if MLNs are bulky.
There are several limitations to this study. First, this was a retrospective study that was conducted at a single institution and that enrolled a relatively small number of patients. That might be why there were no signi cant differences in RFS between Group A (MLN < 5 mm) and Group B (MLN ≥ 5 mm and < 10 mm) or Group C (MLN ≥ 5 mm and < 10 mm). Second, in both univariate and multivariate analyses of RFS, there were no signi cant differences in N stage, which has been considered to be the most powerful independent prognostic factor in stage III colon cancer [23][24][25]. The reason for this discrepancy is unclear. The proportion of N2 cancers was signi cantly higher in Group D (MLN ≥ 15 mm) than in Group A (MLN < 5 mm), which might have resulted in the difference in RFS between the two groups. Therefore, multivariate analysis that included both the N stage and MLN size was performed to minimize this bias.
In conclusion, our ndings indicate that a maximum MLN ≥ 15 mm was signi cantly associated with worse RFS in stage III colon cancer. Bulky MLNs might be a poor prognostic factor in node-positive colon cancer.

Declarations
Ethics approval This study was approved by the institutional review board of Shizuoka Cancer Center hospital (Institutional code, J2019-135).
Consent to participate Informed consent was obtained from all individual participants included in the study.
Consent for publication Not applicable.
Availability of data and material All relevant data are available on request due to privacy/ethical restrictions.
Competing interests The authors declare that they have no competing interest.
Funding The authors declare that they received no funding support for this study. Recurrence-free survival MLN, metastatic lymph node