Effect of Lymph Node Count on Pathological Stage III Rectal Cancer with Preoperative Radiotherapy

Lymph node (LN) status after surgery for rectal cancer is affected by preoperative radiotherapy. The purpose of this study was to perform a population-based evaluation of the impact of pathologic LN status after neoadjuvant radiotherapy on survival. A total of 1,650 patients receiving neoadjuvant chemotherapy in Surveillance, Epidemiology, and End Results Program (SEER)-registered ypIII stage rectal cancer was analyzed. We identified the optimal cutoff for retrieved LNs as 10 (χ2 = 14.006, P < 0.001), which was validated as an independent prognosis factors in a Cox regression model. Further analysis showed that the LN count was only a prognosis factor with the number from 8 to 16(except for 13).After the number 16, the 5-year survival rate decreased gradually. Collectively, our results confirmed that the number of LNs in yp III stage rectal patients was a prognosis factor only with the numbers from 8 to 16(except for 13). Using the total mesorectal excision technique with an adequate pathologic examination, a large number of LNs retrieved (≥17) might indicate worse tumor response grade and poorer survival.


Discussion
The presence of LN metastases in colorectal cancer is well recognized as one of the most important prognostic factors for long-term outcome 9,10 . The total number of LNs retrieved is fundamental in most pathological staging systems for colorectal cancer, including the American Joint Committee on Cancer (AJCC), modified Dukes and Astler and Coller. Inadequate LN evaluation is associated with worse outcomes in terms of tumor recurrence and patient survival, particularly in patients with stage II colorectal cancer [11][12][13][14] . The World Congress of Gastroenterology proposed examining a minimum of 12 LNs to classify stage II colorectal tumors 15   X-tile analysis was performed using patient data, which were equally divided into training and validation sets, from the SEER registry. X-tile plots of the training sets are shown in the left panels, with plots of matched validation sets shown in the smaller inset. The optimal cut-point highlighted by the black circle in the left panels is shown on a histogram of the entire cohort (middle panels), and a Kaplan-Meier plot (right panels). P values were determined using the cutoff point defined in the training set and applying it to the validation set. Figure 1 shows the optimal cutoff point for the ypN (+ ) patients (10, χ 2 = 14.006, P < 0.001). 5-year overall survival if > 40 LNs were analyzed compared with ≤ 10 LNs (P < 0.001); and in patients with N2 stage, the 5-year overall survival rates following analysis of > 35 and < 35 LNs were 71% and 51%, respectively (P = 0.002) 11 . Vather et al. reported that the mean numbers of LNs examined in stage III patients who died or were alive within 5 years was 13.1 vs 14.8, respectively, and this difference was statistically significant (P < 0.001) 17 . Chen et al. showed that the median survival times for colon cancer patients with 1-7, 8-14 and ≥ 15 LN retrieval were 46, 52 and 67 months, respectively (P < 0.001) 18 . However, several studies failed to demonstrate a similar association between survival and LNs harvest in stage III disease 13,14,19,20 . Note that our previous study showed that negative LN count, which does not take positive LN into consideration, was an independent prognostic factor for ypIIIB and ypIIIC rectal cancer patients 21 . However, the relationship between LN count and RCSS in ypIII rectal cancer has not been fully investigated. In all present guidelines for rectal cancer clinical practice, total LN count is the main concern. Hence, in this study, we mainly focused on the prognostic significance of the total LNs count in patients with rectal cancer treated with preoperative radiotherapy. We first used X-tile to identify 10 as the optimal cutoff value, and then it was confirmed as one of the optimal cutoff numbers in an additional one-by-one cutoff value analysis from 5 to 19. The 5-year RCSS rates of patients with N (cutoff number) or more nodes gradually increased when N ranged from 5 to 9, which suggested that inadequate LN retrieval in LN positive rectal cancer patients may also reflect the failure to remove the involved LNs, particularly in IIIB and IIIC stage patients in our study, thus increasing the risk of local recurrence and distant metastasis. In addition, it may be a marker of poor quality surgical or pathologic care, both of Total lymph node count No.    27 . We also found that with a cutoff of less than 7, the number of LNs retrieved was not a prognostic factor. Decreased LN retrieval might reflect improved response to preop-RT rather than inappropriate or suboptimal surgical resection in this setting. Most previously published articles determined LN cutoffs considering only a single value, while they barely used one-by-one analysis. In our study, we found that if the cutoff value was greater than 16, the 5-year RCSS rate in patients with a greater number of LNs (i.e., more than the cutoff) would decrease gradually, losing its prognosis value after the number 17(P > 0.05). Several hypotheses could explain this finding. First, the fact that   harvesting a higher total number of LNs may result in higher detection rate of metastatic LNs with resultant upstaging of cancer, -a widely accepted concept in surgery; Second, previous study showed that greater LN retrieval could indicate a worse preop-RT response 25 . On average, 5 more LNs were retrieved from ypN2 stage patients compared with ypN1 stage patients (15 VS 10), and a poorer tumor regression score results in shorter RCSS rates [25][26][27] . This large population-based study has several potential limitations. First, the SEER database does not include information on the administration of chemotherapy. It is possible that additional or concurrent chemotherapy decreases the number of LNs more than preop-RT alone. Second, individual surgeons, pathologists, and other factors may affect LN harvest, but we cannot adjust for these factors. Finally, SEER database also does not report data on preoperative clinical stage, or tumor regression score, so we cannot analyze the relationship between tumor regression score and LNs retrieval in this study.

5-year CCS
In conclusion, our analysis of the SEER database revealed that the number of LNs retrieved was a prognostic factor only for numbers ranging from 8 to 16(except for 13) with a maximum χ 2 log-rank value of 14.006 at a cutoff of 10. The good tumor response associated with a reduced number of examined  LNs may offset the influence of potential under staging with a cutoff of less than 7. Increased LN retrieval (≥ 17) could indicate worse preop-RT response and poorer RCSS.

Patient selection in the SEER database. Data from the Surveillance Epidemiology and End Results
(SEER) Program of the United States National Cancer Institute, release 2015, were utilized for this study. SEER, a population-based cancer registry, collects cancer incidence and survival data from 18 regional population-based registries covering approximately 26% of the US population. The SEER data contain no identifiers and are publicly available for studies of cancer-based epidemiology and TNM staging of colorectal 22,28 , gastric 29 , esophageal cancer 30 and other cancers. SEER registry patients eligible for this cohort included those with adenocarcinoma, NOS (8150/3), adenocarcinoma in adenomatous polyps (8210/3), villous adenoma (8261/3) or tubulovillous adenoma (8263/3), mucinous adenocarcinoma (8480/3), signet ring cell carcinoma (8490/3) of the rectum diagnosed from 1998 through 2005 and treated with surgical resection. The inclusive study dates were chosen to allow a known treatment sequence of "radiation prior to surgery". The patient age was limited to 18 to 80 years old. Patients diagnosed after 2006 were excluded to ensure adequate follow-up times. Additional exclusion criteria were as follows: no LNs examined pathologically, ypN0 patients, with synchronous distance metastases, and patients who died within 30 postoperative days.
The following information was retrieved from the SEER database: the patient age, sex, race, extension of primary tumor invasion, radiation sequence, total number of LNs examined, number of involved LNs, grade, survival time, and SEER cause-specific death classification. All cases were restaged based on the AJCC-7 staging. The primary endpoint of the study is RCSS which was calculated from the date of diagnosis to the date of cancer specific death. Deaths attributed to the cancer of interest are treated as events and deaths from other causes are treated as censored observation.
Statistical analysis. The LNs cutoff points were analyzed using the X-tile program (http://www. tissuearray.org/rimmlab/), which identified the cutoff with the minimum P values from log-rank χ 2 statistics for the categorical LNs in terms of survival 31 . The relationship between various clinical and histological variables and survival was evaluated using the Kaplan-Meier method. Differences between survival curves were tested for statistical significance by using log rank test. The Cox proportional hazard regression model was used to identify the variables that could independently influence survival in preop-RT rectal cancer patients. The Mann-Whitney U test was used for continuous variables, and the chi-square test was used for categorical variables. The 5-year RCSS rate was estimated from Kaplan-Meier curves. Deaths attributed to the rectal cancer of interest are treated as events and deaths from other causes are treated as censored observation. Statistical analyses were performed with the statistical software package SPSS (Statistical Package for the Social Sciences) for Windows, version 17 (SPSS Inc, Chicago, IL, USA). Two-sided p values of less than 0.05 were considered to be statistically significant.