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Factors Influence Lymph Node Retrieval after Resection for Rectal Cancer

Introduction: Total lymph node (TLN) count is an important qualifier of cancer evaluation and extensive nodal resection has been associated with lower rates of cancer recurrence; allows for more accurate cancer staging and improved survival following resection for rectal cancer. Aim: To examine the adequacy of lymph node sampling in rectal cancer and the influence of the clinicopathological factors and treatment modalities on the total lymph node yield. Methods: This is a retrospective study includes 409 cases of rectal cancer treated by surgical intervention only (126 cases), or surgery after short or long course chemotherapy (75 and 208 cases respectively). Results: TLN count range from (2-50, mean 12.38). Twelve lymph nodes were found in 49.4% of the specimens with or without neoadjuvent chemo radiatherapy (CRT). Patient treated with surgery only have a significant high total LN yield (mean is 15.53) in comparison with (12.99 and 10.25 for short course CRT and long course CRT respectively, P value <0.001). The number of lymph nodes was significantly correlated with the modality of treatment, anterior quadrant involvement, vascular and perineural invasion, pathological staging, Duke's and Mandard staging. On multivariate only perineural invasion, high tie lymph node involvement, Dukes stage, and tumour load were found to be the main predictors of the number of lymph node yield. Conclusion: The neoadjuvent CRT has a significant effect on the adequacy of lymph node sampling and a negative impact on the lymph nodes retrieved. This negative effect was more prominent with the long term rather than the short term CRT. This should be taken in consideration at the N staging of the tumour to avoid down-staging.


Introduction
Bowel cancer is the fourth most common cancer in the UK (2013), accounting for 13% of all new cases. It is the third most common cancer in both males (14% of the male total) and females (11%) separately. There are over 14000 new cases of rectal cancer and 6000 deaths with the disease within the UK every year [1]. Preoperative chemo radiotherapy is now considered standard treatment for patients with a locally advanced rectal cancer and a threatened circumferential resection margin (CRM) [2]. The primary purpose of chemo radiotherapy is to improve local control and resectability and the response to preoperative therapy has been shown to be linked to long-term outcome in several studies [3][4][5].
Pathological staging of colorectal cancer requires adequate resection of the main tumour with the regional lymph nodes by total mesorectal excision (TME). An accurate examination of the surgical specimens is essential for the correct assessment of the lymph node status as inadequate lymph node removal may result in understaging of rectal cancer. The National Cancer Institute (NCI) recommends that at least 12 lymph nodes to be removed 2016 Vol. 1 No. 2: 13 Journal of Adenocarcinoma ISSN 2572-309X for adequate staging of colorectal cancer [6]. This minimum number of retrieved lymph nodes was initially recommended by the World Congress of Gastroenterology held in Sydney in 1990 [7]. Removal of fewer lymph nodes can results in understaging, hence, some patients who would benefit from receiving adjuvant therapy, therefore, would not be offered any [6]. Nevertheless, low number of retrieved lymph nodes is associated with adverse overall survival, higher rates of local recurrence and significant morbidity [8][9][10].
Preoperative radiotherapy is thought to decrease the lymph node yield after surgical excision [11][12][13]. This is probably caused by the immune response and fibrosis in lymph nodes exposed to radiotherapy, which results in diminution in their size, making their identification in the pathology specimen difficult. This study aims to look at the effect of preoperative chemo radiotherapy (CRT) on the pathological criteria of the rectal cancer including the number of lymph nodes retrieved in the mesorectal specimen.

Patient data
This study was performed on a cohort of 409 cases of consecutive, unselected primary operable rectal cancer diagnosed between 2009 and 2010 at Nottingham University Hospital. Patient's clinical history and tumour characteristics including patients' age, sex, site of tumour, histologic grade, tumour necrosis, or fibrosis, depth of tumour invasion beyond the muscularis proprai, high tie lymph node involvement, and the total lymph node (LN) status, vascular (VI) and perineural invasion were obtained from patient's reports.
Hundred and twenty six patients (29%) were treated with surgery only while 282 were treated with long-course preoperative chemo-radiotherapy before surgery for rectal cancer as described previously [14]. The latter group had locally advanced rectal cancer, cT3/4 and/or N+ and were considered inoperable or of borderline resectability due to potential circumferential resection margin (CRM) involvement. Pre-treatment workup consisted of digital rectal examination, sigmoidoscopy and biopsy, barium enema, pelvic magnetic resonance imaging (MRI)/ computed tomography and computed tomography of the chest and abdomen. All patients were discussed at a multidisciplinary team meeting consisting of specialist colorectal surgeons, oncologists, radiologists and pathologist before being referred for neoadjuvant treatment.
The study was approved by the Nottingham University Hospital ethics committee. This group was divided to short and long term CRT that included 75 and 208 patients respectively. Dissection of the specimen was performed according to the departmental standard operating procedure in accordance with the recommendation of the Royal collage of pathologist. Lymph nodes were identified by direct inspection and manual palpation through serial transverse slicing of the mesorectum and sigmoid mesentery. All the identifiable lymph nodes present in the specimen are dissected for histological examination. If less than 12 lymph nodes identified representative sampling from the mesenteric fat surrounding the tumour was performed. Neither fat clearance, nor sentinel node mapping methods were used in this series of patients.
Details of the characterisation of this cohort of the patients according to the treatment modality were described in (Table 1). Tumours were staged (pT stage) according to the TNM staging criteria and Duck's staging system [15].
The response to radiotherapy was graded using a rectal radiotherapy grading system adapted from Mandard et al. grading system [16]. This comprised the following:

Statistical analysis
Statistical analysis was performed using SPSS 21 statistical software. Chi-square test was used to compare clinic-pathological parameters among different treatment modalities. One way ANOVA was used to access the relation between the number of retrieved nodes and categorical variable while spearman correlation test was used to compare the former with numerical variables. Multivariate regression model was used to determine the most significant factors affecting the lymph node yield. All p-values were two-sided, and p<0.05 was considered significant.

Results
This is a retrospective study of a cohort of 409 consecutive cases. The age of the patients was between 20 and 89 years (mean 65.4), which included 260 male and 149 female. The management included surgical excision alone 126 (29%), or a preoperative short or long term chemo-radiotherapy 75 (17.2%), and 260 (59.8%) respectively prior to surgery. Most of the rectal tumours included in the study were moderately differentiated adenocarcinoma (68.5%), with a predominant upper rectal location (35.4%). Forty six patients (10.6%) with a known pT stage were found to have stage pT0 after radiotherapy, with the pT3 stage represent the most predominate stage regard less of the treatment modality (55.5%). It was found that some adverse prognostic parameters as vascular invasion and perineural infiltration and high tie lymph node involvement were more frequent in the patient received long course CRT in comparison with those who went for the surgery or the preoperative short term CRT.

Journal of Adenocarcinoma ISSN 2572-309X
A significant positive correlation was also found between high lymph node yield and upper rectal tumour with anterior quadrant involvement (p value<0.005).
For the group of patient who received neo-adjuvant CRT it was found that the highest number of lymph node dissection was found in those with a poor response to treatment as defined by Mandard grading system (p value is 0.01).
The number of lymph node yield was compared with the number of blocks used for the microscopic assessment of each case. It was found that the using large number of small blocks was associated with high total lymph node (p=0.1). Surprisingly, a reverse relation was identified between using large blocks and the total lymph node yields (p=0.04). However the sum of both the large and the small block used wasn't seems to affect the total lymph nodes ( Table 3).
Multivariate analysis of the factors that have a significant correlation with the number of lymph node showed that only perineural invasion, high tie lymph node involvement, Dukes stage, and tumour load are the factors that can predict the number of lymph node retrieved from an excision specimen ( Table 4).
The lymph nodes were categorized according to their size into five groups. The relation between the size of the node and the number of lymph node yield in different treatment modalities was assessed (surgery only versus neoadjuvant). It was found that in two groups (>0-2 mm and >10-15 mm), the number of lymph node in patient treated with surgery alone is significantly higher than those treated with preoperative CRT (Table 5).

Discussion
Previous studies and guidelines recommended examination of greater number of nodes to provide an adequate staging of tumour [6,[17][18][19]. False negative classification of the lymph node stage will affect patients who might benefit from adjuvant treatment [19].
The number of lymph nodes dissected from rectal specimens was found to be directly proportional to the probability of detecting disease in them [20]. Nevertheless, adequate sampling of lymph nodes decreases the incidence of local recurrences [21,22] and improves the patient overall survival [23,24].
The adequacy of lymph node sampling was defined as a minimum of 12 lymph nodes according to the recommendation of the World Congress of Gastroenterology in Sydney [6] and the NCI guidelines [7].
The number of lymph nodes that should be examined in order to be considered adequate is not universally agreed upon. Various studies have suggested numbers ranging from 7 to 20, based on their individual series [9,20,25,26]. The last edition of TNM classification of the American Joint Committee on Cancer recommended at least 10 to 14 lymph nodes in radical colon and rectum resections without neoadjuvant treatment [27].
In this series the lymph node sampling was adequate in 69.8% of patient treated with surgery alone, and was decreasing from 58.7% to 33.7% with the increase of the course of the neoadjuvent CRT from short term to long term.
In the current study the mean number of lymph nodes excised from the rectal specimen ranged between 2 and 50, mean 12.38, which is in agreement with previous studies and international and national guidelines mentioned above. The wide range of variation may be related to the low number of lymph node retrieval associated with the neoadjuvant CRT. Other factors related to the ability of both the surgeon and the pathologist in excision and assessing the rectal cancer respectively are found to influence the number of lymph node retrieval [28,29].
The Dutch Colorectal Cancer study, which randomly subdivides the patients to receive preoperative short radiotherapy course   Journal of Adenocarcinoma ISSN 2572-309X followed by TME surgery or TME surgery only, found out that there was a significant difference in the number of retrieved lymph nodes [9]. In agreement with previous studies, in the current study the number of lymph node yield after surgery alone overweighed the number retrieved with upfront CRT (mean is 15.53). Interestingly, the effect of the CRT on the number of lymph nodes was time dependent where patient received short term CRT had more lymph nodes than those who revived long term CRT (12.99, and 10.25 in short term CRT and long term CRT respectively, P value <0.005). This contradicts Marijnen et al. who found no down staging after short-term preoperative radiotherapy in rectal cancer patients [11].
Our findings are compatible with Sermier et al. [30] and other studies, that prove the adverse effect of the long term in comparison to the short term CRT on the lymph node retrieval and hence the adequate staging of rectal [31][32][33].
Other clinic-pathological parameters that found to have a significant positive impact on the lymph node yield included upper rectal location which is compatible Marcos et al. finding [20]. Thorn found a higher number of lymph nodes in the mid rectal tumours [34]. Other significant positive association was found with involvement of the anterior quadrant, intravascular and perineural invasion, positive high tie lymph node, high Duke's and pT stages and poor response to neoadjuvent CRT as defined by Mandar, these finding are in agreement with some studies [12,20] but not with others [34,35]. Other histological features as the percentage of tumour load and tumour necrosis had a positive effect of the lymph node number while tumour fibrosis has a significant negative impact.
In this series, the age of the patient did not have any effect on the number of lymph node which come in agreement with previous studies [20,26] but in contrast with others that found higher lymph node yield in younger patients [12,36]. The sex of the patient did not have any association with the number of lymph nodes, however the effect of sex on lymph node number is not consistent in the literature [26,36].
On multivariate only perineural invasion, high tie lymph node involvement, Dukes stage, and tumour load were found to be the main predictors of the number of lymph node yield.
In conclusion, our result provides further evidence to the effect of the widely used neoadjuvant CRT on the number of lymph node retrieved in rectal cancer which was found to be time dependent. Furthermore, the current TNM classification of rectal cancers needs to be improved and take in consideration the effect of neoadjuvent CRT on the number of lymph node retrieved [37]. Despite tumour down staging, pathological postoperative staging remained a very strong prognostic factor for both T and N stages and remains pivotal for selection of patients who may benefit from additional chemotherapy [38]. Nevertheless, the pathological N staging of irradiated specimen should be interpreted with caution.