The Egyptian Journal of Surgery

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 36  |  Issue : 4  |  Page : 340--345

Improved detection of lymph nodes in cases of rectal cancer using combined methylene blue injection and fat clearance compared with fat clearance alone


Ayman Farouk1, Radwa Rashad2,  
1 Department of Surgery, Medical Research Insitute, Alexandria University, Alexandria, Egypt
2 Department of Pathology, Medical Research Insitute, Alexandria University, Alexandria, Egypt

Correspondence Address:
Ayman Farouk
Department of Surgery, Medical Research Institute, Alexandria University, 165 El-Horrya Street, 21561 Alexandria
Egypt

Abstract

Introduction Evaluation of lymph nodes (LNs) in rectal cancer is a fundamental component of all staging systems. Fat clearance and ex-vivo injection of methylene blue into the inferior mesenteric artery are known methods that can improve LN yield in patients with rectal cancer especially after neoadjuvant chemoradiation. Both techniques were widely compared with routine manual palpation of LNs. The question is that ‘Do adding ex-vivo injection of methylene to fat clearance as a single combined technique give further superiority on fat clearance alone regarding detection of nodal harvest and status?’. Patients and methods This study was carried out through comparing clinicopathological data of 40 patients whose specimens were subjected to combined ex-vivo injection of inferior mesenteric artery and xylene fat clearance (group I) with that of 30 patients whose specimens underwent only xylene fat clearance. All patients presented with resectable rectal cancer and have received neoadjuvant chemoradiotherapy. Results There was a statistically significant difference regarding total nodal harvest in group I compared with group II (17.52±6.32 vs. 14.56±5.64; P<0.05). Similarly, detection of at least 12 LNs was statistically different (87.5 vs. 63.3%; P<0.05). However, it was not the case regarding detection of nodal metastases (55 vs. 56.7%), which was not significantly different (P=0.085). Conclusion Using ex-vivo methylene blue injection into the inferior mesenteric artery and xylene fat clearance as a single combined technique shows a significant difference when compared with xylene fat clearance alone regarding total LN harvest and detection of the optimal number of LNs in cases of rectal cancer. However, it did not show such significance regarding detection of nodal metastases.



How to cite this article:
Farouk A, Rashad R. Improved detection of lymph nodes in cases of rectal cancer using combined methylene blue injection and fat clearance compared with fat clearance alone.Egypt J Surg 2017;36:340-345


How to cite this URL:
Farouk A, Rashad R. Improved detection of lymph nodes in cases of rectal cancer using combined methylene blue injection and fat clearance compared with fat clearance alone. Egypt J Surg [serial online] 2017 [cited 2017 Nov 21 ];36:340-345
Available from: http://www.ejs.eg.net/text.asp?2017/36/4/340/218158


Full Text

 Introduction



Lymph nodes (LNs) number and status in colorectal cancer present one of the most important pathologic predictors of patient outcome that can also determines the degree of benefit from adjuvant chemotherapy [1],[2],[3]. Approximately 68% of patients with negative nodal involvement will survive 5 years, compared with only 40% of those with metastases [4]. For accurate staging, the evaluation of at least 12 LNs in colorectal resection specimens is widely reported in clinical guidelines [1],[5],[6],[7],[8],[9],[10]. Neoadjuvant chemoradiotherapy can affect getting the maximal LN harvest with subsequent inaccurate staging and prediction of the patient’s prognosis. Efforts to maximize nodal harvest seem to be a good clinical practice to overcome the inefficient routine manual palpation [8],[11],[12]. Fat clearance techniques are widely used to improve LN harvest particularly in patients receiving neoadjuvant therapy [13]. However, Cohen et al. [14] and Jass et al. [15] reported that fat clearance alone does not improve significantly LN harvest, so there is still a need for further methods to achieve this job. Sanchez et al. [16] modified the fat clearing method by injecting methylene blue into the inferior mesenteric artery of rectal cancer resection specimens, ex vivo, to stain LNs blue followed by fat clearance. These advanced methods showed significant differences regarding LN harvest when compared with routine manual palpation but with little data regarding comparing these methods with each other.

 Patients and methods



This study was carried out through a review of medical records of two groups of patients who underwent elective surgery for resectable rectal cancer with intent to cure after receiving neoadjuvant chemoradiotherapy. All patients were operated at either Department of surgery, Faculty of medicine, or Department of Surgery, Medical Research Institute, University of Alexandria. Group I, as a retrospective study group, included data collected from 40 patients operated between June 2013 and June 2015 as a part of previous research [17]. Specimens of these patients were subjected to ex-vivo injection of inferior mesenteric artery followed by xylene.

Group II, as a prospective study group, included 30 patients who were operated between January 2014 and January 2017 as a part of ongoing study. Specimens of this group were subjected only to xylene fat clearance. Surgical procedures for both groups were done by two expert surgical teams, and pathological studies were done by two expert pathologist one for each group.

Review of medical records involved data regarding history taking, clinical examination, and laboratory and radiological findings. Surgery was performed at least 6 weeks after completing neoadjuvant therapy. Using total mesorectal excision technique, surgical procedures were either abdominoperineal resection or low anterior resection.

Ex-vivo injection of methylene blue was done by the surgeon. After identification of the inferior mesenteric artery stoma in the fresh specimen, cannulation of the artery was done using the plastic tube portion of a standard 16−20 G intravenous catheter, followed by injection of 15–20 ml of methylene blue solution (50 mg diluted with 0.9% saline in the ratio 1 : 3). Subsequently, the specimens were fixed in formalin for 24 h. Serial sectioning of the mass was done together with mouting of radial, proximal, and distal margins. The regional fat was dissected and cut into less than 0.5-cm sections and processed in ascending concentration of alcohol, then placed overnight in xylene. After clearing, meticulous picking of visible LNs was preformed followed by cutting, staining, and examination by microscopy ([Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6]). In group II, specimens were subjected to the previous steps without injection of methylene blue.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

Statistical analysis

It was performed using IBM SPSS statistics for Windows (version 20.0; IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percent. It was compared using χ2-test. Numeric data were expressed in mean±SD and compared using student t-test or the Mann–Whitney rank-sum test, depending on the results of the distribution test. Statististical significance was considered at P 0.05 or less.

 Results



Both groups were matched according to demographic, anatomical, surgical, and tumor histopathological data without statistical significance ([Table 1]). A total number of 701 (17.52±6.72) LNs were identified in group I compared with 437 (14.3.7±5.63) LNs in group II, which was a statistically significant difference (P=0.019).{Table 1}

At least 12 LNs were identified in 35 (87.5%) patients of group I compared with 19 (63.3%) patients in group II, which was also statistically significant (P<0.001). Metastases were detected in 22 (55%) patients in group I and 17 (56.7%) patients in group II with (P=0.988) ([Table 2]). Retrieved LNs were measured and categorized according to their size. The difference in nodal harvest between the two groups was most pronounced and statistically significant in LNs measuring up to 2 mm and more than 2 to up to 4 mm categories. However, for the LNs in the more than 4 mm categories, the difference was statistically insignificant ([Table 3]).{Table 2}{Table 3}

 Discussion



For decades, fat clearance techniques were adopted by many centers to improve nodal harvest in cases of colorectal cancer [18],[19],[20],[21]. Methylene blue injection into the superior rectal or the inferior mesenteric artery has been introduced as a simple effective alternative technique [22],[23],[24]. Each of these methods was widely compared with the routine manual palpation showing significant differences regarding nodal harvest especially after neoadjuvant chemoradiation. Fat clearing techniques have been claimed to be a time consuming method, so addition of methylene blue injection technique to fat clearance may help in identification of LNs by intense blue staining of them, decreasing the time consumed in LNs identification [16]. Sanchez et al. [16] reported evident significant difference regarding LN harvest comparing combined ex-vivo methylene blue injection and fat clearance with manual palpation of surgical specimens of rectal adenocarcinoma in patients receiving neoadjuvant chemoradiotherapy. In the present study, we tried to demonstrate the possible superiority of using combined methylene blue injection and fat clearance in comparison with the popular technique of using only fat clearance taking into consideration the superiority of both techniques on routine manual palpation regarding total nodal harvest especially after neoadjuvant therapy. Both groups were matched regarding demographic, anatomical, surgical, and tumor histopathological criteria.

In the present study, the total nodal harvest was statistically superior in group I (17.52±6.72 vs. 14.3.7±5.63; P=0.031). This can be explained by the visualization and detection of a significant higher number of smaller LNs in group I. This is an evident finding regarding LNs measuring up to 2 mm and more than 2 to up to 4 mm categories, which was not the case regarding larger LNs categories. This increase in individual nodal harvest reflects a significant detection of an optimal number of at least 12 LNs in group I compared with group II (87.5 vs. 63.3%; P<0.05).

Although there was a presence of a significant difference in total nodal harvest and detection of the optimal number of LNs, the difference between the two groups regarding the rate of patients with nodal metastasis was not statistically significant (55.00 vs. 56.70%; P=0.98), with also no significant difference regarding the mean of affected nodes (9.82±6.74 vs. 8.65±5.40; P=0.08). Taking into consideration that fat clearance technique by itself has improved detection of total and metastatic nodes, so it may be accepted to say that no further significant detection of metastatic nodes could be achieved using other methods, and subsequently, nonsignificant difference in this study can be easily explained. However, there are several studies that revealed absence of a significant difference regarding detection of nodal metastases even when these studies compared advanced techniques with routine manual palpation of LNs [22],[25],[26]. Parson et al. [25] in their SEER database reported evident increase in LN harvest over time which was associated with the outcome but with no increase in LN positivity.

Markle and colleagues studied a group of 669 cases of colorectal cancer using special methods to improve nodal harvest (methylene blue injection alone in 559 cases, fat clearance alone in 55 cases, and a combined methylene blue injection followed by fat clearance in another 55 cases). Although they compared this group with a cohort of 663 historical cases using only conventional manual dissection, they reported no significant difference regarding nodal positivity rates, which were even absolutely identical at 37% (P=0.98), with only trends toward higher rates of nodal metastases in subgroups of high-grade cancers and rectal cancers without neoadjuvant therapy [22]. They compared their results with Ricciardi et al. [26], who conducted a detailed analysis of more than 120 000 cases, and concluded that the poor nodal harvest did not significantly cause detection of lower rate of metastases. Märkl et al. [22], depending on their sequential LN preparation and examination, detected the first metastatic node among the first nine detected node in close proximity with the tumor in 86% of cases.

In another publication, Märkl and colleagues found that in only two of 81 cases the largest metastatic node was less than 4 mm whereas the largest LN in all other cases was large enough to be missed during routine examination. They concluded that the pathologists may show a poor performance regarding detecting high number of LNs, but they are much more effective regarding crucial nodes [27]. Herrera et al. [28] noticed that though metastases in LNs from rectal adenocarcinoma occur frequently in small LNs (≤5 mm), but mostly of perirectal distribution rendering them easily expected by the pathologists. Kim and colleagues even reported that fat clearance did not increase metastatic LNs yield in both neoadjuvant and nonneoadjuvant groups. This might reflect the fact that one certified pathologist who is specialized in colorectal disease can perform precise pathologic assessment by manual dissection, and this may explain their relatively small additional benefit of fat clearing in detecting more metastatic LNs [29]. Back to our study, the significance of small LNs was the key to explain absence of significant difference regarding detection of nodal metastases though significant difference regarding total nodal harvest. Analysis of detected metastatic LNs in group I revealed that categories of LNs size less than 2 and 2–4 mm contained only 2.32 and 5.56 % of total metastatic nodes, respectively, though these two size categories were responsible for the significant difference regarding total nodal harvest and detection of optimal number of LNs.

 Conclusion



Though adding methylene blue injection technique to widely used fat clearance method gave superiority over using fat clearance alone regarding total nodal harvest and detection of optimal number, it did not show similar statistical significance regarding detection of nodal metastases. Nevertheless, we recommend this combined technique as it can achieve a sufficient nodal harvest in patients with rectal cancer treated with neoadjuvant chemoradiation, as this total harvest is strongly associated with outcome of the patients.

Limitations of this study include the small number of cases and absence of randomization between the two groups. Moreover, although being experts, presence of two pathologists is another limitation.

Acknowledgements

The authors are thankful to Dr. Karim Atteia for his effort in collecting the data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Compton CC, Fielding LP, Burgart LJ, Conley B, Cooper HS, Hamilton SR et al. Prognostic factors in colorectal cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000; 124:979–994.
2Baxter NN, Virnig DJ, Rothenberger DA, Morris AM, Jessurun J, Virnig BA. Lymph node evaluation in colorectal cancer patients: a population-based study. J Natl Cancer Inst 2005; 97:219–225.
3Gray R, Barnwell J, McConkey C, Hills RK, Williams NS, Kerr DJ. Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomised study. Lancet 2007; 370:2020–2029.
4Hermanek P, Merkel S, Hohenberger W. Prognosis of rectal carcinoma after multimodal treatment: ypTNM classification and tumor regression grading are essential. Anticancer Res 2013; 33:559–566.
5Tepper JE, O’Connell MJ, Niedzwiecki D, Hollis D, Compton C, Benson AB et al. Impact of number of nodes retrieved on outcome in patients with rectal cancer. J Clin Oncol 2001; 19:157–163.
6Schofield JB, Mounter NA, Mallett R, Haboubi NY. The importance of accurate pathological assessment of lymph node involvement in colorectal cancer. Colorectal Dis 2006; 8:460–470.
7Swanson RS, Compton CC, Stewart AK, Bland KI. The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined. Ann Surg Oncol 2003; 10:65–71
8McDonald JR, Renehan AG, O’Dwyer ST, Haboubi NY. Lymph node harvest in colon and rectal cancer: current considerations. World J Gastrointest Surg 2012; 4:9–19.
9Sobin LH, Greene FL. TNM classification: clarification of number of regional lymph nodes for pNo. Cancer 2001; 92:452.
10Li Destri G, Di Carlo I, Scilletta R, Scilletta B, Puleo S. Colorectal cancer and lymph nodes: the obsession with the number 12. World J Gastroenterol 2014; 20:1951–1960.
11Le Voyer TE, Sigurdson ER, Hanlon AL, Mayer RJ, Macdonald JS, Catalano PJ et al. Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT-0089. J Clin Oncol 2003; 21:2912–2919.
12Wright FC, Law CH, Berry S, Smith AJ. Clinically important aspects of lymph node assessment in colon cancer. J Surg Oncol 2009; 99:248–255.
13Wang H, Safar B, Wexner SD, Denoya P, Berho M. The clinical significance of fat clearance lymph node harvest for invasive rectal adenocarcinoma following neoadjuvant therapy. Dis Colon Rectum 2009; 52:1767–1773.
14Cohen SM, Wexner SD, Schmitt SL, Nogueras JJ, Lucas FV. Effect of xylene clearance of mesenteric fat on harvest of lymph nodes after colonic resection. Eur J Surg 1994; 160:693–697.
15Jass JR, Miller K, Northover JM. Fat clearance method versus manual dissection of lymph nodes in specimens of rectal cancer. Int J Colorectal Dis 1986; 1:155–156.
16Sanchez W, Luna-Perez P, Alvarado I, Labastida S, Herrera L. Modified clearing technique to identify lymph node metastases in post-irradiated surgical specimens from rectal adenocarcinoma. Arch Med Res 1996; 27:31–36.
17Karim A, Mohamed E-L, Suzan H, Walid E, Mohammed FA. Evaluation of the effect of ex-vivo methylene blue into the inferior mesentric artery with fat clearance to improve lymph node harvest in neoadjuvantly treated rectal cancer [Thesis]. Alexandria: Department of Surgery, Faculty of Medicine, University of Alexandria; 2015
18Scott KW, Grace RH. Detection of lymph node metastases in colorectal carcinoma before and after fat clearance. Br J Surg 1989; 76:1165–1167.
19Hernanz F, Garcia-Somacarrera E, Fernandez F. The assessment of lymph nodes missed in mesenteric tissue after standard dissection of colorectal cancer specimens. Colorectal Dis 2010; 12:e57–e60.
20Prabhudesai AG, Dalton R, Kumar D, Finlayson CJ. Mechanised one-day fat clearance method to increase the lymph node yield in rectal cancer specimens. Brit J Biomed 2005; 62:120–123.
21Richter D, Lorenz D, Isemer FE, Braun S, Fisseler-Eckhoff A. Acetone treatment of lymph node preparations in staging colorectal specimens. Pathologe 2007; 28:269–272.
22Märkl B, Schaller T, Krammer I, Cacchi C, Arnholdt HM, Schenkirsch G et al. Methylene blue-assisted lymph node dissection technique is not associated with an increased detection of lymph node metastases in colorectal. Mod Pathol 2013; 26:1246–1254.
23Martijnse IS, Dudink RL, Kusters M, Rutten HJ, Nieuwenhuijzen GA, Wasowicz-Kemps DK. Patent blue staining as a method to improve lymph node detection in rectal cancer following neoadjuvant treatment. Eur J Surg Oncol 2012; 38:252–258.
24Reima H, Saar H, Innos K, Soplepmann J. Methylene blue intra-arterial staining of resected colorectal cancer specimens improves accuracy of nodal staging: a randomized controlled trial. Eur J Surg Oncol 2016; 42:1642–1646.
25Parsons HM, Tuttle TM, Kuntz KM, Begun JW, McGovern PM, Virnig BA. Association between lymph node evaluation for colon cancer and node positivity over the past 20 years. JAMA 2011; 306:1089–1097.
26Ricciardi R, Madoff RD, Rothenberger DA, Baxter NN. Population-based analyses of lymph node metastases in colorectal cancer. Clin Gastroenterol Hepatol 2006; 4:1522–1527.
27Märkl B, Rößle J, Arnholdt HM, Schaller T, Krammer I, Cacchi C et al. The clinical significance of lymph node size in colon cancer. Mod Pathol 2012; 25:1413–1422.
28Herrera L, Villarreal JR. Incidence of metastases from rectal adenocarcinoma in small lymph nodes detected by a clearing technique. Dis Colon Rectum 1992; 35:783–788.
29Kim IK, Lim BJ, Kang J, Kim SA, Kang D, Sohn SK et al. Clinical impact of fat clearing technique in nodal staging of rectal cancer after preoperative chemoradiot. J Korean Surg Soc 2013; 85:30–34.