Optimal Lymph Node Harvest in Neoadjuvantly Treated Colorectal Cancer Using Methylene Blue Assisted Lymph Node Dissection Technique

[N A J Med Sci. 2013;6(2):51-56. DOI: 10.7156/najms.2013.0602051]

Bruno Märkl, MD; Tina Schaller, MD; Ines Krammer, MD; Claudio Cacchi, MD; Hanno Spatz, MD

Lymph node staging is still of crucial importance in rectal cancer. However, the recommendation to evaluate at least 12 lymph nodes for a sufficient staging is often not achieved in daily practice. This problem is even more pronounced after neoadjuvant treatment. The methylene blue assisted lymph node dissection technique (MBLND) has been shown to improve lymph node harvest in gastrointestinal cancers very effectively. A retrospective analysis enrolling 54 cases was performed to investigate the effect of MBLND in neoadjuvantly treated rectal cancer cases. Two historical collections evaluated using conventional dissection technique (n = 47) and fat clearance (FC) technique, respectively served as controls (n = 12). The lymph node harvest was highly significantly improved in the methylene group compared to fat clearance and conventional dissection with mean LN numbers of 29 ± 11, 21 ± 13, and 9 ± 4 (P < 0.001), respectively. Insufficient LN harvest occurred in 72 % of the control group cases, in 17 % of the FC-group and in no case of the Methylene group. Node positivity was found in 24%, 25% and 28% in the MB-, FC- and control group, respectively. MBLND is a highly effective method guaranteeing an optimal lymph node harvest in colorectal cancer even under the difficult conditions of preoperative chemo-radiation. In our hands it was even more effective than a fat clearance technique.

Key Words: lymph node, colorectal cancer, methylene blue, neoadjuvant

INTRODUCTION
Exact lymph node (LN) staging is of crucial importance in colorectal cancer. The prognosis and further therapy stratification are mainly influenced by the result of the histopathological LN evaluation.1 Moreover, the number of investigated lymph nodes is widely accepted as surrogate marker for the quality of the surgical resection.2 The number of at least 12 LNs has been established as a minimum for a sufficient evaluation.3 Many studies, however, have shown that this number is very often not achieved in daily routine.4-7 As a consequence the topic of improving the LN harvest has been addressed by many investigators. Different fat clearance techniques have been used repetitively to address this problem.8-13 The effectiveness of these techniques has been proved many times. However, costs, time delay, need of potentially toxic substances and labor intensity are disadvantages of all these protocols. These factors are probably the reason of the limited acceptance of these

techniques. In 2007 we introduced methylene blue assisted lymph node dissection technique (MBLND) as a simple method to improve the lymph node harvest in rectal cancer.14,15 In the following, the technique has been adapted for its usage in colon and gastric cancer.16,17 Using this technique the LN yield is dramatically improved compared to the standard manual method, as could be shown in prospective and randomized studies.15,16

The number of harvested LNs is influenced by different factors. The surgical technique and the pathologist´s diligence of LN dissection are two of these. The immunological reaction to the tumor and the application of preoperative radiation therapy are another two.18,19 Particular the latter is something experienced in daily practice and documented in many studies.20-24

In order to investigate the effectiveness of MBLND in neoadjuvantly treated cancers we performed a retrospective study enrolling cases where MBLND was used and building a control group with conventional dissected cases.

Table 1. Clinicopathological characteristics.

  MB-group
(n =54)
FC-group (n=12) Co-group
(n = 47)
Age ± SD 63±12 58 ± 11 65 ±10
Gender (f:m) 0.4 : 1 0.7 : 1 0.4 : 1
low grade 37 (69%) 8 (67%) 33 (70%)
high grade 10 (19%) 3 (25%) 7 (15%)
ypT0 7 (13%) 1 (8%) 7 (15%)
ypT1 2 (4 %) 2 (17%) 4 (9%)
ypT2 18 (33%) 3 (25%) 21 (45%)
ypT3 27 (54%) 3 (25%) 15 (32%)
ypT4 0 3 (25%) 0
ypM1 3 (6%) 3 (25%) 0
LN-number mean ± SD 29 ± 11 21 ± 13 10 ± 4
Cases with insufficient LN-number 0 (0%) 2 (17%) 34 (72%)
ypN1 6 (11%) 2 (17%) 9 (19%)
ypN2 7 (13%) 1 (8%) 4 (9%)
Number of positive LN 58 23 41

METHODS
Patients
The study group (MB-group) consisted of total 54 cases (53 rectal cancers and 1 colon cancer) which had surgery in the time between 2007 and 2009. 47 rectal cancers initially treated between 2003 and 2004 evaluated with conventional technique (Co-group) served as control group. A second control group consisted of 12 cases from the year 2007 when a fat clearance protocol was used to facilitate the lymph node detection (FC-group). The main clinicopathological characteristics are given in Table 1. The two groups were well balanced.

Methylene blue assisted lymph node technique
The technique has been carried out as detaily described before.14,16 In brief: after receiving the specimens in fresh status the inferior mesenteric artery was identified and the ligation or clip was cut away. Then the artery was cannulated using a standard i.v.-catheter (17 – 20 gauges) without steel mandarin and a syringe containing the methylene blue solution (Merck, Darmstadt, Germany; diluted 1:3 with 0.9% saline) was connected. After that 15 – 20 ml of the solution were gently injected. A successful injection was indicated by an immediate turn to blue of the serosal layer. The specimens were then fixed in 10% buffered formalin over-night. On the next day the mesenteric fat was dissected from the rectal wall after cutting out representative samples from the tumor region and the resection margins. The fat was then palpated in order to identify larger lymph nodes. Then the whole tissue was thoroughly sliced in 3 to 5 mm distances. The LNs within the fat were easily to detect because of their light blue colour. LNs with diameters ≥ 5mm were bisected and both parts were embedded. LNs that were cut in half during slicing were also embedded completely. Double or multiple counting was avoided by previous documentation.

Fat clearance technique
After fixing and conventional LN dissection the remaining adipose tissue was dehydrated by increasing concentrations of isopropanol (70% and 100%) and clearance in xylene. Then the fat was screened again for remaining LNs which could be identified as white nodules whithin the transparent fat.

Statistics
Dependent on sample size dichotomous data were compared using Chi-square test or Fisher’s exact test. Metric data were analyzed with the One Way RM ANOVA test. A p-value < 0.05 was considered significant. Mean values are given with ± 1 standard deviation. All statistical analyses were calculated using the Sigma-Plot 11.0 software package (Systat Software, Richmond, USA).

Figure 1. Technique of methylene blue injection. A) Rectal specimen and instruments needed for methylene blue injection. B) Identification of the vascular pedicel and cutting off the clip. C) Short longitudinal opening of the vessel facilitates cannulation. D) Cannulation of the artery using a standard i.v.-catheter without mandarin (multiple usage is possible). E) and F) The serosal and the mucosal layer turn into blue immediately after the injection of the first milliliters of the methylene solution.

RESULTS
The injection was easy and successfully performed in all cases of the MB-group. The detection was facilitated by the light blue staining of the LNs (Figure 1).

The lymph node harvest was highly significantly (p < 0.001) improved in the MB-group compared to the FC-group and the Co-group with mean LN numbers of 29 ± 11, 21 ± 13 and 9 ± 4, respectively (p < 0.001) (Figure 2A). Insufficient LN harvest occurred in 72 % of the control group cases, in 17% of FC cases and in no case of the Methylene group (P < 0.001). The node positivity was similar in all groups (P = 0.917; MB-group: 13 cases, 24%; FC-group: 3 cases, 25%; Co-group: 13 cases, 28%). The mean numbers of positive LNs in nodal positive cases were 4 ± 4 (Range: 1 – 13), 8 ± 10 (Range: 2 – 19), 3 ± 3 (Range: 1 – 11) for the MB-, FC- and Co-group, respectively (P = 0.436).

Figure 2. MB = methylene blue group, FC = fat clearance group, Co = control group; A) Mean LN numbers of the different groups. Error bars indicate 1 standard deviation. B) Rate of sufficient LN harvest (≥ 12 LNs) dependent on the group.

CONCLUSION
Lymph node staging is an important part of the histopathological assessment in colorectal cancer.1 The number of 12 LNs is recommended as minimum for an adequate investigation by the Union against Cancer (UICC).3 Nevertheless, there is still an on-going controversial debate about the minimal number of LNs that has to be retrieved for an optimal staging. The recommendations in the literature vary between 9 and more than 30 LNs.4,25-28 It is well known that the LN retrieval is poor in daily practice. Large studies discovered that the number of 12 nodes is very often not achieved.4-7 Several factors have been identified that potentially influence the number of harvested LNs in colorectal cancer.18 Especially difficult seems the situation under the conditions of neoadjuvant radiation therapy where sufficient LN staging is given only in a minority of cases.20-24 Since decades many different fat clearance protocols have been published to provide a tool for an improved LN harvest. Recently, two major variations of this technique have been established aiming at embedding of the complete mesenteric adipose tissue.8-13,25,26 All these protocols are hampered by the disadvantages of higher costs, time delay, necessity of potentially toxic substances and increased labor intensity. These points are probably the reasons why the technique has not been widely used despite intensive attention in the literature. Nevertheless, fat clearance has been proved to be an effective tool to facilitate the LN dissection. Moreover, fat clearance can also be applied secondary when the primary dissection failed to deliver a proper number of nodes.

Recently, we introduced intra-arterial methylene blue injection as a simple and very effective method to improve the LN harvest in gastrointestinal cancers.14-16 Meanwhile its usefulness has been confirmed by several other groups.27-31 As mentioned before, neoadjuvant radiation therapy often causes insufficient LN harvest in a majority of cases. It was

the aim of this study to investigate whether MBLND is an adequate method to solve this problem. Our results show a dramatically improved LN yield (Figure 2A). Insufficient harvest could be avoided in all cases. In comparison, usage of the conventional technique was associated with an insufficiency harvest of 72%. Even with the FC technique the threshold of 12 LNs was not achieved in 17% of the cases (Figure 2B). Moreover, former studies revealed that the time needed for the dissection using MBLND is in fact shorter than using conventional technique or FC.

This study is clearly hampered by its retrospective design and comparison of the study group with a collection of historical control cases. Therefore, several important points could not be controlled and we cannot rule out that our results are confounded by factors like changes in preoperative imaging diagnostic, surgical or radiation technique and performance of surgeons and pathologists. Nevertheless, it seems very unlikely that the reported superiority with a rate of sufficient LN harvest of 100% in the methylene group is the result of one or more bias. Taking into account that the methylene blue injection is additionally a helpful tool to evaluate the integrity of the mesorectal fascia, MBLND can be seen as an ideal method to facilitate the histopathological assessment of rectal cancer specimens.

However, our data also show that the nodal positive rate was similar in all groups. This could be with no doubt the consequence of the small case numbers in this study. In any case this finding leads us to the question of the clinical significance of evaluating a high number or at least 12 LNs in colorectal cancer. Many studies have shown that the outcome in colon cancer – recently also in rectal cancer – is strongly associated with the number of investigated LNs.32-36 Stage migration is the widely accepted explanation for this effect that is also known as Will-Rogers-phenomenon.37 The main point of this thesis is that a high number of investigated LNs prevents from understaging caused by missing LN metastases. We and others reported upstaging after secondary dissection in single cases.15,16 Only the study of Jepsen et al. revealed a significant higher metastases rate due to the usage of advanced lymph node dissection like MBLND.28 Abbassi-Ghadi et al. could not identify an increased detection rate of LN metastasis using special techniques for LN dissection in their systematic review.38 Nevertheless, we now have doubts about the correctness of the thesis that the better prognosis associated with high harvested LN number is the result of a stage migration effect. In a recently published paper we could show that the occurrence of large LNs is associated both with increasing numbers of detected LNs and favorable outcome.19 This indicates that an immunological effect might be the real reason for the above mentioned phenomenon.19 Other authors found no differences concerning the lymph node positivity rate dependent on the number of harvested LNs.36,39 Questioning the sense of the 12 lymph node rule, two recently published papers report no clinical benefit in cases of adequate LN harvest after neoadjuvant therapy in comparison to insufficiently staged cases.20,22

If this is true what is then the rational for MBLND in colorectal cancer? In our opinion establishing the technique is very easy, it is fast, cheap and it guarantees the detection of all relevant LNs in an individual specimen. Using MBLND not only warrants a standardized process ruling out an insufficient staging caused by the pathologist, it also seems to eliminate pathologist variability.15 Keeping in mind that the LN harvest is a quality indicator for the surgical technique this is of particular importance.

___________________________________________________________________________

Bruno Märkl, MD;* Tina Schaller, MD; Ines Krammer, MD; Claudio Cacchi, MD; Hanno Spatz, MD
Institute of Pathology, Klinikum Augsburg, Augsburg, Germany
Department of General, Visceral and Transplantation Surgery, Klinikum Augsburg, Augsburg, Germany

*Corresponding Author: Institute of Pathology, Klinikum Augsburg, Stenglinstrasse 2, 86156 Augsburg, Germany. Tel: 0049 821 4003199, FAX: 0049 821 400173199. (Email: Bruno.Maerkl@klinikum-augsburg.de)

CONFLICT OF INTEREST
The authors have no conflict of interests to disclose.

ACKNOWLEDGEMENTS
The authors are thankful to Barbara Kresse and Kai-Uwe Hebick for the data management.

___________________________________________________________________________

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