Methylene Blue Assisted Lymph Node Dissection in Colon Specimens. A Prospective, Randomized Study

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1 Anatomic Pathology / Methylene Blue Assisted Lymph Node Staging Methylene Blue Assisted Lymph Node Dissection in Colon Specimens A Prospective, Randomized Study Bruno Märkl, MD, 1 Therese G. Kerwel, MD, 2 Hendrik G. Jähnig, MD, 1 Daniel Oruzio, MD, 3 Hans M. Arnholdt, MD, 1 Claus Schöler, MD, 2 Matthias Anthuber, MD, 2 and Hanno Spatz, MD 2 Key Words: Methylene blue; Fat clearance; Colon cancer; Lymph node staging Abstract Recently, we introduced ex vivo intra-arterial methylene blue injection into the inferior mesenteric artery as a novel method to improve lymph node (LN) harvest in rectal cancer. We have now adapted this method to the other segments of the colon. A total of 60 cases were enrolled. Primary LN dissection was followed by fat clearance and a secondary dissection. The mean ± SD primary LN harvest differed highly significantly with 35 ± 18 and 17 ± 10 LNs in the methylene blue stained and unstained groups, respectively. Primary insufficient LN harvest occurred in 8 cases of the unstained group and in only 1 case of the methylene blue stained group (P =.0226). After secondary dissection, upstaging was seen exclusively in the unstained group. The time/ln ratio differed significantly with 0.9 and 0.6 min/ln in the unstained and methylene blue stained groups, respectively. Intraarterial methylene blue injection is recommended as a routine technique in the histopathologic study of colon cancer. Lymph node (LN) assessment is a crucial part of the histopathologic staging of colon cancer. Stage I and II cases need no further therapy after oncologic resection. In contrast, stage III cancers, which are defined by LN metastases, are generally treated with adjuvant chemotherapy. 1,2 The 5-year survival rates drop from 83% for stage II to 60% for stage III cancers. 3 This decrease highlights the importance of accurate LN assessment. Despite that, the optimal method for staging LNs continues to be debated. There is a lack of consensus in the literature on how many LNs should be assessed. 4-7 To further complicate the issue, multiple advanced techniques for LN staging have been developed; however, their roles are not clearly defined. Other strategies include techniques such as fat clearance to increase the LN harvest 8-10 and newer methods that are used to improve the detection rate of metastases. Recent developments include techniques such as sentinel node biopsy, 11 immunohistochemical analysis, and polymerase chain reaction. 8,12 Widespread use of these techniques, however, is limited by an increase in time effort, costs, and the need for special facilities. Despite an emphasis on accurate LN staging, the reality is that the minimal 12 LNs as recommended by the International Union Against Cancer for a colorectal specimen is often not achieved in practice. 7,13-15 We recently published a pilot study with a novel technique to increase LN harvest in rectal cancer by intra-arterial injection of methylene blue. 16 This technique was originally developed to assess the integrity of the mesorectal fascia after total mesorectal resection. 17 During routine assessment of such rectal specimens, we observed that LNs were stained blue within the fat and, therefore, were very easy to detect. After demonstrating improved LN harvests in rectal specimens, we adapted this method for the other colon segments. The present Am J Clin Pathol 2008;130:

2 Märkl et al / Methylene Blue Assisted Lymph Node Staging study was performed to compare this novel method with the standard technique in a prospective and randomized manner, with special consideration of the time and technical aspects. Materials and Methods We prospectively enrolled 60 consecutive cases in 9 months and randomized them to the methylene blue stained or the unstained group. The inclusion criterion was a curative resection of any part of the colon and the upper rectum for histologically proven or suspected malignancies. Exclusion criteria were palliative and emergency resections. All specimens were brought to pathology immediately after resection in a fresh and unfixed state. Depending on the result of randomization, the specimens were immediately opened, washed, and stretch-fixed in 10% buffered formalin overnight or injected with methylene blue followed by fixation zfigure 1z. Injection was carried out by 2 pathologists in the department (B.M. and H.J; B.M. had previous experience with approximately 30 specimens, and H.J. had no experience at the start of the study). The learning curve is very small for left colon specimens and requires a few cases for right colon specimens. For the injection, the main artery was identified and the clip or ligature was cut off. The artery was opened longitudinally for a length of about 10 mm to facilitate cannulation with a standard 16- or 17-gauge intravenous catheter without a steel mandrin. To seal the catheter in the artery, a Randomization Methylene Injection Pathology specimen in fresh state Formalin fixing Lymph node dissection (primary) Fat clearance Unstained Lymph node dissection (secondary) zfigure 1z Algorithm of different steps. The clock indicates time measurements. clamp was fixed beside the artery in parallel orientation. The success of the gentle injection of 15 to 20 ml of methylene blue solution (50 mg diluted with 0.9% saline; ratio, 1:3) can be observed by instantaneous blue staining of the specimen s serosal layer zimage 1z. The time for this procedure was measured and the performance judged in 4 categories: (1) easy, (2) minor difficulties, (3) major difficulties but successful, or (4) not successful. Following injection, the specimens were handled in the same way as in the unstained group. After fixing, representative sections of the tumor region were cut out and embedded. The paracolic fat was then dissected. Larger LNs detectable by palpation were cut out immediately. Next, the fat was sliced and stretched to reach thin layers. The cut surfaces were then screened for LNs. LNs in the methylene blue stained group were stained blue and, therefore, easy to detect (Image 1). A second LN preparation was done in all cases after fat dehydration with increasing concentrations of isopropanol (70% and 100%) and clearance in xylene. Time for the primary and secondary LN preparation was measured. After paraffin embedding, 3-µm thin sections were cut and stained with H&E. The slices were then screened for metastases. Maximum diameters of LNs and metastases were measured using a digital camera with a calibrated software system (Progress C10, Jenoptik, Jena, Germany) and grouped into 6 categories (<1 mm, 1-4 mm, >4-6 mm, >6-8 mm, >8-10 mm, and >10 mm). The significance of differences between groups was calculated by using the Mann-Whitney rank sum test for measured values with failed normality and the χ 2 test for clinical characteristics. All data were processed using SigmaStat 3.5 software (Systat Software, Richmond, CA). A P value of less than.05 was considered significant. Results Comparison of group characteristics ztable 1z showed a nonsignificant higher frequency of male sex (23 vs 16) and locally advanced cases (24 vs 19) in the methylene blue stained group. The other parameters, including the distribution of cancer location, were well balanced. Methylene Blue Injection Methylene blue injection was successfully performed in all but 1 case. An extravasate of the dye caused by an arterial leakage occurred in a right hemicolon specimen. Because the staining of the LNs failed, the case was excluded from further analysis. The conventional investigation was not influenced negatively. Minor difficulties arose in 3 right hemicolon resections, 2 sigmoid specimens, and 1 left hemicolon specimen. The difficulties were due to problems with finding the main artery 914 Am J Clin Pathol 2008;130: Downloaded 914 from

3 Anatomic Pathology / Original Article A B C D E F zimage 1z Methylene blue injection technique in sigmoid colon specimen and staining results. A, Longitudinal opening of the artery over 10 mm after removing the clip or the ligature. B, Cannulation of the artery using a 16-gauge intravenous catheter without steel mandrin. C, Advancing the catheter and sealing in the artery with a clamp that is fixed beside the artery in parallel orientation. D, Connecting the syringe with the catheter and injection of the methylene blue solution. The serosal layer turns blue during the injection. E, Two blue-stained lymph nodes on cut surface with diameter of 2-3 mm (arrows). F, Further improved visibility of blue-stained lymph nodes after fat clearance. Am J Clin Pathol 2008;130:

4 Märkl et al / Methylene Blue Assisted Lymph Node Staging ztable 1z Group Characteristics in 59 Cases of Colon Cancer and inhomogeneous staining results. Because of the inhomogeneous staining, a second injection was done into a distal artery. Major problems occurred in only 1 case in which the injection procedure was prolonged and took 7 minutes in a right hemicolon specimen. Primary LN Dissection Methylene Blue Stained Unstained Mean ± SD age (y) 66 ± ± 9 Female 7 14 Male Right hemicolon Left hemicolon 2 3 Sigmoid colon/upper rectum Inflammatory 1 0 Adenoma high-grade intraepithelial neoplasia 0 2 pt1 2 4 pt2 3 5 pt pt4 4 0 Vascular invasion The mean ± SD numbers of LNs in the primary dissection differed highly significantly () with 35 ± 18 LNs (range, ) in the methylene blue-stained group and 17 ± 10 LNs (range, 1-42) in the unstained group zfigure 2z. The greatest differences were seen in the 3 smallest size groups (<6 mm) with total LN numbers of 832 and 391 in the unstained and methylene blue stained groups, respectively () zfigure 3z. Insufficient LN harvest with less than 12 detected LNs occurred significantly (P =.026) more often in the unstained group, with 8 cases compared with only 1 case in the methylene blue stained group. Two of these cases were T stage 1, 2 were stage 2, and the remaining 5 cases were stage 3. Metastases were found in 8 and 10 cases of 28 malignant cases in the methylene blue stained and unstained groups, respectively. Of these total 18 cases, 3 (17%) showed insufficient LN harvest. The total numbers of metastasized LNs were 34 and 30 in the methylene blue stained and unstained groups, respectively. Of the metastasized LNs, 62% of the methylene blue stained group and 57% of the unstained group were smaller than 6 mm. No metastases were found in LNs smaller than 1 mm. Secondary LN Dissection The mean ± SD additional LN yield after fat clearance was 8 ± 7 (range, 0-28) and 5 ± 4 (range, 0-18) in the methylene blue stained and unstained groups, respectively (P >.05) (Figure 2). Additional metastases were found in 1 case in the methylene blue stained group compared with 4 cases in the unstained group. Upstaging occurred exclusively in the unstained group. A total of 3 cases were upstaged: 1 case changed from N0 to N1, and 2 cases were upstaged form N1 to N2. Two of these cases, including the primary N0 case, showed sufficient LN harvest, with 14 and 17 LNs from the primary dissection. Mean LN Number (1/x) After Primary Dissection After Secondary Dissection LN Number (x/1) P =.121 P =.001 P =.01 P =.1 <1 1-4 >4-6 >6-8 >8-10 >10 LN Diameter (mm) zfigure 2z Mean lymph node harvest in unstained (white bars) vs methylene blue stained (black bars) group with highly significant differences after primary and secondary dissection. Error bars indicate the SD. zfigure 3z Lymph node (LN) size distribution in the unstained (white bars) and methylene blue stained (black bars) groups. 916 Am J Clin Pathol 2008;130: Downloaded 916 from

5 Anatomic Pathology / Original Article Time Effort for Primary and Secondary LN Dissections Time effort is defined as the sum of time for injection (if applicable) and dissection and preparation of the LNs. The mean ± SD primary time effort in the methylene blue stained group was 17.8 ± 7.1 minutes (time per LN, 0.6 minute). The average time needed for injection was 2.4 minutes. In comparison, the mean ± SD time effort for preparation in the unstained group was 10.9 ± 4.1 minutes, resulting in a mean time/ln ratio of 0.9 minute. The total time effort and the time/ LN ratio differed significantly with P values of <.001 and.02 zfigure 4z. The mean ± SD times for secondary LN preparation after fat clearance were 16.0 ± 9.3 and 18.7 ± 9.9 minutes in the methylene blue stained and unstained groups, respectively (P =.21). Time/LN Ratio (min/1) P <.002 zfigure 4z Mean time/lymph node (LN) ratio (min/ln) in the unstained (white bar) vs methylene blue stained (black bar) group with a significantly shorter time in the methylene blue stained group. Error bars indicate the SD. Discussion Despite the recommendation of the International Union Against Cancer to examine at least 12 LNs for sufficient staging in colorectal cancer, it is difficult to reach this number in practice. 7,13,15,18,19 Furthermore, the minimal number of LNs is still the subject of a broad discussion in the literature, and the recommended numbers range from 8 to 30 LNs. 5,13,18,20 Swanson et al 21 showed a very impressive correlation between increasing numbers of screened LNs and improved 5-year survival rates in T3N0 colon cancer cases. Cases with fewer than 8 LNs had a prognosis similar to cancer with positive LNs, whereas the 5-year survival of patients with 15 to 30 LNs was equal to T2 cancers. 21 Similar correlations have also been established by other authors. 12,22,23 Therefore, different concepts have been developed to improve LN harvest and accuracy of staging. One group of techniques follows a strategy to heighten sensitivity of metastasis detection in a single or in all collected LNs. This group includes the sentinel LN technique, immunohistochemical analysis, and polymerase chain reaction. 8,12,24 The aim of fat clearance techniques is to increase the number of collected LNs. Until now, several protocols for fat clearance have been published and have all clearly improved LN harvest. 8,10,25 Despite proven superiority over conventional techniques, widespread use of these techniques seems to be limited by the necessary additional costs, time, labor, and potentially harmful substances. We recently reported ex vivo intra-arterial methylene blue injection into the inferior mesenteric artery as a novel technique to improve LN harvest in rectal cancer. 16 Compared with the conventional technique in a historical control group, the LN harvest was almost doubled with a mean LN count of 27. We have now adapted this technique to other colon segments and performed a prospective and randomized study with consideration for the time effort and technical aspects. In concordance with the situation in rectal cancer, the mean ± SD number of LNs was doubled in the methylene blue stained group compared with the unstained group (35 ± 18 LNs and 17 ± 10 LNs, respectively). Although the unstained group showed an already high mean LN harvest, insufficient LN harvest after primary dissection occurred in 8 cases (27%). In contrast, only a single case in the methylene blue stained group (3%; P =.026) showed an insufficient LN harvest with 10 LNs. Despite a higher rate of locally advanced cases and a dramatically improved LN harvest in the methylene blue stained group, the identification of metastasized LNs after primary and secondary dissection was higher in the unstained group, with 10 vs 8 and 11 vs 8 cases, respectively. However, this difference is not statistically significant and is probably caused by chance. Upstaging after fat clearance was found in 3 cases (10%) exclusively in the unstained group. This is in accordance with the rates reported in other studies. 5,8,10 Kim et al, 26 in contrast, found no upstaging from N0 to N1 and no upstaging in LN+ cases with more than 12 investigated LNs after entire submission of residual mesenteric tissue. 26 They therefore doubt that this technique adds any additional information. It is hence worth emphasizing that in our study, 2 of the 3 upstaged cases showed primary sufficient LN harvest, including 1 case that was upstaged from N0 (0/17) to N1 (1/24). According to our findings, the recommended minimum number of 12 examined LNs might be too low. Although Haboubi et al 9 found on average 58 LNs per specimen by using the fat clearance technique, lower counts have been reported by others with mean numbers between 19 and ,25,27 Our own results in the unstained group after additional fat clearance (mean ± SD, 22 ± 11 LNs) are on the Am J Clin Pathol 2008;130:

6 Märkl et al / Methylene Blue Assisted Lymph Node Staging lower end of this range. This might be explained by the fact that this technique was not routinely used in our laboratory and the investigators were not experienced in it. Although the primary dissection produced an already much higher number of LNs in the methylene blue stained group, there was a nonsignificant trend toward finding more LNs after secondary preparation compared with the unstained group (8 ± 7 vs 5 ± 4). Further improved visibility of methylene blue stained LNs after fat clearance is the most likely explanation (Image 1). In this study, there was no change in the clinical stage after applying fat clearance following a primary dissection with methylene blue injection. Therefore, we believe that an LN dissection with the aid of intra-arterial methylene blue injection does not need to be combined with a fat clearance technique to further increase the LN yield. The mean ± SD total time effort for primary dissection was significantly higher in the methylene blue stained group, with 17.8 ± 7.1 minutes, than in the unstained group, with 10.9 ± 4.1 minutes. However, the mean time per LN was significantly lower in the methylene blue stained group (0.6 vs 0.9 minute). This finding indicates that the higher absolute time effort in this group is caused by the injection and, more important, by the greater number of LNs that had to be cut out. Considering that this already favorable time/ LN ratio of the methylene blue stained group includes the time for the injection, it seems clear that the higher LN harvest is not an effect of a potentially more careful dissection. Compared with rectal specimens and other regions of the colon, the injection was slightly more difficult in right hemicolon specimens in which a second injection into a more distally located artery was more often necessary. Nevertheless, the mean ± SD time needed for performing the methylene blue injection was short, 2.4 ± 1.4 minutes. The injection was successful in all but 1 case, in which an extravasate occurred. For practice, it is important to mention that the injection can also be performed in the operating room. Furthermore, the injection after a short formalin fixing of less than 4 hours is possible and successful. Finally, this prospective and randomized study proves that intra-arterial methylene blue injection is a very simple and highly effective technique to improve LN harvest in colon specimens. Because conventional techniques cannot guarantee adequate LN staging, we recommend methylene blue assisted LN dissection as a routine technique in the histopathologic examination of colorectal specimens. From the 1 Institute of Pathology, 2 Department of Visceral Surgery, and 3 Medical Clinic II, Klinikum Augsburg, Augsburg, Germany. 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