Evaluation of ex-vivo methylene blue injection into the inferior mesenteric artery to improve lymph node harvest in rectal cancer

Evaluation  of ex-vivo methylene blue injection into the inferior mesenteric  artery to improve lymph node harvest in rectal cancer

Khaled S Abbas(a), MD, PhD; Suzan M Helal  (bJ, MD, PhD; Walid El Shazry(a), MD, PhD; HeshamA Elwan(a), MS; Muhammad S Ellabishi(a), MD, PhD

(a) Department of Surgery, University of Alexandria, Egypt.

(b) Department of Pathology,  University of Alexandria, Egypt.

Purpose:  Exact lymph  node  staging is essential  for prognosis  estimation  and  treatment stratification in rectal cancer. Recent  studies showed that ex-vivo injection of methylene  blue dye into the inferior mesenteric artery of rectal cancer specimens might improve lymph node harvest. This study evaluates pathological lymph node assessment using this recent technique compared to conventional pathological techniques.

Methods:Methylene  blue solution was injected ex-vivo into the inferior mesenteric artery of

25 rectal cancer specimens and lymph node assessment was performed after formaline fixing overnight. The results were compared to data obtained from a control group of25 rectal cancer specimens which underwent conventional pathological lymph node assessment.

Results: Methylene  blue injection was successfully performed in all patients in the stained group. A total number of 383 (15.32 ±4.28) and 157 (6.28 ±2.79) lymph nodes were detected in the stained and unstained groups respectively (p< 0.001). The difference was most pronounced in lymph nodes measuring -:!:_4  mm in diameter (p< 0.001). Metastases  were found in 154 (6.16

±6.76) and 37(1.48 ± 2.86)  lymph nodes occurring in 18 and 10 patients in the stained and unstained  groups, respectively  (p=  0.003). Lymph node ratio (LNR) was calculated  for the patients who were finally staged as stage III showing no significant difference between the two groups.

Conclusions: Ex-vivo  methylene  blue injection  into  the  inferior  mesenteric  artery  is  a simple, easy and safe method that significantly improves lymph node harvesting in rectal cancer, especially small-sized lymph nodes.

Key words: Methylene blue, lymph node, rectal cancer.

Introduction:

In  Egypt,  colorectal cancer  is the  fourth most  commonly  diagnosed  cancer   in  both men and women.l Rectal cancer comprises approximately 30%  of  the  malignancies arising  in the  large bowel.2  Total mesorectal excision  (TME)  is the standard treatment of cancer rectum  and the total number of lymph nodes harvested in the surgical resection specimen  greatly  impacts   staging   accuracy and prognosis.3-4 TNM stage of rectal cancer with  exact  nodal  staging  correlates with survival  rates  and  prognosis estimation and

it is the foundation on which all treatment regimens  are based. 5

Complete  surgical   resection   for  stage   I rectal  cancer  is considered curative  therapy, and  the   five-year   relative   survival   rate  is around  90%.  However, stage  II and III rectal cancers  have five-year  survival  rates of only

67 and  54%,  respectively.6  A hypothesis for

the  relative   decrease  in  survival   in  stage  II rectal  cancer  is  that  some  patients  may  be under staged  as node-negative when they  are actually node-positive.7 Moreover,  in many patients who are not treated with preoperative

radiation or radio-chemotherapy the decision for adjuvant chemotherapy is made by the lymph   node   status.8,9   Evidence   suggests that patients, particularly in stage II rectal cancer,  with  a reduced  lymph  node harvest have a worse prognosis. In addition, 5-year overall survival and disease-free survival are significantly reduced in patients with low lymph node harvest.1D-11 Also, several studies have demonstrated a similar association between survival and lymph node harvest in stage III disease.12-13

Therefore,     Lymph    node     assessment is   an  essential   part  of  staging   in  rectal cancer.  In  fact,  nodal  involvement   is  the single most important prognostic factor in colorectal  carcinoma.10,14 The  accuracy  of (N)  staging  increases  with  the  number  of lymph nodes examined_15 Numerous studies have demonstrated  an improvement in the overall  survival  and/or disease-free  survival of colorectal cancer patients with increasing number of lymph nodes retrieved for examination.16,17 Furthermore,  the  number of  lymph  nodes  examined  in  rectal  cancer is considered an independent factor for locoregional disease whether metastases are present or not.18

There is debate regarding the optimal number oflymph nodes required for adequate staging. The evaluation  of at least 12 lymph nodes following  colorectal  cancer  resection is widely cited in clinical guidelines. This number was first proposed in 1990 by the Working Party Report to the World Congress of  Gastroenterology  in  Sydney.19 However, this is not a scientific biological figure and is a grade C recommendation  based on level III­ IV evidence.2D-22

Ex-vivo       intra-arterial       mJection       of methylene  blue solution in order to improve the lymph node harvested in the pathological examination  of colorectal  cancer specimens was  initially   introduced   by  Bruno  Miirkl et  a1.23 The  injected  methylene  blue  stains arterial  blood  vessels  and  capillaries.  This is  one  of  the  main  reasons  for  choosing methylene   blue   solution   to   improve   the visualization   of  lymph  nodes  because  the density  of vessels is much  higher  in lymph

nodes in comparison  to the surrounding  fat. It is not entirely clear how the solution passes into the lymph nodes when injected into the specimens  ex-vivo  after  the  blood  stream is  disjuncted.  The  most  likely  mechanism could be the  increase of interstitial  pressure caused by the injection and therefore induced lymphatic fiow24,25 several studies concluded that this method is effective, simple and time and cost effective.23-25

This study evaluates pathological lymph node   assessment   using   ex-vivo   injection of methylene blue dye into the inferior mesenteric artery of rectal cancer specimens compared to conventional pathological techniques.

Patients and methods:

This study was carried out on 50 patients with resectable rectal cancer who were allocated   into  two   groups,   stained   group with ex vivo methylene blue injection then pathological assessment and unstained group with conventional pathological assessment. Patients      who      underwent      emergency surgery, palliative surgery and patients with recurrent  rectal  carcinoma  were  excluded. All the patients  included  in this  study were subjected to history taking, thorough clinical examination  and collection of available data from the investigations. Total mesorectal excision (TME) was done to patients with middle and lower rectal tumors while patients with   upper   rectal  tumors   were   subjected to tumor specific mesorectal excision. All specimens were sent fresh to pathology after randomly  assigned  to  undergo  injection  of the inferior mesenteric artery with methylene blue dye (stained group) or a gross pathologic dissection of the lymph nodes (unstained group).

The  inferior  mesenteric   artery  of  each

specimen of the stained group was cannulated ex-vivo  by the surgeon with  a standard  16-

20G   intravenous   catheter   using   only  the plastic tube portion, followed by the injection of 15 to 20 ml of methylene blue solution (50 mg diluted with 0.9% saline in the ratio 1:3). Subsequently,  all  specimens  were  fixed  in

10% buffered formalin for 24 hours. (23-25)

Figures (1-3).

Beginning from the proximal end, the specimens were cut in 5 to 7 mm thick slices until the end of the tumor region was reached and  representative    areas   were   embedded using  the  whole  mount  technique.  The  fat of  the  remaining  parts  was  then  dissected and examined by palpation. The fatty tissues were then sliced and stretched to create thin, transparent layers. The cut surfaces were screened for lymph nodes.

After paraffin embedding,  3 11m sections

were cut and stained with hematoxylin and eosin. Slides were then examined for the number and size of lymph nodes and the presence of metastases.

Statistical analysis: Data were fed to the computer and analyzed using IBM SPSS software  package  version  20.0.  Qualitative data were  described  using  number  and percent.   Quantitative   data  were  described using mean and standard  deviation, median, minimum and maximum.

Comparison    between    different   groups

regarding  categorical   variables   was  tested using Chi-square test. When more than 20% of the  cells  have expected  count  less  than

5, correction for Chi-square was conducted using Fisher's Exact test or Monte Carlo correction.

For normally distributed data, comparison

between  two  independent  population  were done  using  independent  t-test  while  more than two population were analyzed F-test (ANOVA) to be used. For abnormally distributed data, comparison between two independent  population  were done using Mann  Whitney   test   Correlations   between two  quantitative  variables  and ordinal  data were assessed using Spearman coefficient. Multivariate Linear regression was assessed. Significance test results are quoted as two­ tailed  probabilities.   Significance  of  the obtained results was judged at the 5% level.

Results:

This study included 50 patients with resectable  rectal  cancer  who  were  divided into two groups, stained group whit ex vivo methylene  blue  injection  then  pathological

assessment and unstained group with conventional pathological assessment. 56% (14) in each group were males with mean age

51.68 ±11.60 years (range, 25-74) and 51.04

±13.45  years (range,  26-74)  for the stained and unstained groups, respectively. The mean body  mass  index  (BMI)  was  27.56  ±1.94 for  the  stained  group,  compared  to  27.16

±1.65 for the unstained  group. There was no statistically significant differences between both groups in demographic data, tumor site, histological criteria and preoperative staging. In the stained group, 17 patients (68%) received neoadjuvant therapy, while in the unstained group, 18 patients (72%) received neoadjuvant therapy. Low anterior resection (LAR) was performed  in 18 patients (72%) in the stained group and 17 patients (68%) in the unstained group and rest of the patients underwent abdominoperineal resection. Operations were performed by 5 colorectal consultant surgeons and pathological assessment  was  performed  by  two pathologists. Differences between the two groups  regarding  the  surgeons  performing the  operations  and  pathologists   examining the resection spectmens were statistically insignificant.

Although     there      was     no      significant

difference between both groups regarding preoperative  clinical  nodal  staging  (eN)  (P

0.844), this difference become statistically significant  after  pathological  nodal  staging (pN) (p= 0.003). In the stained group, 14 patients (56%) were finally staged as pN2, while in the  unstained group,  only 3 (12%) patients were staged as pN2 and 15 patients (60%) were finally staged as pNO Table (1). On studying the comparison between eN and pN in each group, a statistical significant difference was identified only in the stained group (p= 0.009) Table (2).

In the stained group, a total of 383 lymph

nodes were identified with a mean of 15.32

±4.28,  compared  to  a total  number  of  157 lymph   nodes   identified   in  the   unstained group   with   a  mean  of  6.28   ±2.79.   The difference   between   the   two   groups   was highly statistically  significant (p< 0.001). At least  12 lymph  nodes  were  identified  in 21

out of25 patients (84%) in the stained group. However, in the unstained group, adequate nodal harvest was found in only 3 out of 25 patients (12%) Table (3).

Metastases   were   found   in   154   lymph nodes in 18 out of 25 patients  (72%) in the stained group with a mean of 6.16 ±6. 76 and in 37 lymph nodes in 10 out of 25 patients (40%) in the unstained group with a mean of

1.48 ±2.86. The difference between the two groups was statistically significant (p= 0.003) Table (3).

LNR  was  calculated  for  these  patients with  positive  lymph  nodes  and  a  mean  of

0.53 ±0.32 and 0.41 ±0.32 were found for the stained and unstained groups respectively, the difference was not statistically significant. Lymphovascular   invasion   was  detected  in

16 out of25 patients (64%) and 15 out of25

patients (60%) in the stained and unstained groups respectively.

The  lymph   nodes  were   measured   and

categorized  by size. The difference  in nodal harvest between the two groups was most pronounced  in  lymph  nodes  measuring  ::;4 mm in diameter. The difference for the <2 mm and 3-4 mm categories was statistically highly significant (p< 0.001). Forlymph nodes in the

5-6 mm category there was also a statistical significant difference between the two groups (p= 0.008). However, for the lymph nodes in the  >6 mm category the difference  between the stained and unstained groups was not statistically significant Figure (4).

On      studying     the     relation      between

neoadjuvant therapy and lymph node harvest in the two groups, the effect of neoadjuvant therapy  on  reducing  the  nodal  harvest  was statistically   significant  only  in  the  stained group (p< 0.001) with a mean nodal harvest of  19.88  ±2.95  in the  patients  who  didn't receive   neoadjuvant   therapy   compared   to

13.18  ±2. 90  in  the   patients  treated   with

neoadjuvant  therapy. In the unstained group there was no significant difference  in lymph nodes harvest (p=O.752) in patients who received   neoadjuvant   therapy    and   those who directly underwent surgery (6.17 ±3.17 vs6.57 ±1.62).

In the present study, out of 17 patients in

the stained group who received neoadjuvant therapy   13  patients  (76.5%)  had  adequate nodal   harvest.   Whereas,   none   of  the   18 patients in the unstained group who received neoadjuvant therapy had adequate nodal harvest.

The  relationships  of  other  factors  with

lymph node harvest were also studied as these factors can affect lymph node retrieval. These factors include surgeon variation, pathologist variation, type of the operation, tumor site, histological type and grade of the tumor and lymphovascular invasion. However, none of these  factors  had  a  statistically  significant effect on nodal harvest neither in the stained nor in the unstained groups.

Linear regression for the effect of neoadjuvant therapy and methylene blue injection    on    lymph    node    harvest    was studied  revealing  a  more  powerful  impact of methylene blue injection on nodal harvest than neoadjuvant therapy Table (4).

Discussion:

It was proved by Wong etal that accuracy of lymph node status increases with the number of  examined  lymph  nodes.26 Additionally, Swanson  et  al.  showed  a  very  impressive linear correlation between increasing numbers of lymph nodes examined and 5-year survival rates in T3NO colorectal cancer.27

Many   studies  tried   to   improve   lymph node harvest by increasing sensitivity of metastasis detection using sentinel lymph node technique,     immunohistochemical    analysis and  polymerase  chain  reaction  (PCR).28-30

Immunohistochemical  1s expensive  and failed to prove a significant difference concerning   3-year   disease-free   survivaPl In contrast, confirmation of micrometastasis using RT-PCR showed  a significant  smaller

3-year   disease-free   survival   and   reduced

overall survival in positive patients. Fat clearance protocols are an effective way to improve lymph node recovery. Despite that, many  of  these   protocols  are  difficult  and time   consuming.   Even  more   problematic is the additional necessity of poisonous chemicals that need to be handled.  Both are circumstances   that   limit  their   widespread

Figure (1): Identification of the DvfA.

Figures (2-3): Injection of methylene blue solution into the DvfA.

I•Slained •Uns ained I

<2mm                  3-4mm

.S-6n1m                  mm

Figures (4): Comparison between both groups according to size ofL.Ns harvest.

use.32,33

A  new  and  simple  teclmique  was desctibed to stain lymph nodes in rectal cancer specimens by ex vivo methylene blue injection into the inferior mesenteric arte1y making them easier to detect by conventional pathological dissection. This teclmique was

first described by Markl et a1.23

Neo-adjuvant therapy, smgeon and pathologist vruiation and type of operations ru·e   factors  that  affect  number  of  lymph nodes hru·vested. In this study there was no significant difference between the two groups regru·ding those factors.34·38

Table (1): Comparison between the studied groups regarding clinical and pathological nodal

staging.

Table (2): Comparison between clinical and pathological  nodal staging in each group.

rs: Spearman coefficient

*: Statistically significant at p :S 0.05

Table (3): Comparison of lymph node harvest in both groups.

t: Student t-test

MW: Mann Whitney test

Table (4): Linear regression for total L.Ns.

R= 0.840, R2= 0.706, F= 56.320*, p< 0.001

In the present study, the process of methylene blue injection into the inferior mesenteric artery was simple, easy and safe method as it was successful in all patients in the stained group with an average time of less than 4 minutes. In addition to the availability and low cost of methylene blue dye possesses no hazardous or poisonous effects. This coincides with the results of similar studies assessing  this technique.  Kerwel  et  al. described  this technique  to  be simple, time and cost-effective and should be reproducible in other institutions, particularly where inadequate nodal harvests are problematic.39

This  study   shows   improvement   in  the mean  lymph  node  harvest  in  the  stained group as compared with the unstained group (15.32  ±4.28 vs.  6.28  ±2.79, p<  0.001).  In

84% of patients in the stained group, lymph node recovery was more than 12 whereas this was achieved in only 12% of patients in the unstained   group.  This  major  improvement in nodal harvest following methylene blue staining of lymph nodes was similarly found by other  published  studies. for example,  in the original  retrospective  study by Miirkl et al. including 24 rectal cancer specimens, the average  number  of lymph  nodes  examined was 27 ±7 and 14 ±4 for the stained and unstained  groups, respectively.23 An average lymph node harvest of 30 ±13.5 for the methylene blue stained group compared to 17

±11 for the unstained  group were found in a

prospective study by Kerwel et al. involving

50 patients with primary resectable rectal cancer.39 A study by Klepsyte et al. including

40 rectal cancer specimens also demonstrated an   improvement   in   lymph   node   harvest following  methylene  blue  staining  with  an average lymph  node numbers  per specimen

of  18  ±5  and  14  ±6  in  the  stained  and unstained   groups,  respectively.24 However, on comparing the results of the present study with  other  published  results,  it seems  clear that nodal harvest in this study is the lowest among all, whether before or after methylene blue  lymph  node  staining.  This  indicates  a more obvious need to improve lymph node harvests  aiming  at  a  more  accurate  rectal cancer staging.

Looking  at  the  additional  lymph  nodes

identified in the stained  group, the  increase was  attributed  to  identifying  more  of  the small  lymph   nodes.  Kerwel   et  al.  found similar  results  as  the  largest  improvement was  found  in  size  groups  between  1  and

4  mm  causing  a  shift  in  size  distribution

toward smaller  nodes.39 The significance of the smaller  lymph nodes is that studies have reported that 66 to 78% of metastatic lymph nodes are smaller than 5 mm.28,40-42

Otherfindingin thisstudywas improvement of the number of metastatic lymph nodes identified  in the  stained  group.  Kerwel  et al.,  found   no  significant   improvement   in the detection of nodal metastasis. This was attributed  to  the  finding that  nodal  harvest in  the  unstained  group   was  already   very good and well above the reported averages obtained with conventional techniques.39

To     date,     thorough      examination      of postoperative   specimens   in  trial  to  obtain the   maximum   number   of   lymph   nodes is  the  optimal  method  for  accurate  nodal staging.     Several     authors     claim   that     an insufficient      number             of           identified               and examined  lymph  nodes is  one of  the  main causes  of understaging  in colorectal  cancer. The stage migration theory  is based on this

concept.43  In the  present study, nodal stage

migration was significant in the stained group (p=0.009). Two patients  in the stained  group were  preoperatively staged  as node-negative and   ended   up  to  be  node-positive  and   5 other  patients  were  upstaged  from  N1 to N2 after pathological assessment. While in the unstained group, only one case was upstaged from  NO to Nl.

A  study   by   Markl   et  al.  involving  53 rectal and 1 colon cancer patients who had received  neoadjuvant therapy demonstrated that adequate  lymph node harvest can be achieved after methylene blue staining  even in patients  treated  with  neoadjuvant therapy. The mean number oflymph nodes was 29 ±11 and 10 ±4 for the methylene blue stained  and unstained  groups,   respectively.44   Similarly, in the present study, there was a significant improvement in nodal harvest  in patients received    neoadjuvant   therapy    in   stained group  13.18 ±2.90 compared to unstaining group  6.17 ±3.17,  (p< 0.001).

Baxter  et al. have  reported  that only  20% of  patients  treated  with  neoadjuvant therapy have  adequate nodal  sampling.34  In the present study, 76.5% of patients in the stained group  who received  neoadjuvant therapy had adequate  nodal   harvest   whereas,   none   of the 18 patients  in the unstained group  who received   neoadjuvant therapy  had  adequate nodal harvest.

Lymph   node   harvest   in  the  patients   in the stained group who received neoadjuvant therapy was significantly lower than  patients who    didn't    receive    neoadjuvant  therapy (13.18 ±2.90   vs.   19.88   ±2.95,   p<  0.001), which  is consistent with published  studies documenting a reduced  lymph node harvest in patients treated with neoadjuvant therapy. 34-36

Interestingly, a similar trend was not observed in the unstained group. This finding maybe attributed to the assumption that neoadjuvant therapy doesn't cause the lymph  nodes to disappear but rather  become  smaller  and therefore more difficult to detect.44 However, in the present  study, methylene blue injection proved  to  have  a more  powerful  impact  on nodal harvest than neoadjuvant therapy.

Conclusion:

Ex-vivo  methylene blue injection into the inferior  mesenteric artery  is  a simple,  easy and safe method that significantly improves lymph node harvesting in rectal  cancer, especially small-sized lymph  nodes. This improvement in nodal harvest can be even achieved in patients  treated  with neoadjuvant therapy.  This eventually leads to a more accurate rectal cancer staging via improved detection of metastatic lymph nodes.

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