doi:10.1111/codi.12681
Original article
Intra-arterial methylene blue injection into ex vivo colorectal cancer specimens improves lymph node staging accuracy: a randomized controlled trial D. W. Borowski*, B. Banky*†, A. K. Banerjee*‡, A. K. Agarwal*, M. A. Tabaqchali*, D. K. Garg*, C. Hobday§, M. Hegab¶ and T. S. Gill* *Department of Colorectal Surgery, North Tees and Hartlepool NHS Foundation Trust, University Hospital of North Tees, Stockton-on-Tees, UK, †Department of Surgery, St Borbala Hospital, Tatabanya, Hungary, ‡Department of Surgery, Raigmore Hospital, Inverness, UK, §Department of Pathology, Northumbria Healthcare NHS Foundation Trust, North Tyneside General Hospital, North Shields, UK and ¶Department of Histopathology, North Tees and Hartlepool NHS Foundation Trust, University Hospital of North Tees, Stockton-on-Tees, UK Received 16 January 2014; accepted 15 April 2014; Accepted Article online 9 June 2014
Abstract Aim A randomized controlled trial was carried out to study the effect of a recently proposed technique of ex vivo intra-arterial methylene blue injection of the surgical specimen removed for colorectal cancer on lymph node harvest and staging.
the intervention was particularly observed in the patients who underwent preoperative neoadjuvant radiotherapy [median 30 nodes (12–57) vs 11 (7–15); P = 0.011; proportion of cases with < 12 nodes 0/5 vs 5/8 (62.5%), P = 0.024].
Method Between May 2012 and February 2013, 100 consecutive colorectal cancer resection specimens in a single institution were randomly assigned to intervention (methylene blue injection) and control (standard manual palpation technique) groups before formalin fixation. The specimen was then examined by the histopathologist for lymph nodes.
Conclusion Ex vivo intra-arterial methylene blue injection increases lymph node yield and can help to reduce the number of cases with a lower-than-recommended number of nodes, particularly in patients with rectal cancer having neoadjuvant treatment. The technique is easy to perform, cheap and saves time.
Results Both groups were similar for age, sex, site of tumour, operation and tumour stage. In the intervention group, a higher number of nodes was found [median 23 (5–92) vs 15 (5–37), P < 0.001], with only one specimen not achieving the recommended minimum standard of 12 nodes [1/50 (2%) vs 8/50 (16%), P = 0.014]. However, there was no upstaging effect in the intervention group [23/50 (46.0%) vs 20/50 (40.0%); P = 0.686]. With a significantly lower number of nodes harvested in rectal cancer, the positive effect of
Keywords Colorectal cancer, lymph node harvest, methylene blue, staging, randomized controlled trial What does this paper add to the literature? A new technique of ex vivo intra-arterial injection of methylene blue to improve lymph node harvest has recently been described. In this study, we assessed its efficacy in a randomized controlled trial and confirmed the efficacy of the technique to improve lymph node harvest in colorectal cancer.
Introduction Correspondence to: D. W. Borowski, Department of Colorectal Surgery, North Tees and Hartlepool NHS Foundation Trust, University Hospital of North Tees, Hardwick, Stockton-on-Tees TS19 8PE, UK. E-mail:
[email protected] The trial data was in part presented at the annual scientific meeting of the Association of Coloproctology of Great Britain and Ireland, Liverpool, UK, 1–3 July 2013.
According to current guidance [1,2], a minimum number of 12 lymph nodes should be examined in all colorectal cancer surgical specimens to maximize the identification of regional lymph node metastases [3]. As a consequence the number of harvested lymph nodes is being used as a benchmark for clinical audit
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to compare the quality of individual service providers [4]. Considerable variability between hospitals [5–7], surgeons [8] and histopathologists [9] has been observed, in addition to the known patient-related factors that positively influence lymph node harvest such as younger age [6,7,10,11], lower body mass index [6], lower comorbidity index [7], right-sided location [10–12] and greater tumour size [6,12]. Manual palpation of the surgical specimen is the standard technique used by histopathologists, but this may miss smaller nodes [13] and it is known that nodes smaller than 5 mm in diameter may account for up to half of metastatic nodes present [14]. In order to improve the lymph node harvest and reduce the non-patient-related variability, specimen preparation techniques such as fat clearance with xylene and alcohol [15] or acetone compression [16] have shown significant improvement in the number of lymph nodes harvested [15,17]. Fat clearance is time consuming, expensive and involves potentially hazardous substances. Studies on sentinel lymph node sampling of the colorectal region have failed to prove the efficacy of this technique in avoiding the examination of all lymph nodes in cases of node-negative disease [18,19]. Recently, a simple ex vivo injection of methylene blue solution into the feeding artery of the specimen has been described, resulting in significantly better lymph node detection in rectal [20] and colon cancer [21] compared with manual palpation. In this study, we randomly assigned 100 consecutive specimens from colorectal resections to either routine manual lymph node dissection or to postoperative
intra-arterial injection of methylene blue. The aim was to determine whether methylene blue injection, in our hands, could improve the lymph node harvest and N staging.
Method Between May 2012 and February 2013, 100 consecutive colorectal cancer surgical specimens were randomly assigned either to formalin fixation followed by manual dissection (control group) or to injection of methylene blue into the highest ligated artery in the operating theatre immediately after removal, followed by formalin fixation and manual dissection (intervention group). The CONSORT diagram is shown in Fig. 1. Randomization was carried out after removal of the specimen in the operating room, using sealed pre-prepared envelopes containing the study allocation (50:50). Previous specimen allocations and interim data were not known to the surgeons. According to the review by the internal research board, due to the ex vivo nature of the study intervention, no specific ethical approval or individual patient consent was required. All patients underwent standard colorectal resection either by laparoscopic or open surgery, following discussion by the multidisciplinary colorectal team. Whilst most patients had preoperative histological confirmation of malignancy, a few had been planned for surgery on the basis of suspicious radiological findings and were included in the analysis. All patients received standard peri-operative care, in most cases in a well-established enhanced recovery programme.
Patients undergoing planned colorectal resection of a suspected cancer following multidisciplinary team discussion N = 100
Randomised to intervention n = 50 Ex-vivo intra-arterial Methylene blue injection and formalin fixation n = 50 Intention to treat analysis n = 50 Benign n = 1 colectomy n=3
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Additional analysis n = 46
Randomised to control n = 50
Standard formalin fixation n = 50
Intention to treat analysis n = 50 Additional analysis n = 48
Benign n=2
Figure 1 CONSORT diagram outlining the trial.
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Methylene blue preparation
Statistical analysis
Following completion of surgical resection, all specimens in the intervention group were laid out on a table and the main feeding artery (or arteries) was carefully dissected at its highest point of ligation. It was then cannulated with a 16–20 gauge intravenous catheter which was secured with a surgical ligature (Fig. 2a). A pre-prepared solution of 50 mg methylene blue, diluted in 30 ml of sodium chloride (0.9%), was injected until dye was visible on the surface of the specimen (Fig. 2b). The specimen was subsequently fixed in formalin (10%) for 24 h and sent to the laboratory for routine histopathological examination. In the control group, the specimens were treated identically except for the methylene blue injection. No additional fat clearance methods were employed. Lymph nodes were confirmed only when a capsule was detected histologically.
All study data were securely stored on the hospital’s internal server, with adherence to the data protection principles of the National Health Service, England. Data were entered in Microsoft Excel 2003 (Microsoft Corporation, Redmond, WA, USA) spreadsheets and statistical analysis was carried out with SPSS version 20 (IBM SPSS, Armonk, New York, USA). From previously available data [22] we calculated the minimum sample size required to demonstrate a statistically significant difference with a power of 95% to be 36 in each group. With an annual workload of more than 150 colorectal cancer resections in our hospital, a total number of 100 cases in a 12-month period was judged to be a realistic target to establish our own practice and allow for a potential learning-curve effect. Data were analysed on an intention-to-treat basis. Patient-related and pathological variables were considered in univariate analysis for association with each of the outcomes. Significance was taken at the P ≤ 0.05 level. The chi-squared test and chi-squared test for trend were used for categorical variables and Student’s t test for continuous variables having a parametric distribution (e.g. age). For non-parametric data (e.g. lymph node yield) the Mann–Whitney U and Kruskal–Wallis tests were used. To establish the effect of the methylene blue intervention on lymph node detection, linear regression modelling was performed with multivariate adjustment for patient- and tumour-related factors.
(a)
Results Patient characteristics (b)
Patient age was normally distributed with a median age of 70 years (35–92). There was a slight male predominance (57/100, 57.0%). Both groups were similar with respect to patient age, sex, site of tumour, operation, surgical access, final Dukes stage, presence of synchronous tumour(s) and metastatic disease at surgery (Table 1). There was a slightly higher proportion of right-sided tumours in the control group and more subtotal colectomy specimens in the intervention group.
Surgeon and pathologist variability
Figure 2 Cannulation of the feeding artery and preparation of methylene blue injection (a) and appearance of the specimen following injection (b).
Five surgeons and seven pathologists were involved with the study. There was no significant variation in the number of lymph nodes harvested between individual surgeons (Fig. 3a) and the number of lymph nodes detected between individual pathologists (Fig. 3b). There was no significant difference in the number of
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Age (median) Sex (%) Female Male Comorbidity (%) ASA I ASA II ASA III ASA IV Tumour site (%) Right colon Left colon Rectum Operation (%) (Extended) Right colectomy Left hemicolectomy Sigmoid colectomy/high anterior resection Low anterior resection APER (Sub) total colectomy Access (%) Open Laparoscopic conversion Laparoscopic Dukes stage (%) A B C D Benign histology Synchronous tumour (%) Not present Present Distant metastases (%) Not present Present
Control
Intervention
P
71 years (38–91)
69 years (35–92)
0.871 0.841
21 (42.0) 29 (58.0)
Table 1 Clinical details of the control (n = 50) and intervention groups (n = 50).
22 (44.0) 28 (56.0) 0.942
4 23 18 5
(8.0) (46.0) (36.0) (10.0)
5 26 143 5
(10.0) (520.0) (286.0) (10.0) 0.348
22 (44.0) 12 (24.0) 16 (32.0)
16 (32.0) 16 (32.0) 18 (36.0)
22 (44.0) 3 (6.0) 17 (34.0)
16 (32.0) 6 (12.0) 21 (42.0)
6 (12.0) 2 (4.0)
2 (4.0) 2 (4.0) 3 (6.0)
5 (10.0) 4 (8.0) 41 (82.0)
6 (12.0) 44 (88.0)
14 13 17 4 2
8 17 20 4 1
0.195
0.123
0.596 (28.0) (26.0) (34.0) (8.0) (4.0)
(16.0) (34.0) (40.0) (8.0) (2.0) 0.727
46 (92.0) 4 (8.0)
45 (90.0) 5 (10.0)
46 (92.0) 4 (8.0)
46 (92.0) 4 (8.0)
1.0
APER, abdomino-perineal excision of the rectum.
cases randomized to the intervention group for surgeons [surgeon 1, 6/18 cases (33.3%); 2, 16/26 (61.5%); 3, 11/25 (44.0%); 4, 10/16 (62.5%); 5, 7/15 (46.7%); P = 0.307] and pathologists [pathologist 1, 10/22 cases (45.5%); 2, 2/8 (25.0%); 3, 8/15 (53.3%); 4, 13/22 (59.1%); 5, 11/18 (61.1%); 6, 2/7 (28.6%); 7, 4/8 (50.0%); P = 0.525]. Lymph node harvest
The median overall lymph node harvest was 18 (5–92). The number of nodes harvested was significantly higher in the methylene blue group [median 23 (5–92)]
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compared with the control group [median 15 (5–37); P < 0.001; Fig. 4].The proportion of lymph-node-positive disease was not significantly greater in the methylene blue injection group (23/50, 46.0%) compared with the control group (20/50, 40.0%; P = 0.686). In the control group there were eight (two node-positive) cases with less than 12 detected nodes (8/50, 16.0%) and only one (node-positive) in the intervention group (1/50, 2.0%; P = 0.014). Thus, node positivity in the accurately staged control group was 20/44 (45.5%), compared to 23/50 (46.0%, P = 0.887). Three patients were found to have benign disease and three in the methylene blue group had a total
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(a)
Number of harvested nodes
100
80
60
* *
40
20
0 1
2
3 Surgeon
4
5
(b) 100 Number of harvested nodes
*
Figure 3 Overall surgical lymph node harvest by surgeon (a) and lymph node detection by histopathologist (b). *Indicates extreme values.
80
60
40
20
0 1
2
3
4 Pathologist
Figure 4 Lymph node detection in control (left) and intervention (methylene blue) groups (right); Poisson distribution is shown.
6
7
Methylene blue
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0 10
8
6 4 Frequency
2
0
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2
4 6 8 Frequency
Number of harvested nodes
Number of harvested nodes
No dye/control
5
10
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colectomy. Analysis of the data in the remaining 94 cases confirmed the higher lymph node detection in the methylene blue group [median 23 (5–92)] compared with the control group [median 15 (5–37); P = 0.001]. Patient and tumour characteristics
The number of examined lymph nodes in all patients was highest in patients with right-sided colon cancer [median 20.5 (12–37)] compared with left colon [median 18.5 (10–48)] and rectal cancer [median 14.5 (5– 92)]. In right-sided cancers no significant increase in the number of harvested lymph nodes with injection of methylene blue was found, in contrast to left colon and rectal specimens (Table 2). For all tumour sites, the number of detected lymph nodes showed a weakly positive association with the size of the primary tumour (LN = 15.9 + 0.261 9 size (mm), P = 0.010; R2 = 0.068) and reduced with the patient’s age (LN = 45.5 0.327 9 age (years), P = 0.001; R2 = 0.107). There was also an association between the patient’s level of comorbidity and lymph node harvest [ASA I, median 28 (10–92); ASA II, 18 (5–57); ASA III, 15 (5–26); ASA IV, 19.5 (12–56); Kruskal–Wallis P = 0.004], but there was no significant difference between males and females [median 16 (5–92) vs 20 (5–57), P = 0.625]. In multivariate regression analysis, adjusting for the patient’s age, sex, ASA grade, tumour site and size, methylene blue injection was significantly associated with a higher lymph node yield (Table 3).
Table 2 Number of harvested lymph nodes in relation to tumour site.
Right colon Control Intervention Left colon Control Intervention Rectum Control Intervention All cases Control Intervention *Mann–Whitney U.
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Number of lymph nodes (median)
Range
19.5 21.0
12–37 12–31
13.5 27.0
10–21 12–48
12.5 20.0
5–28 5–92
P* 0.895
< 0.001
0.003
< 0.001 15.0 23.0
5–37 5–92
Table 3 Multivariate regression model demonstrating the effects of tumour, patient and methylene blue intervention on lymph node detection (n = 100); R2 = 0.362.
B* Age (per annum) 0.243 Male gender 1.073 (vs female) ASA grade (vs ASA I) II 10.28 III 14.43 IV 9.90 Tumour site (vs right colon) Left colon 0.629 Rectum 2.339 Tumour size 0.174 (per mm) Methylene blue 6.514 intervention (vs control) Constant 38.2
Standard error (B)
b**
P
0.113 2.256
0.223 0.043
0.033 0.636
4.029 4.562 5.574
0.415 0.541 0.242
0.012 0.002 0.079
2.845 2.918 0.061
0.023 0.089 0.277
0.826 0.425 0.005
2.194
0.263
0.004
7.94
< 0.001
Bold values indicate significant associations. *B is the unstandardized regression coefficient. **b is the standardized regression coefficient.
Effect of neoadjuvant treatment on rectal cancer
Of the 34 cases with rectal cancer, 11 (32.4%) had had preoperative long-course chemoradiotherapy and two (5.9%) had short-course radiotherapy. There was no significant difference in the number of nodes between the patients who had no neoadjuvant treatment [median 16.0 (5–92)] compared with those who did receive treatment [median 14 (7–57); P = 0.420]. Amongst the patients who had no neoadjuvant treatment, there was no significant effect associated with the injection of methylene blue [median 18.0 (5–92)] compared with the control group [median 13.0 (5–28); P = 0.185]. In contrast, amongst patients who underwent neoadjuvant treatment there was a higher lymph node yield seen in the intervention group [median 30 (12–57, n = 5) vs control, 11 (7–15, n = 8); P = 0.011]. Particularly amongst patients who had neoadjuvant treatment, there was a significant reduction in the proportion of cases with less than 12 lymph nodes when methylene blue was used [control, 5/8 (62.5%) vs intervention, 0/5 (0%); P = 0.024].
Discussion M€arkl et al. have recently proposed the ex vivo intraarterial injection of methylene blue following colorectal cancer resection as a simple technique to aid the histo-
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pathological detection of mesocolic [21] and mesorectal [20] lymph nodes. In the present randomized controlled study a significant increase in the number of detected and examined lymph nodes was found following methylene blue injection compared with the standard manual palpation technique. The technique was also shown to help the detection of lymph nodes in specimens where palpation identified insufficient nodes to reliably exclude node-negative disease. In particular this includes rectal cancers receiving preoperative neoadjuvant treatment, known to be associated with lower nodal yield [6]. The presence of lymph node metastases has long been one of the most powerful prognostic predictors of colorectal cancer [23] and influences the indication for adjuvant chemotherapy. The number of examined lymph nodes is independently associated with survival both in node-positive and node-negative disease, a relationship that seems not to be sufficiently explained by staging inaccuracies [8,24,25]. The suggested minimum total number of lymph nodes has varied considerably between six and 21 nodes [17,25–35], but many clinical guidelines have taken Nelson’s extrapolation to recommend a minimum of 12 nodes to make regional nodal spread unlikely in 90% of cases [3]. In clinical practice, however, this number is not often achieved [4,10,33,36], and there is therefore a potential for the understaging of patients who are actually at risk of under-treatment. To increase lymph node detection, several fat clearance techniques have been successfully introduced [15,17], but their widespread use appears to be limited due to cost, time consumption and the use of potentially harmful substances [21,37]. In comparison, the methylene blue technique is cheap, requires no additional histopathologist time and can be easily learned and included in routine operating theatre practice. From our own experience, some specimens may require a ‘second-look’ or fat clearance technique to maximize the number of nodes found, but using methylene blue is likely to result in significant time and cost savings while optimizing accuracy. A significant reduction in the time spent by the manually examining pathologist has been reported [21] and this may be of great benefit in institutions where there is pressure on resources and the recommended minimum number of nodes is not regularly achieved. In our control group 12% of specimens were staged N0 with fewer than 12 negative nodes examined compared with no case of N0 stage in specimens with fewer than 12 negative nodes in the intervention group. This agrees with the observation of M€arkl et al. [21] that 26% in the unstained control were potentially understaged compared with only 3% when methylene blue was used.
Methylene blue to improve lymph node staging accuracy
In contrast with the 10% upstaging reported by M€arkl et al. [21] in the unstained group following additional secondary fat clearance, we found no difference in the proportion of node-positive disease between the two groups, indicating that involved lymph nodes generally would have been be found by manual palpation. However, the 6% higher rate of node-positive specimens in the intervention group might indicate a significant difference in a larger sample size. All five participating surgeons are part of the colorectal cancer multidisciplinary team and regularly perform laparoscopic surgery for suitable patients. All patients with colon cancer were operated according to the principle of total mesocolic excision [38], and patients with mid and low rectal cancers had a full total mesorectal excision [39] or extra-levator abdomino-perineal excision [40]. Our study population is comparable to most colorectal cancer patient cohorts in the UK and this allowed us to examine some of the previously described factors affecting lymph node harvest in both study arms. Analysis confirmed the previously described positive effect of younger age [6,7,10,11], right-sided tumour site [10–12] and the size of the primary tumour [6,12]. Potentially, there may have been some collinearity between age and comorbidity, but conclusive evidence was beyond the remit of this trial and would require a much larger sample size. In the group of rectal cancers, there was no significant effect on the overall lymph node number whether or not preoperative neoadjuvant treatment was given. This contrasts with the finding of Mekenkamp et al. [6] but is not surprising given the small sample size. Whilst this study was powered to detect the effect of methylene blue injection on the overall lymph node harvest, it is possible that any effect due to the detection of small positive lymph nodes may have been missed due to the sample size. It did include three patients who underwent more than a segmental colorectal resection and three with no preoperative histological confirmation of a cancer diagnosis who ultimately had a benign diagnosis. Although this appears not to have affected the overall findings and conclusion, future studies must take account of this possible source of bias. Ex vivo intra-arterial injection of methylene blue achieved a significantly higher overall lymph node yield and a significant reduction in the proportion of cases with fewer than the minimum recommended number of examined nodes. Particularly in the group of rectal cancer patients having neoadjuvant treatment, methylene blue injection led to a significant reduction in the number of cases with fewer than 12 examined negative nodes, compared with control. The technique is easy to perform, cheap and can save time for the histopatholo-
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gist. A more widespread adoption of the technique is recommended and further evaluation in larger patient population studies to determine the potential for upstaging and the effect on long-term survival.
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Author contributions D.W. Borowski: study concept/design, acquisition of data, analysis and interpretation, writing. B. Banky: study concept/design, acquisition of data, analysis and interpretation, writing. A. K. Banerjee: acquisition of data, analysis and interpretation. A. K. Agarwal: acquisition of data, writing. M. A. Tabaqchali: acquisition of data, writing. D. K. Garg: acquisition of data, writing. C. Hobday: study concept/design, acquisition of data, writing. M. Hegab: acquisition of data, analysis and interpretation, writing. T. S. Gill: study concept/design, acquisition of data, analysis and interpretation, writing.
10
11
12
13
Conflict of interest The authors declare no competing or conflicting interest, and have no financial benefit from this work.
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Methylene blue to improve lymph node staging accuracy
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