Colorectal cancer (CRC) is the second most common cause of cancer death worldwide [1]. Currently, the cornerstone of treatment is surgery with curative intent, and as such, en bloc removal of the local lymph node (LN) field is a key aspect of the operation [2]. Operative perfection of lymph node clearance, called complete mesocolic excision (CME) in the case of colon lesions, and total mesorectal excision (TME) in the case of rectal tumors, is associated with survival benefit and local recurrence reduction [3, 4]. Accurate lymph node staging based on histopathologic examination of each available lymph node (or as many nodes as possible) is recommended both as a prognostic tool and decision basis of adjuvant chemotherapy [2, 5, 6]. In 2009, the Union for International Cancer Control (UICC) recommended at least 12 lymph nodes to be harvested and reported, in order to adequately exclude node-positive disease [7]. Differentiating between stage II and stage III colorectal cancer is paramount, as most stage II CRC requires no adjuvant treatment, while surgical removal of stage III cancers with at least one metastatic lymph node or presence of extranodal tumor deposits should usually be followed by chemotherapy [2]. In the case of insufficient lymph node retrieval from the surgical specimen, node-negative tumors leave some uncertainty regarding the reliability of a pN0 stage. Incorrect classification may lead to inadequate postoperative management either way, which might contribute to a worse prognosis [8]. The estimated understaging rates may be as high as 6% in some studies [9, 10]. In particular, the subset of patients who undergo pre-operative chemo-irradiation may have fewer lymph nodes due to the effect of neoadjuvant treatment (NAT), but may also have better outcomes despite suboptimal lymph node harvest and examination [11]. Other studies have previously demonstrated that an increased number of histopathologically examined lymph nodes seems to be associated with better long-term survival [8, 12, 13].

The obvious need for accurate lymph node staging has led to the development of several techniques to improve the lymph node yield. The traditional visualization and manual palpation of the mesocolon for lymph nodes is performed first by the pathologist. However, 80% of mesorectal lymph nodes are smaller than 3 mm, and at least half of metastatic lymph nodes are smaller than 5 mm [14, 15]. Additional methods employed by the pathologist include fat clearance techniques with xylene, alcohol or acetone, which can improve visualization of lymph nodes within the mesocolic or mesorectal fat. Although these methods have been demonstrated to increase lymph node harvest, they can be time-consuming, expensive and harmful for personnel due to toxic components [16, 17]. In some countries, pathologist assistants are employed to enhance the number of dissected lymph nodes, but due to increased costs, this strategy may be unaffordable in some healthcare systems [18].

Methylene blue (MB) injection into the main supplying artery of the removed colorectal specimen is a low-cost, simple and non-hazardous maneuver [19–21]. It results in accumulation of the blue dye in the vessels, lymphatic channels and lymph nodes, providing the pathologist with a good color contrast between nodes and surrounding fat tissue. This ex vivo method was first introduced by Märkl et al. in 2007, and multiple groups have adopted this approach considering its feasibility and affordability [9, 20–33]. Our study aimed to examine the method of MB injection in a randomized setting within an Eastern European healthcare system. The primary outcome of the study included the overall LN harvest, and the proportion of accurately staged patients. The secondary aim was to analyze the effect of lymph node staging on 5-year overall survival of recruited patients.

There has been a controversial discussion in the literature about the necessary number of dissected lymph nodes during colorectal cancer pathological evaluation. The cut-off threshold varied from 6 to 20 extracted lymph nodes in different studies [34]. Conventionally, the UICC, the American Joint Committee on Cancer, the National Quality Forum, National Comprehensive Cancer Network and the College of American Pathologists recommend a minimum number of 12 lymph nodes for accurate staging, as the quality measure of both surgical lymph node clearance and pathological workup [34]. On the other hand, a recently published high volume study proposed a minimal cut-off value above 21 lymph nodes [8]. Regardless of the recommendations, the majority of dissected colorectal specimens does not reach the minimum recommended lymph node count, especially in cases of rectal cancer after neoadjuvant chemo-irradiation [12, 35, 36].

Our results are largely consistent with those found in the literature. Application of this method might result in an increase of lymph node yield [9, 19–33]. Although a tendency for increment could be observed in each subgroup, a statistically significant improvement was documented among rectum cancer cases but not for colonic cases.

Furthermore, methylene blue staining provided a significant increase in the rate of sufficient (≥12) lymph node detection in our study, as a quality measure for pathological work-up. In the control group, lymph node harvest reached the minimal number in less than half of all cases, while with MB staining, two-thirds of the cases were sufficiently reported. This effect was again more pronounced in the rectal cancer subgroup. Reviewing the related articles from the literature, the rate of insufficient lymph node harvest could possibly be diminished, even close to zero, with the help of MB staining [9, 20–24, 27].

Lymph node staging accuracy is a complex term, covering the minimal number (≥12) of dissected lymph nodes, and N1c (with extranodal tumor deposit) stages and others with at least one positive lymph node (N1–2). In our experience, the rate of accurate lymph node staging by methylene blue injection was markedly improved, but this tendency did not reach the level of statistical significance. In further subgroup analysis, lymph node staging accuracy significantly improved among rectal cancer cases without neoadjuvant therapy, while no statistical difference was found in the colon cancer subgroup. Similar findings have been reported by other scientific groups [20, 21, 24, 27]. Regarding the small lymph nodes of the colorectal mesentery, there is great deal of inconsistency. Some authors, like Märkl et al. reported that most of the metastatic lymph nodes are simply noticeable by pathological examination [21]. Similarly, in the analysis of Reima et al., only 8% of the patients had metastasis in small-diameter (≤4 mm) lymph nodes, meaning that small lymph nodes may not be subject of major clinical interest [27]. On the other hand, other authors state that the majority (or at least half) of metastatic lymph nodes are located in lymph nodes smaller than 5 mm [37–39]. In these cases, methylene blue staining might help in the detection of these otherwise difficult-to-retrieve small metastatic lymph nodes.

Recently, a few trials have reported a significant increment of the metastatic lymph node count during the methylene blue technique, compared to the conventional lymph node dissection, leading to stage migration or upstaging [9, 30, 31]. For example, Jepsen et al. observed a significant increase in the rate of positive lymph nodes, resulting in an upstaging from UICC stage I or II to stage III, however in advanced cancer (T3/4), no upstaging was detected [31]. In addition, Liu et al. published a significantly raised rate of node positive lymph nodes resulting in an upstaging in 4 cases out of 66 [9]. In our study, in spite of the improved number of examined lymph nodes, this was not observable. Our experimental setting however was not designed to point out nodal stage migration.

It is well established that neoadjuvant chemo-irradiation negatively affects lymph node yield in rectal cancer cases, as preoperative treatment leads to fibrosis and lymph node size decrease in the affected area, causing a lack of properly dissectible lymph nodes [9,40–42]. However, this phenomenon may not negatively influence oncological outcomes, if we consider the decreased number and size of mesorectal lymph nodes as a favorable marker of neoadjuvant treatment effectivity [29]. This might be in line with the observation reported by Gurawaila et al., as fewer retrieved mesorectal lymph nodes correlated with effectiveness of oncotherapy after NAT [11].

Methylene blue injection has been successfully applied in the pathological investigation of rectal cancers in a few recent studies [20, 24, 29, 32]. Borowski et al., Klepšytė et al. and Münster et al. found that the use of MB injection led to a higher lymph node yield among patients who have received NAT, and the number of cases with insufficient lymph node detection was reduced, while no increase of the lymph node recovery in the subgroup of rectal cancer without preoperative radio-chemotherapy was observed [20, 29, 32]. Similar results were obtained in our research. In our experience, the use of MB injection showed a clinically relevant impact on the total lymph node count, the rate of sufficient (≥12) lymph node extraction, and lymph node staging accuracy in patient subgroups with rectal cancer.

Lymph node yield has a significant association with long term survival [43]. This is probably due to accurate nodal staging and the appropriate indication of adjuvant chemotherapy. Even one positive lymph node can cause a staging shift from stage II to stage III CRC, leading to different postoperative treatment [44, 45]. While stage II CRCs do not always require adjuvant treatment, stage III cancers are followed by adjuvant chemotherapy, as long as the patient is fit enough to complete it [44, 45]. Therefore, an insufficient lymph node extraction might contribute to a worse prognosis [8]. Although several methods have been introduced to increase the number of the dissected lymph node yield, most of them lack information on direct survival benefit [16–22, 24, 26, 27, 29–32]. We found that MB injection might be associated with improved 5-year overall survival among patients with colorectal cancer, undergoing colorectal resection. Similarly, Liu et al. have also investigated the overall survival after methylene blue injection in rectal cancer. However, their study showed no long-term benefit [9]. We observed no difference between the control and the MB arms until 24 months. After a period of a 2-year follow-up, Kaplan-Meier survival curves split up, and at 5 years postoperatively, the difference reached the level of statistical significance. Furthermore, in our stratified subgroup analysis, data suggested that cases with early CRC could profit the most from the MB staining method. The difference between the findings of Liu et al. and our group might be explainable by the alternation of adjuvant oncotherapy strategies among insufficiently staged cases. While Liu et al. reported that their colorectal multidisciplinary team rostered these patients for adjuvant chemotherapy, in our practice, we prefer not to treat patients with less than 12 negative lymph nodes. The necessity of adjuvant chemotherapy in stage II with insufficient (≤12) lymph node removal has a controversial interpretation in the literature. Current colorectal cancer guidelines recommend that early CRCs with less than 12 examined lymph nodes should be considered high-risk for recurrence, and are associated with increased mortality, and therefore, adjuvant chemotherapy should be administered among these cases [44, 45]. Despite this recommendation, the majority of patients could not be given adjuvant chemotherapy due to advanced age and associated comorbidities, which would limit the overall tolerance during chemotherapy [46]. Considering these features, methylene blue injection could play a major role in the appropriate lymph node detection, reducing uncertainty of staging resulting in under- or overtreatment.

In our study, application of the methylene blue staining technique resulted in an increment of examined lymph node yield, and an improvement of lymph node staging with a 5-year overall survival benefit of patients with colorectal cancer undergoing colorectal resection. In conclusion, methylene blue staining technique seems a reasonable approach to be adapted for routine use in healthcare systems with limited staff and expenditure.