The lymph nodes harvested by the surgical procedure are almost 15-20 nodes per case. Pathologists take 1 or 2 section for each lymph node and stained with Hematoxylin and Eosine (H&E;) stain. It is not feasible to screen all lymph nodes by using advanced pathologic techniques. Thus the researchers try to find sentinel lymph node(s) popularized by Giuliano [5] in melanomas. Sentinel lymph node is the first node to collect the lymphatic drainage from primary tumor and is most likely to include metastasis. Bilchik et al. [6] and Saha et al. [7] have utilized the SLN mapping in CRCs. The aim of this study is to show whether viability of lymphatic mapping in CRC improves staging by advanced pathologic techniques.

Table 1: The patients’ operational characteristics and tumor localization

Ex-vivo SLN mapping technique
Ex-vivo sentinel lymphatic mapping was admitted as the main attempt of this study. Our technique was similar to that of Wood et al. [8]. After the surgical procedure was completed, the specimen was instantly taken to an extra table in the operating room. It was performed just after the specimen was taken out. The colonic specimen was incised longitudinally on the antimesenteric side. The rectal specimen was incised on the anterior border across the mesorectum. Lymphatic mapping was employed on the specimen by using 1 ml 1% Patent blue V dye (Guerbet Lab., France) subserosally and submucosally around the tumor (peritumoral site was employed) by using tuberculin syringe. After 5-7 minutes of massage with little circulatory movements on the lesion, the dye moved into the lymphatic paths to the SLN(s) in the mesentery. By low level diathermy, sharp dissection of lymphatic path(s) to the SLN(s) was existent under more care. Each sentinel lymph node was removed from the basin and marked before the specimen was submitted for pathologic appraisal (Figure 1).

Fig 1: A photograph of the specimen which was prepared with dying

Histopathologic procedure
Pathologic analysis entailed routine microscopic examination of the tumor, margins and LNs. Lymph nodes were manually dissected from the mesenteric fat. No chemical clearance method was employed. Each identified LN and SLN more than 5 mm was bisected and embedded in paraffin. Single section was routinely performed. Slices were stained by H&E; staining. If the result (after two faces of the LN bigger than 5 mm and only one face for LN smaller than 4 mm was observed) was negative all SLN’s paraffin blocks were sectioned in multiple slices of 4 microns thick. Slices apart from each other 200 microns in length were also stained by H&E; stain in second step of pathological evaluation. When no metastasis was experienced in multi-sectioned slices further analysis as immunohistochemical (IHC) staining was utilized to search metastasis and/or MM.

Immunohistochemical staining
A single paraffin section was stained with antibodies (Pan-Keratin AE1/3, CAM 5.2®, Beckton-Dickinson, San Jose, California - 35 bH11; prediluted Vantana Medical System Inc. Tuscon, AZ).

Then H&E; and IHC staining and multi-sectioning were employed for LNs harvested as SLN. Isolated tumor cells, micrometastasis and any metastatic foci was evaluated in the thinner level of the SLNs. A false negative SLN was described as a SLN containing no tumor cell while one or more LNs in the specimen were positive for tumor. Upstaging was determined as pN1 in the patients with SLN stained by IHC while those LNs had no metastatic deposit by using H&E; stain.

By using IHC staining technique 2 patients had MM in the SLN(s) despite of the fact that there was no metastasis and MM after H&E; staining and multi-sectioning. The exact stage of CRC in 2 patients was upstaged (11.7%). Then the chemotherapy protocol was changed. In the 4 patients with no SLN harvested one had no LNs in the tumoral lymphatic basin. In the remaining 3 patients, two without SLN had metastasis in the non-SLN (from tumoral basin via H&E; stain), which was evaluated as a false negative result (10%) (Table2).

Table 2: The overall results in this study

Joseph et al. [11] stated that about 40 LNs in the tumoral lymphatic basin should be evaluated to make a true staging for T1-T2 CRC. Furthermore it was declared by UICC that 12 LNs must be examined in depth in a specimen’s lymphatic basin for this purpose [12]. Some authors such as Bilchik [12], Joseph [11] and Koren [13] stated that all LNs taken from the basin were not evaluated enough in routine pathologic manner. Because there have been more than 15-20 LNs coming with each specimen detailed examination of those requires more time and that is not cost effective. In routine pathologic examination about 85- 90% of harvested LNs set down not investigated [6,11-13]. Joosten et al. [14] in 1997 declared the sentinel lymph node mapping in CRC in the meeting of Society of Surgical Oncology. The authors such as Wood [8] and Feig [15] dealing with the SLN biopsy (SLNB) then suggested that SLNB has been a sensitive and analytical process of pathologically staging patients with CRC. SLNB of the basin achieved 93-95% of accuracy by using advanced pathologic techniques. Though multiple sectioning and IHC staining are too time-consuming and expensive for examination of all LNs, these ultrastaging pathologic techniques could be cost effective for 2 to 4 SLNs. But debate on regarding both accuracy and significance of SLNB is present. In CRC, SLNB is used to improve staging unlike in breast cancer and melanoma in which SLNB is used to evade unnecessary radical lymphatic dissection. The advantages achieved in patients with breast cancer and melanoma that abolishing the morbidity of regional LN dissection besides improved staging of disease with MM may not have a significant impact in patients with CRC [14-16]. More recent studies, in CRC, have reported decreasing survival in the patients with MM, which were appraised as pN1 but isolated tumor cell (ITC) was received as pN0 [3,4,12,17,18]. At the same time, the authors such as Adell, Broll, Cutait and Jeffers [19-22] stated the presence of MM had no significant effect on survival. But survival in true node negative stage II CRC could be better than that with MM.

Pathological assessment of regional LNs gives the most important knowledge for decision making regarding adjuvant therapy in CRC providing a survival benefit for patients with positive LNs but no benefit to those with negative LNs. SLNB for CRC provides an efficient means of scrutinizing the regional lymph node basin more in the patients with CRC. Thus, it better evaluates the stage of the CRC and determines the patients having benefit from additional therapy [10,23,24].

In this study we did perform SLN mapping by using blue dye. SLNs were exposed in 37 patients (90.2%). The mean number of SLN was 3. Upstaging of CRC was revealed in 2 patients by IHC staining (11.7%). False negative result were also revealed in 2 patients (10%) (Table 3). The reasons of false negativity may depend on small number of patients in this series, atypical localization of SLNs, or whole invasion of SLN by tumor cells that does not allow blue dye. Four patients in first 10 cases in the study had no SLN exposed. In fact no LN was assessed in 1 of 4 patients. That should be correlated with inattentive surgical techniques or erroneous pathological cleansing methods. SLNB indicating the basin with 90% of accuracy was found in this study.

Table 3: The comparison of the outcomes between several studies and ours

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