**3. Histological analysis**

One of the main advantages that the SLN technique offers pathologists is the ability to select and minimise the number of lymph nodes to study, which enables a more exhaustive analysis and search for microscopic metastatic involvement.

#### **3.1 Intraoperative value**

Classically, the most widely used method for analysing SLNs has been the intraoperative assessment with haematoxylin-eosin (H&E) of imprint cytology performed in fresh samples and in frozen sections with rapid Diff-Quik staining. Intraoperative examination is questionable due to its limitations, both in the processing and in the results. Its success depends greatly on the collaboration between the surgeon and pathologist [74]. An intraoperative assessment can only examine a small portion of the SLN, has low sensitivity (56–67%) for the intraoperative detection of metastases and has an FNR of 20–30%, which makes the procedure inadequate and unsafe for the patient [75]. The results of Kim et al. [76] indicate an intraoperative understaging of 24%. For Ballester et al. [77], the rate was 43.7%, with little assessment of low-volume metastases. Additionally, the frozen sections distort the lymph node tissue, thereby precluding lymph node ultrastaging [30] and precluding the detection of micrometastatic involvement in the case of initially negative lymph nodes [75]. Currently, the guidelines of the National Comprehensive Cancer Network (NCCN) do not recommend the routine intraoperative assessment of SLNs, except when there is high suspicion and a nonsentinel lymph node [5]. However, other authors have emphasised the importance of intraoperative study to avoid reoperations, especially in high-risk tumours [78].

#### **3.2 Ultrastaging**

Conventional histological examination of a nonsentinel lymph node involves a single section along the lymph node's major longitudinal axis and H&E staining, with deeper levels or application of immunohistochemistry (IHC) at the pathologist's discretion [30].

Occasionally, the only evidence of extrauterine disease is the presence of metastases in the SLN. Considering the poorer prognosis associated with the detection of lymphatic metastases, a much more exhaustive analysis is justified [74, 79]. Therefore, pathological assessment by the ultrastaging of the SLN is the most important advance in the SLNB technique [80].

#### *3.2.1 Histological ultrastaging and immunohistochemistry*

The histological ultrastaging procedure includes a protocol for the series of microscopic examinations of the SLN block fixed and imbedded in paraffin, with the addition of the immunohistochemical analysis with cytokeratin measurement (pan-cytokeratin kits AE1/AE2 or anti-CK19), thereby increasing the sensitivity [80]. There are no formal evidence-based regimens for the pathology assessment of SLNs in EC, which entails considerable variability among institutions and,

### *Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

therefore, debatable results [30]. The study by Euscher et al. [81] compared two methods of ultrastaging by histological microsection, achieving increased detection of metastatic SLN in 32% of patients, with a mean metastasis size of 0.24–0.38 mm, with no differences in the detection rate by ultrastaging method. The algorithm proposed by the MSKCC consists of an initial assessment by H&E and, if negative, performing two adjacent 5-μm slices at two levels separated by 50 μm in the paraffin block, applying H&E staining and pan-cytokeratins AE1/AE3 in each slice [82]. Holloway et al. [48] performed three slices per level, two of which were then stained with H&E and one with AE1/AE3. Other authors have performed four levels with six slices at 40 μm intervals (levels 1 and 2 with H&E and levels 3 and 4 with AE1/AE3) or five levels (levels 1, 3 and 5 with H&E and 2 and 4 with AE1/AE3) [30] (**Figure 1**).

Cytokeratin-19 (CK-19) is a protein of the intermediate filament responsible for the structural integrity of epidermal epithelial cells, which in normal conditions is not expressed in the lymphatic tissue and is expressed abnormally in more than 90% of cells by EC [83, 84]. CK-19 is a biomarker directly related to the capacity for tumour dissemination in EC, with high sensitivity and a capacity for discriminating between metastatic and nonmetastatic lymph nodes or areas of lymphovascular invasion [84, 85].

Ultrastaging is a more complex procedure, requiring significant dedication, and has the added risk of high intraobserver and interobserver variability. The diagnostic categories of the American Joint Committee on Cancer (AJCC) are applied for breast cancer, with the following classifications for SLN: negative (<200 individual tumour cells or tumour cell aggregates <0.2 mm in size, including the presence of isolated cytokeratin-positive tumour cells [ITC]), micrometastatic (size ≥0.2 and <2 mm) (μM) or macrometastatic (>2 mm) (MM) [86]. The term low tumour volume includes the ITC and μM categories.

The ultrastaging of SLN has improved the validity of the technique and detects an additional 5–15%, with a high rate of low-volume lymph node disease (approximately 50% of patients with metastatic SLN), which would not be identified with the conventional technique. Ultrastaging represents an overall mean increase of 25% (range, 10–60%) in detecting metastatic lymph nodes [80, 84].

#### **Figure 1.**

*The SLN algorithm for surgical staging of endometrial cancer. Obtained from NCCN Guideline in Endometrial Carcinoma, Version 4.2019: 'Principles of evaluation and surgical staging when SLN mapping is used, Figure 4: The SLN algorithm for surgical staging of endometrial cancer'. SLN = sentinel lymph node; LND = lymph node dissection.*

Analysis by histological microsection and H&E detects 6.9% more patients with metastatic SLN, while the inclusion of IHC provides an additional 4.5% (82.6% with low tumour volume), which represents 12.6% of patients with metastatic SLN [82]. In the multicentre retrospective study by Raimond et al. [87] with 136 lowintermediate risk cases, the detection of SLN and the ultrastaging analysis increased the detection of metastatic lymph nodes threefold over lymphadenectomy (16.2 vs. 5.1%; p = 0.03); 11% of the cases were μM and 5.1% were MM, with an FNR of 0 (95% CI 0–1.6%). In the study, 6.1% of the metastatic SLN were detected by histological microsection, and 10.1% were detected by IHC. All MM cases were diagnosed by histological microsection, and 73.3% of the μM cases were diagnosed by IHC readings. Thus, by applying ultrastaging, the SLNB restaged 50% of the patients included in the study to an European Society of Medical Oncology (ESMO) high risk (14.7% of the sample), thereby changing the adjuvant therapy compared with women with negative or unassessed lymph nodes (p < 0.001). In the study by Hagen et al. [88], 75% of patients with metastatic SLN were detected with histological microsection and an additional 25% with IHC. For Desai et al. [89], 50% of the metastatic SLNs were detected by IHC.

A number of authors have debated and hypothesised a different tumour biology and therefore a different behaviour between tumours with MM and μM. For other authors, μM metastases appear early and are reflections of isolated metastases in type 2 histology but would be metastases of late evolution in tumours with low oncologic aggressiveness [97]. It has also been suggested that μM metastases could represent an *intermediate state* between negative lymph nodes and positive lymph

One of the aforementioned advantages of the SLNB technique in early stages is avoiding the implementation of lymphadenectomy [87]. However, the therapeutic benefit of completing the lymphadenectomy when faced with the finding of metastatic SLN with low tumour volume remains unknown, and its systematic implementation is not justified [83, 98, 99]. The FIRES trial performed postoperative radiological studies (with scanner or positron emission tomography-computed tomography [PET-CT]). When the findings showed voluminous residual metastases, the proposed treatment was surgical cytoreduction or a change in the adjuvant

In recent years, research has been conducted on the possible influence of adjuvant therapy and the prognosis for patients with low-volume lymphatic metastases [100]. There is still no evidence from prospective randomised studies on cases with detected low-volume metastatic tumours, resulting in heterogeneity among the published studies [57]. Moreover, it is important to determine the benefit provided to these patients by combining CT and/or adjuvant RT, considering the scarce survival benefit obtained. The study by Plante et al. [101] analysed the impact of adjuvant therapy on survival. Thirty-five percent of the patients with ITC in the SLN underwent CT and external beam RT, 32% only underwent external beam RT or vaginal brachytherapy and 32% underwent follow-up. The overall survival at 3 years in the group with ITC was 95.5%, with no differences compared with the patients without metastatic lymph nodes or with μM in the SLN (87.6 and 85.5%, respectively). However, the low tumour volume SLN was superior to the SLN with

Significant differences have not been observed between the prognosis of patients with only SLN excision versus those with lymphadenectomy (disease-free survival [DFS] at 3 years of 94.9 vs. 96.8%; p = 0.35). However, it has been observed that the patient group with metastatic SLN increases the portion of adjuvant therapy received (27.1 vs. 10.8%; p < 0.001) [102]. In the retrospective series by Raimond et al. [87], neither the absence of metastases in SLN, the detection of metastatic SLN nor the presence of μM in the SLN represented an improvement in DFS ([hazard ratio (HR), 0.89; 95% CI 0.42–1.90; p = 0.77], [HR, 0.82; 95% CI 0.18–3.64; p = 0.8] and [HR, 0.46; 95% CI 0.03–7.42; p = 0.59], respectively). When comparing negative lymphadenectomy with the detection of lymphatic metastases, there were no differences in DFS (HR, 1.13; 95% CI 0.34–3.76; p = 0.84) or overall

Yabushita et al. [92] showed that the detection of μM and SLN was an independent factor for recurrence in early stages of EC. Kim et al. [76] measured a tendency to late distant metastasis when the SLN was metastatic for μM. For Todo et al. [97],

nonlymphogenic or extrapelvic recurrence. For Kim et al. [76], these ITCs did not represent increased relapses. In the study by Erkanli et al. [103], DFS and overall survival were significantly lower in patients with μM (p < 0.05), while the presence of ITCs appeared to have no effect on survival. In another study by Todo et al. [104], the presence of low tumour volume was an independent factor for

extrapelvic relapse (RR, 17.9), with 20% lower survival (overall survival of 71.4 vs. 91.9% and DFS of 55.6 vs. 84%; p = 0.074) and a tendency towards late relapse

ITCs in the paraaortic area were not associated with a greater risk of

radiation therapy (RT) to include the paraaortic area [75].

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

nodes for MM [98].

MM (58.5%; p < 0.001).

**187**

survival (HR, 1.29; 95% CI 0.30–5.59; p = 0.73).

#### *3.2.2 Molecular ultrastaging*

The one-step nucleic acid amplification method (OSNA) is a validated technique for breast cancer and enables a quantitative, systematic, automated, nonobserverdependent analysis for detecting lymph node metastases [74, 84]. The application of this method always requires prior confirmation of CK-19 expression in the tumour tissue [90, 91]. The application of radiocolloid or dye does not interfere with the process. The number of mRNA copies of CK-19 corresponds to the size of the metastatic foci present in the SLN [92], such that the results are visualised in four separate categories: negative (<102 copies of mRNA/μL), ITC (102 to <sup>&</sup>lt;2.5 <sup>10</sup><sup>2</sup> copies of mRNA/μL), <sup>μ</sup>M (≥2.5 <sup>10</sup><sup>2</sup> to <sup>&</sup>lt;<sup>5</sup> <sup>10</sup><sup>3</sup> copies mRNA/μL) and MM (><sup>5</sup> <sup>10</sup><sup>3</sup> copies mRNA/μL) [93].

The results of the Breast Complete Lymphadenectomy OSNA Study for Enhanced Review-I (B-CLOSER-I) [94] indicate that histopathology (compared with molecular detection) significantly underestimates the rate of metastases of axillary lymph nodes.

Preliminary results of its application in EC indicate superior diagnostic accuracy compared with conventional ultrastaging, although further research is needed. By applying the OSNA method, the study by Nagai et al. [84] achieved sensitivity of 93.3%, specificity of 99.5%, a negative predictive value (NPV) of 99.5% and a correlation of 99.1%. In the study by López-Ruiz et al. [85] of 34 patients with chronic diseases and 94 analysed SLNs, the OSNA method detected a larger portion of additional low tumour volume metastases, with diagnostic capacity (sensitivity of 100%, specificity of 87.5%, accuracy of 88.3% and an FNR of 2.8%).

### **4. Approach and prognosis for metastatic sentinel nodes**

The increase in detection of lymphatic metastases resulting from the introduction of ultrastaging is mainly at the expense of the detection of low tumour volume. The NCCN guidelines [5] and the consensus of The Society of Gynecologic Oncology (SGO) [30] recommend the study of SLNs by ultrastaging, indicating that the significance of low-volume lymph node involvement is still uncertain and have not established an optimal treatment approach [56, 95, 96].

A number of authors have debated and hypothesised a different tumour biology and therefore a different behaviour between tumours with MM and μM. For other authors, μM metastases appear early and are reflections of isolated metastases in type 2 histology but would be metastases of late evolution in tumours with low oncologic aggressiveness [97]. It has also been suggested that μM metastases could represent an *intermediate state* between negative lymph nodes and positive lymph nodes for MM [98].

One of the aforementioned advantages of the SLNB technique in early stages is avoiding the implementation of lymphadenectomy [87]. However, the therapeutic benefit of completing the lymphadenectomy when faced with the finding of metastatic SLN with low tumour volume remains unknown, and its systematic implementation is not justified [83, 98, 99]. The FIRES trial performed postoperative radiological studies (with scanner or positron emission tomography-computed tomography [PET-CT]). When the findings showed voluminous residual metastases, the proposed treatment was surgical cytoreduction or a change in the adjuvant radiation therapy (RT) to include the paraaortic area [75].

In recent years, research has been conducted on the possible influence of adjuvant therapy and the prognosis for patients with low-volume lymphatic metastases [100]. There is still no evidence from prospective randomised studies on cases with detected low-volume metastatic tumours, resulting in heterogeneity among the published studies [57]. Moreover, it is important to determine the benefit provided to these patients by combining CT and/or adjuvant RT, considering the scarce survival benefit obtained. The study by Plante et al. [101] analysed the impact of adjuvant therapy on survival. Thirty-five percent of the patients with ITC in the SLN underwent CT and external beam RT, 32% only underwent external beam RT or vaginal brachytherapy and 32% underwent follow-up. The overall survival at 3 years in the group with ITC was 95.5%, with no differences compared with the patients without metastatic lymph nodes or with μM in the SLN (87.6 and 85.5%, respectively). However, the low tumour volume SLN was superior to the SLN with MM (58.5%; p < 0.001).

Significant differences have not been observed between the prognosis of patients with only SLN excision versus those with lymphadenectomy (disease-free survival [DFS] at 3 years of 94.9 vs. 96.8%; p = 0.35). However, it has been observed that the patient group with metastatic SLN increases the portion of adjuvant therapy received (27.1 vs. 10.8%; p < 0.001) [102]. In the retrospective series by Raimond et al. [87], neither the absence of metastases in SLN, the detection of metastatic SLN nor the presence of μM in the SLN represented an improvement in DFS ([hazard ratio (HR), 0.89; 95% CI 0.42–1.90; p = 0.77], [HR, 0.82; 95% CI 0.18–3.64; p = 0.8] and [HR, 0.46; 95% CI 0.03–7.42; p = 0.59], respectively). When comparing negative lymphadenectomy with the detection of lymphatic metastases, there were no differences in DFS (HR, 1.13; 95% CI 0.34–3.76; p = 0.84) or overall survival (HR, 1.29; 95% CI 0.30–5.59; p = 0.73).

Yabushita et al. [92] showed that the detection of μM and SLN was an independent factor for recurrence in early stages of EC. Kim et al. [76] measured a tendency to late distant metastasis when the SLN was metastatic for μM. For Todo et al. [97], ITCs in the paraaortic area were not associated with a greater risk of nonlymphogenic or extrapelvic recurrence. For Kim et al. [76], these ITCs did not represent increased relapses. In the study by Erkanli et al. [103], DFS and overall survival were significantly lower in patients with μM (p < 0.05), while the presence of ITCs appeared to have no effect on survival. In another study by Todo et al. [104], the presence of low tumour volume was an independent factor for extrapelvic relapse (RR, 17.9), with 20% lower survival (overall survival of 71.4 vs. 91.9% and DFS of 55.6 vs. 84%; p = 0.074) and a tendency towards late relapse


*The results of 13* 

**189**

*that includes patients with high-risk endometrial*

*[108] The average of the collected variables is estimated. Results of two* 

*already included in their data. The word*

*article.*

*N = patients included; SLN = sentinel lymph node; DR = detection rate; BL = bilaterality;*

*= with or without; (%) = proportion; Σ = summation;*

*1All three FNR cases had*  *2The data are reflected after application*

*metastasis.*

*3The estimation of the FNR is performed from the total of negative* 

*4The predictive capacity is estimated only based on cases with SLN detected bilaterally.*

*5Null FNR with Se and NPV of 100% after the retrospective*

*6The FNR would be 4.3% after adjusting by MSKCC algorithm.* *7Surgical detection with dual technique: radiotracer and dye. ICG increases the overall detection rate to 93% and bilaterality of 78%. Cervical injection increases Se to 93% and detection rate to 86%.*

**Table 3.** *Analysis of recent studies: Detection rate, predictive capacity, false negative rate and paraaortic*

 *drainage of the sentinel node.*

*methodologically*

 *comparable*

 *studies are cited; studies include more than a hundred of cases, except for the one of Ehrisman et al. [107] because it is one of the recently published studies*

 *cancer. The studies include cases with all histological type and two papers only high-risk cases, the one mentioned by Ehrisman et al. [107] and Soliman et al.*

*"Various" indicates that various injection methods or tracer* 

*meta-analyses,*

 *which have not been added in the estimation, are attached to the possibility that the previously mentioned studies were*

 *Se = sensibility; NPV = negative predictive value; FNR = false negative rate; FN = false negative; (+) = metastasis; w/o*

*99mTc-nanocolloid*

 *albumin; MB = methylene blue; ICG = indocyanine*

*standard deviation; 99mTc =*

> *(<sup>μ</sup> σ) = mean*

> > *nonendometrioid*

 *histology, one with paraaortic positive drainage. There were no cases of isolated paraaortic drainage.*

 *of the MSKCC proposed surgical algorithm. Without applying the algorithm: Se of 85.1%, VPN of 98.1% and FNR of 14.9%. One FNR case with isolated paraaortic*

*lymphadenectomies;*

 *application*

 *of the MSKCC algorithm.*

 *it does not include negative SLN and positive* 

*lymphadenectomy,*

 *in which case it would be a FNR of 9.1%.*

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

*combinations*

 *were collected. The blank box indicates that this information*

 *has not been mentioned in the*

 *green; PAO = paraaortic.*


*Analysisofrecentstudies:Detectionrate,predictivecapacity, falsenegativerateandparaaorticdrainageofthesentinelnode.*


*green.*

 *(<sup>μ</sup> σ) = mean* 

*standard deviation;*

 *=*

 *albumin; MB = methylene blue; ICG = indocyanine*

#### **Table 4.**

*MM =* 

*macrometastasis;*

 *w/o = with or without; (%) = proportion; Σ = summation;*

*Analysis of recent studies: proportion of patients with metastatic sentinel node, proportion of single metastatic sentinel node and distribution by type of metastases detected.*

(49 vs. 16.5 months; p = 0.066) compared with the patient group without lymphatic

a prognostic biomarker in terms of survival, but whether the presence of ITCs should be used as such, regardless of other clinical-pathological risk factors, is still

**5. Recommendations for the clinical application of selective sentinel**

The published results of SLNB in EC have shown good diagnostic performance. SNLB is a promising and safe technique from the oncological point of view [10]. Given the mixed results, which have failed to show a therapeutic benefit [9], and the lack of long-term results [5], an appropriate interpretation is recommended

As with lymphadenectomy and adjuvant therapy, SNLB can be safely omitted for low-risk patients (endometrioid histology: IA G1, IA G2) [9, 56]. For intermediaterisk (endometrioid histology: IA G3, IB G1, IB G2) patients, SNLB has not shown a clear benefit in survival when performing systematic lymphadenectomy. This group does, however, have a greater risk of lymphatic involvement and typically undergo

adjuvant therapy. SLNB in both patient groups has been shown to detect 2

more cases of metastatic EC than lymphadenectomy, without changing the disease prognosis [87, 110]. These results justify extending the technique to low to intermediate risk, with the recommendation of including it in an algorithm or surgical protocol that includes the implementation when faced with failure of the technique. The potential benefit of detecting metastatic SLN in low-risk patients is however diluted by the low incidence of cases and the high proportion of low tumour volume. It appears we need to clarify the role of SLNB and the effect of low tumour volume,

Two randomised clinical trials did not show that lymphadenectomy changed

In terms of applying SLNB to high-risk patients, the greatest debate concerns paraaortic drainage, with a greater proportion of undetected metastatic paraaortic nonsentinel lymph nodes. A study by Naoura et al. [117] analysed 180 patients and

study, it was much more likely that the high-risk subtype (7 vs. 28%; p = 0.03) and

The FIRES study [75] on 385 patients represents the largest prospective series to date and included low and high-risk patients, achieving a sensitivity of 97.2% and

tive series did show this change [114, 115]. The implementation of pelvic and paraaortic lymphadenectomy has been systematised due to the higher rate of lymphatic metastases and poorer prognosis associated with their detection. In contrast, sceptics of lymphadenectomy state that routine nodal assessment of high-risk patients rarely changes the recommendations for adjuvant therapy. EC (especially in high-risk cases) is not just a disease confined to the pelvis [51]. Patients with high-risk EC have a higher probability of recurrence and recidivism with or without detection of lymphatic metastases and should undergo systemic therapy regardless of the nodal state [116]. Nevertheless, published results on high-risk patients have shown no differences compared with those expected from studies that also include low- to intermediate-risk cases, with the same incidence of paraaortic metastasis estimated by lymphadenectomy. Survival data have shown no differences [30], which would assume that the benefit of SNLB in high-risk patients would be

μM can represent

–3 times

–113], although retrospec-

< 0.001). In this

metastasis. The conclusion of these studies is that the presence of

**4**).

[4, 29], considering SNLB a technique under study [79].

which is more frequently detected in this group [87, 110].

overall survival or relapse rates in high-risk patients [111

equivalent to lymphadenectomy alone [107, 108].

**191**

achieved a much higher FNR in the high-risk group (2.3 vs. 20%; p

the nonendometrioid type (8 vs. 29%; p = 0.02) were poorly assessed.

unknown [30] (**Tables 3** and

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

**node biopsy**

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

(49 vs. 16.5 months; p = 0.066) compared with the patient group without lymphatic metastasis. The conclusion of these studies is that the presence of μM can represent a prognostic biomarker in terms of survival, but whether the presence of ITCs should be used as such, regardless of other clinical-pathological risk factors, is still unknown [30] (**Tables 3** and **4**).
