This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.
Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been recommended for low-risk endometrial cancer (EC) patients by authoritative international guidelines, but it has not been performed broadly in China and worldwide. This work aims to describe detailed SLN mapping and biopsy procedures to promote the clinical application. SLN mapping and postoperative pathologic ultrastaging were conducted in a patient with low-risk EC using indocyanine green (ICG) dye to track the SLNs under laparoscopy and resecting them completely for ultrastaging. In conclusion, this protocol describes details of ICG injection, and SLN mapping and biopsy in EC patients based on the experiences gained during clinical practice.
Endometrial cancer (EC) is one of the most common diseases in gynecological oncology, and its incidence is rising1,2. Surgery is the first-line treatment for early-stage EC3,4. The evaluation of lymph node metastasis is an essential part of surgical staging in EC. The Gynecologic Oncology Group (GOG) study 33 demonstrated that lymph node metastases are associated with poor prognosis5.
As a new and essential technique for evaluating lymph node metastasis, sentinel lymph node (SLN) mapping and biopsy have emerged in recent years and have been recommended to be employed for patients with apparent uterine-confined EC according to the National Comprehensive Cancer Network (NCCN) guidelines for uterine cancer6,7,8,9,10. SLN mapping has also been extensively applied in tumors such as breast cancer11, lung cancer12, thyroid cancer13, and melanoma14. Pathologic ultrastaging has achieved good performance in colorectal and gynecological cancer15,16,17 and is recommended by the authoritative European guidelines18. Although principles of SLN mapping for EC staging have been provided in international guidelines10,19, there are no detailed manipulations of surgery in other present works.
This work presents the protocol for detailed SLN mapping and biopsy with ICG in a 28-year-old female who had been clinically diagnosed with early-stage EC, thus, improving and promoting the diagnosis of patients.
All surgery procedures related to the patient described here were approved by the Ethics Committees of Obstetrics & Gynecology Hospital of Fudan University in Shanghai, China. Informed consent was acquired from the patient.
1. Indications for SLN mapping with ICG in EC patients
2. Patient preparation
3. Fluorescent dye preparation
4. ICG injection
5. Intraoperative SLN identification
6. Intraoperative SLN biopsy
7. Postoperative SLN ultrastaging20
The patient in the present case was a 28-year-old female with irregular vaginal bleeding for 2 years, and she was diagnosed with an abnormality of the endometrium 4 months ago. Transvaginal ultrasound examination revealed a heterogeneous endometrial thickness with an adnexal mass. Abdominopelvic magnetic resonance imaging (MRI) demonstrated a 51 mm x 56 mm x 88 mm mass with a clear boundary within the uterine cavity under a high T2WI signal in a local hospital. Then, she was evaluated by hysteroscopy, and the endometrial lesion was dissected. The pathological results showed grade 1 endometrial cancer. This patient came to the hospital for further treatment with a strong fertility requirement. After a comprehensive evaluation (including another MRI examination that showed an irregular signal of endometrium in hospital, Figure 4) and signing informed consent, she experienced fertility preservation treatment. However, the second hysteroscopy in the hospital revealed superficial myometrial invasion, a contraindication of fertility preservation. Ultimately, after the patient was informed of her condition, she decided to undergo a total hysterectomy + bilateral salpingectomy + SLN mapping.
The cervix was injected with diluted ICG at 3 and 9 o'clock positions superficially and deeply, respectively (Figure 2). Then, the lymphatic vessels and SLNs were fluorescently labeled, which allowed for their recognition under various color modes (green for fluorescence mode and blue and red for color-segmented mode) in the Pinpoint Endoscopic Fluorescence Imaging system (Figure 3). Subsequently, the pathology department conducted H&E and IHC staining of SLN (Figure 5 and Figure 6) and ultrastaging of SLN. The staging results revealed a negative metastasis output.
Figure 1: Antiseptic preparation scope of SLN mapping in EC with laparoscopy. The black arrow indicates mid-axillary lines. The numbered red circles indicate the places of four trocars on the abdominal wall. Circle No. 2 is for the 10 mm trocar. The rest of the circles are for 5 mm trocars. The distance between circles No. 2 and 3 is around four fingerbreadths. Please click here to view a larger version of this figure.
Figure 2: Illustration of the SLN mapping in EC. (A) Anatomical diagram of the SLN in the pelvis. The dark green line represents the direction of the lymphatic drainage. Arrows indicate SLN lymph nodes in this case (yellow arrow presents the lymph node located between the right external and internal iliac vessels, brown arrow demonstrates the SLN in the left obturator foramen, and black arrow shows enlarged lymph node on the left iliac vein). Blue flat line indicates clock directions. (B) Injection spot locations in the cervix. ICG = indocyanine green. Green dots indicate the injection spots. The blue square shows the 3 and 9 o'clock positions of the ectocervix. (C) Enlarged cervix diagram: green dots indicate superficial and deep cervical injections. Please click here to view a larger version of this figure.
Figure 3: Intraoperative imaging of the SLN in EC. (A) Fluorescence mode. The green arrow indicates the SLN under fluorescence mode. (B) Color-segmented fluorescence mode. The yellow arrow indicates the lymph node under color-segmented mode. (C) Florescence mode of sentinel lymph nodes of this case. (D) Color-segmented fluorescence mode of sentinel lymph nodes of this case. HD: High definition; CSF: Color-segmented fluorescence. Please click here to view a larger version of this figure.
Figure 4: The MRI of EC patient. (A) Irregular signal of the endometrium located in the lower uterus cavity (arrows) on T1WI. (B) Irregular signal of the endometrium (arrows) on T2WI. Please click here to view a larger version of this figure.
Figure 5: H&E staining of EC and IHC staining of SLN. (A) H&E staining of myoinvasion in EC. (B) H&E staining of SLN. (C) IHC staining of AE1/AE3 (AE1/AE3 may stain myofibroblasts and smooth muscle cells and indicate the residual tumor cells) in SLN. 2.5x and 5x refer to the magnification under the microscope. Please click here to view a larger version of this figure.
Figure 6: IHC staining of EC patients. (A) IHC staining of estrogen receptor (ER) with five magnifications. (B) IHC staining of progesterone receptor (PR). (C) IHC staining of P53. (D) IHC staining of Ki67. (E) IHC staining of MLH1. (F) IHC staining of MSH2. (G) IHC staining of PMS2. (H) IHC staining of MSH6. All the images are captured with 5x magnification. Please click here to view a larger version of this figure.
Table 1: Comparison between tracers of blue dye, ICG, and Tc99. Please click here to download this Table.
SLN mapping and biopsy is a more selective and tailored lymph node dissection approach that has been applied in the clinic for nearly 20 years. In the field of EC, SLN mapping and biopsy have been increasingly recommended by several guidelines due to their high diagnostic efficiency in early-stage EC, achieving overall and bilateral detection rates of 89%-95% and 52%-82%, respectively, with a sensitivity of 84%-100% and negative predictive value of 97%-100%22. The present study reports a typical EC patient who performed SLN mapping and described the SLN protocol in detail from ICG injection to SLN biopsy.
SLN mapping is feasible with various dyes. Previous research on lymph node staging of breast and endometrial cancers applied blue dye (usually methylene blue or isosulfane blue (ISB), radiocolloid, or both23,24,25,26), reaching good performance and accuracy. As shown in white light, the blue dyes mentioned above have been extensively used due to their convenience and the least complex equipment requirement. However, the ICG dye had higher SLN detection rates (83%) than the ISB (64%)26. Thus, ICG is considered a better option for successful detection. Comparison between different dyes is stated in Table 1 according to the SGO consensus27.
In addition to tracker dyes, injection positions also affect detection rates of SLN mapping. Dye injection in the cervix cannot wholly reflect the lymphatic drainage, though the procedure is relatively simple and popular in clinical trials28,29. A hysteroscopic injection requires more skill and is located near the tumor lesion. The myometrial site is challenging to access intraoperatively, resulting in a low negative predictive rate of 87.50% and a false-negative rate of 33.30%30. In studies using cervical injection, the sensitivity range was 62.5%-97.5%, while myometrium injection was 66.70%-94.10%31. A systematic review including 55 eligible studies revealed an overall detection rate of 81% (95% confidence interval is from 77 to 84)32. Double detection works better than single detection, regardless of the injection location33. In the ongoing clinical study (NCT04276532), where 92 EC patients were enrolled, the SLN mapping detection rate was as high as 91.3%, with a total sensitivity of 73.3%. Combining cervical and fundus injections reached a higher para-aortic detection rate (40.4%) than cervical injections alone (4.4%), indicating that the combined injection is more efficient.
There are many possible reasons for insufficient SLN detection. Anatomically, the Naboth cyst and postsurgical abnormal pelvis affect mapping accuracy. Excessive adipose tissue near the cervix and cervical stenosis due to aging also lead to failed mapping. Factors such as body mass index (BMI), ISB dye use, and enlarged nodes are associated with SLN mapping failure34. Controversially, one prospective study including 110 EC patients reported that obesity and the presence of lymph node metastases were not associated with detection failure35. Lymphovascular space invasion might lead to failure of SLN mapping as well.
International guidelines consider SLN mapping and biopsy as necessary procedures10,18. However, there are debates about whether to conduct SLN mapping in intermediate-high risk EC patients. Patients with severe carcinoma, clear cell carcinoma, and carcinosarcoma have a higher risk of node metastasis36. On the contrary, some trials have determined that SLN mapping is a safe alternative to systematic lymphadenectomy because of lower complication occurrence37,38 and equivalent overall survival37. More high-quality clinical trials are required to determine the optimal inclusion standards.
In conclusion, SLN mapping and biopsy are better options than systematic lymphadenectomy to make the operation as simple and minimally invasive as possible. It can even improve the pathological stage of the disease by using a pathological ultrastaging method to guide the postoperative adjuvant therapy more accurately. For maximizing the specificity and sensitivity of SLN detection, this protocol provides tips for SLN mapping and biopsy to ensure a better outcome of the surgery.
The authors have nothing to disclose.
This work was supported in part by grants from the National Natural Science Foundation of China (81772777), Shanghai Science and Technology Commission Medical Guidance Project (18411963700), Clinical Research Plan of SHDC (No. SHDC2020CR4079); Shanghai Pujiang Talents Project (17PJ1401400). We thank the timely help given by Fenghua Ma from radiology department for MR images and Chao Wang from pathology department for pathologic images from Obstetrics and Gynecology Hospital of Fudan University.
2-mL syringe | Becton, Dickinson and Company, USA | 301940 | |
Coagulation forceps | Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instrument Factory | D0A010 | |
Fluorescence microscope | Olympus | IX73 | |
Harmonic scalpel | Soering GmbH | SONOCA190 | |
Hematoxylin-eosin staining | Beyotime Biotechnologies Corporation,CN | C0107 | |
Immunohistochemical cytokeratin staining | MXB Biotechnologies Corporation, CN | Kit-0020 | |
Indocyanine green (ICG) | Dandong Medical Innovation Pharmaceutical Corporation. | 3599-32-4 | |
Pararaffin | Sangon biotech Co., Ltd. | A601889 | |
Pinpoint Endoscopic Fluorescence Imaging system | Novadaq Technoloies Bonita, Springs, FL | NA | |
Propofol Injection | Fresenius Kabi Deutschland GmbH D-61346 Bad Homburg v.d.H., Germany | H20030124 | |
Silk Braided non-absorbable suture | Shanghai Ethicon | SA854G | |
Silk thread | Beijing AKIN AIEN International Medical Technology Co.,Ltd. | VCPP71D | |
Suction-Irrigation Tube | Shanghai SMAF | YX980D | |
Toothless forceps | Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instrument Factory | J41010 | |
Trocar | ETHICON Co,. Ltd, USA | B10LT, B5LT |