Sentinel Lymph Node Detection in Early-stage Ovarian Cancer Using Radionuclide Technetium-99m Sulfur Colloid - A Feasibility Study

Introduction

Ovarian cancer is the 8^th most common cancer worldwide and a leading cause of morbidity and mortality. It is the third most common cancer in Indian women. Indian national cancer registry program has estimated 46,126 cases in the year 2022, with a cumulative risk of 1 in 133 Indian women and projected 49,644 new ovarian cancer cases by 2025.[1]

Approximately 30% of newly diagnosed epithelial ovarian cancer (EOC) patients present with early-stage disease. For these patients, surgical staging that includes pelvic and para-aortic lymphadenectomy is crucial for determining recurrence risk and guiding decisions on adjuvant treatment. However, nearly 15% of patients initially thought to have early-stage EOC are later upstaged due to lymph node involvement.[2]

While chemotherapy and maintenance therapy can significantly benefit node-positive patients, the therapeutic value of lymphadenectomy, particularly in node-negative patients, remains a subject of debate.[3] Pelvic and para-aortic lymphadenectomy is linked to significant comorbidities such as vascular and nerve injuries, increased blood loss and operative time, lymphocyst formation, lymphorrhea, and lower limb lymphedema.[4]

In recent years, sentinel lymph node (SLN) mapping has emerged as a less invasive alternative, reducing morbidity in other gynecologic cancers.[56] Despite its potential, the role of SLN mapping in early-stage EOC remains under study, and while existing research has shown it to be feasible and safe, unanswered questions persist about optimal tracer selection, injection techniques, and detection rates. The study aimed to assess the feasibility of using Tc-99 m sulfur colloid for SLN detection in early-stage EOC and to evaluate its diagnostic accuracy.

Materials and Methods

This prospective observational study was carried out at a tertiary care center in North India, with the participation of the departments of Obstetrics and Gynaecology, Nuclear Medicine, and Pathology. Patient recruitment occurred over a 10-month period (from May 2022 to February 2023) after receiving approval from the Institute Ethics Committee (AIIMS/IEC/22/215 dated April 22, 2022). The study was registered with the Clinical Trials Registry of India. Eligible participants were 18 years or older, diagnosed with apparent stage I or II EOC, or had suspicious ovarian masses scheduled for hysterectomy and/or salpingo-oophorectomy. Written informed consent was obtained from all participants. Exclusion criteria included evidence of spread to abdominal cavity on preoperative imaging, allergies to the tracers, suspicious lymph node involvement on imaging, previous pelvic or para-aortic lymphadenectomy or lymph node sampling, and pregnancy or lactation.

Patients were managed peri-operatively according to enhanced recovery after surgery protocol which included preoperative counseling, optimized hydration, avoidance of prolonged fasting, use of multimodal analgesia, prevention of hypothermia, early postoperative mobilization, and resumption of oral intake as early as tolerated. After performing thorough quality control with labeling efficacy of >95%, Tc-99 m sulfur colloid was prepared in the department of Nuclear medicine 1 h before surgical incision and was sent to operating room.

Tc-99 m Sulfur Colloid Preparation: Filtered 99m Technetium-Sulfur Colloid (100–220 nm particle size) was prepared using a five-vial kit obtained from the Board of Radiation and Isotope Technology, Mumbai. Freshly eluted sterile 99 mTc-Sodium Pertechnetate (Na^{99 m}TcO4) was reconstituted with components A, B, and C from the kit. The mixture was boiled in a water bath for 3–5 min, cooled for 15 min, and filtered using a micropore filter (220 nm pore size). Quality control was performed using thin-layer chromatography to ensure a labeling efficiency of over 95%. The prepared radiopharmaceutical was used immediately for injection.

All the procedures were done with midline vertical incision, adnexal mass exposed, peritoneal cytology taken and abdominal cavity examined. Baseline radioactivity was evaluated at the opposite thigh and at the liver surface. Tc-99 m sulfur colloid 37 mBq (one mCi) (1 mL) was injected subperitoneally at each of two sites; in the infundibulopelvic ligament and ovarian ligament at their attachments to the ovary. For bilateral tumors, tracer was injected on both sides. In cases where large masses or adhesions obscured the injection sites, the ovarian mass was first excised, and the tracers were then injected subperitoneally at the stumps of the utero-ovarian and infundibulopelvic ligaments. The retroperitoneum was opened, and SLNs identified. After a 10-min wait following tracer injection, a handheld gamma probe (SG04 Model: MK482HN/A, Crystal Photonics, Germany) was used to locate the SLNs [

SLN underwent ultrastaging and were sliced at 2 mm intervals across the short axis. The SLNs were then fixed in formalin, embedded in paraffin, and sectioned at 50 µM intervals. Two sections were stained with h and e, and a third section was examined for pan-cytokeratin AE1/AE3 via immunohistochemistry. SLN were categorized as negative or positive, with positive nodes further classified into isolated tumor cells (ITCs) (single cells or clusters ≤0.2 mm), micrometastases (0.2–2 mm deposits), and macrometastases (deposits >2 mm). Non-SLNs were evaluated following standard protocol. Follow-up for each patient extended to 30 days’ postsurgery.

Data analysis was conducted using STATA version 17.0 (Stata Corp (2017) Stata Statistical Software: Release 15. College Station, TX, USA: StataCorp LLC.), with descriptive statistics such as mean, standard deviation, and range calculated for normally distributed data. For nonnormal data, median and interquartile range (IQR) were reported. Frequency and percentage were used to represent categorical variables. Diagnostic accuracy metrics were calculated to evaluate the performance of SLN mapping in detecting metastases. Results of comprehensive complete nodal staging were considered gold standard and accuracy of SLN procedure was evaluated. Diagnostic accuracy metrics, including sensitivity, specificity, positive predictive value, and negative predictive value, were calculated.

Results

Epidemiology

Ten cases underwent the SLN detection procedure. The baseline characteristics are presented in Table 1. The median CA125 level was 312.5 U/mL (range: 14–1420). There were 9 cases of malignancy and one case had fibroma. Of the cases with malignancy, five cases had high grade serous adenocarcinoma, two cases each of mucinous and endometrioid adenocarcinoma. There were five cases with bilateral adnexal masses. There were three cases with tracer injection performed postadnexal mass excision. The summary of tracer injection and SLN localization is shown in Table 2. The radioactivity baseline and during SLN localization are shown in Supplementary Table 1. The sites of SLN localization are shown in Figure 1.

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Figure 1 Sentinel lymph node detection *(alphabets denote nodal stations and numbers denote number of cases). A: paracaval supra-mesenteric, B: Inter-aortocaval supra-mesenteric, C: para-aortic supra-mesenteric, D: paracaval infra-mesenteric, E: Inter-aortocaval infra-mesenteric, F: para-aortic infra-mesenteric, G: right common iliac, H: left common iliac, I: right external iliac, J: left external iliac, K: right obturator, L: left obturator

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Supplementary Table 1 Radioactivity at baseline, at sentinel lymph node and at the nodal basin after sentinel lymph node excision

Parameters	Radioactivity (n=10)
	n	Mean±SD	Median (IQR)	Range
Baseline radioactivity	10	2.1±0.99	2 (1–3)	1–4
Radioactivity at SLN (in vivo)
Pelvic
Right	4	620.75±447.61	542.5 (241.75–1078.0)	168–1230
Left	5	319.2±394.60	130.0 (111.0–622.0)	102–1020
Para-aortic	8	312.00±231.82	249.5 (169.5–326.25)	148–860
Radioactivity at nodal basin after SLN excision
Pelvic
Right	4	25.25±9.57	25.5 (16.5–33.75)	16–34
Left	5	27.00±33.90	15.0 (7.5–52.5)	5–87
Para-aortic	8	21.38±16.60	13.5 (8.5–36.25)	5–50
Radioactivity of SLN ex vivo
Pelvic
Right	4	375.25±178.26	383.5 (199.75–542.5)	154–580
Left	5	226.8±236.35	100.0 (92.0–425.0)	90–640
Para-aortic	8	259.25±163.00	180.0 (152.5–322.5)	150–624

SLN: Sentinel lymph node, SD: Standard deviation, IQR: Interquartile range

Table 1 Baseline characteristics

Parameters	Cases (n=10), n (%)
Age (years), mean±SD	48.2±10.66
BMI (kg/m2)
Underweight (<18.5)	2 (20)
Normal (18.5–22.9)	1 (10)
Overweight (23–24.9)	2 (20)
Obese (≥25)	5 (50)
ECOG performance status
0	2 (20)
1	6 (60)
2	2 (20)
ASA grade (ASA physical status classification system)
ASA grade I	5 (50)
ASA grade II	5 (50)
Parity
Nullipara	1 (10)
Multipara	9 (90)
Menopausal	5 (50)
Co-morbidities
Diabetes mellitus	2 (20)
History of tuberculosis	1 (10)
Hypertension	1 (10)
Past lower abdominal surgeries (hysterectomy)	1 (10)

SD: Standard deviation, ASA: American Society of Anesthesiologist

Table 2 Sentinel lymph node detection and localization

Parameters	Tc–99m sulfur colloid (n=10), n (%)
Injection postovarian mass excision	3 (30.00)
Site of injection
Utero-ovarian ligament
Not possible (posthysterectomy)	1 (10.00)
Unilateral	6 (60.00)
Bilateral	3 (30.00)
Infundibulo-pelvic ligament
Unilateral	7 (70.00)
Bilateral	3 (30.00)
Detection rate	10 (100.0)
Location of SLN
Pelvic alone	3 (30.00)
Para-aortic alone	3 (30.00)
Both	4 (40.00)
Specific location of SLN*
External iliac	5
Obturator	2
Common iliac	2
Para-aortic inframesenteric	3
Para-aortic supramesenteric	0
Para-caval inframesenteric	3
Para-caval supramesenteric	2
Time for SLN detection (min)	14.2±4.89 (10–20)

*The locations of SLN are more than number of cases as few had bilateral and both pelvic and paraaortic LN detection. SLN: Sentinel lymph node

Discussion

The concept of SLN was first described by Gould et al. for parotid gland carcinoma and SLNB was first utilized for early-stage melanoma and has subsequently evolved and is being used in various disciplines including gynecologic malignancies.[89]

Vulvar cancer was the initial gynecologic malignancy investigated for SLN mapping and biopsy due to its superficial position and well-defined lymphatic drainage patterns. Following multiple studies confirming the feasibility and precision of this approach in early-stage vulvar, endometrial, and cervical cancer, it has become a viable substitute for complete lymphadenectomy.[10] However, its utilization in ovarian cancer poses difficulties because the technique for tracer injection and the lymphatic drainage system of the ovaries are more intricate compared to other gynecological tumors.[4]

In the present study, the overall SLN detection rate was 100%, with SLNs identified in 60% of cases in both the pelvic and para-aortic regions, 30% exclusively in the para-aortic region, and 10% exclusively in the pelvic region. Ultrastaging detected two cases with ITCs and the false negative rate was 0%. Hassanzadeh et al. investigated SLN detection in 35 patients with pelvic masses, comparing cortical and sub-peritoneal Tc-99 m-Phytate injection. SLNs were identified in 21 of 25 patients (84%) with sub-peritoneal injections. SLNs were located in the para-aortic region in 21 patients, in both the pelvic and para-aortic regions in two patients, and in the pelvic region alone in two patients. Among three patients with lymph node involvement, all had positive SLNs, resulting in a false negative rate of 0%.[11]

Tc-99 m S colloid used in combination with indocyanine green (ICG) or blue dye has become a standard of care in cervical, endometrial and vulvar cancers.[12] A systematic review and meta-analysis on SLN in early ovarian cancer by Agusti et al. reported nine patients (8%), with Tc-99 m S colloid used alone, achieving a detection rate of 88.9% (95% confidence interval [CI], 58.2% to 100%; I² = 0%). When combined with patent blue, the tracer was used in 20 patients (17.7%) and showed a detection rate of 80.9% (95% CI, 22.1% to 100%; I² = 82.5%). In addition, in 30 patients (26.5%) who received a combination of Tc-99 m S colloid with ICG, the detection rate reached 100% (95% CI, 94% to 100%; I² = 0%) with no significant difference in detection outcomes across tracer types.[4]

The radiation safety concerns associated with sentinel node mapping have been explored and the radiation dose to patients remains very low, as there is minimal systemic absorption of the tracer, and the injection site is excised during surgery. In addition, radiation exposure for surgical, nuclear medicine, and pathology staff is well within the International Commission on Radiological Protection threshold limit.[11]

Consistent with our results, studies using Tc-99 m S colloid had a waiting time of 10–15 min after injection for SLN detection.[411] However, studies that exclusively use ICG, tracer migration is observed immediately postinjection.[4]

In the present study there were two cases with ITCs in SLN. In one case, a pelvic SLN was negative while a nonpelvic lymph node tested positive. However, the para-aortic SLN in the same patient was also positive, ensuring that the SLN mapping procedure accurately identified metastatic disease with no false negatives [Supplementary Table 2]. The SLN procedure is promising as it has good sensitivity and specificity and no false negative rate. However, the detection rates have a wide variation ranging from 27% to 100%. In the systematic review and meta-analysis, the pooled negative predictive value reached 100%. This is likely the most reliable estimate to take into account when evaluating the diagnostic accuracy of SLN procedure in patients with early-stage ovarian cancer, as the primary anticipated advantage of using SLNs in these cases is to prevent unnecessary systematic lymphadenectomy in instances where nodes are confirmed as negative. However, a recent multicenter prospective phase II trial on SLNB in early-stage ovarian cancer by Nero et al. with a sample size of 169 patients with apparent early-stage ovarian cancer reported that SLN mapping was successful in 58.6% of patients, with a sensitivity of 73.3% and specificity of 100%. Notably, 26.7% of node-positive cases were missed by SLNB, and 35% of node-positive cases were detected solely through ultrastaging. These findings underscore that SLN mapping is not a substitute for lymphadenectomy but may complement it, particularly when ultrastaging is utilized.[2]

As previously elucidated in the context of other cancer sites, the implementation of a clearly defined SLN algorithm that goes beyond merely removing the identified SLNs has the potential to enhance sensitivity and reduce the rate of false negatives. Consequently, it appears crucial to delineate the pathways of ovarian lymphatic drainage and establish an algorithm, which may involve conducting lymphadenectomy in cases where there is no tracer migration in a particular nodal region.

To the best of our knowledge, this is the first study evaluating the role of SLN and ultrastaging in early-stage EOC from the Asian continent. Standardized technique and site for tracer injection was better defined. Robust quality control of Tc-99 m Sulfur colloid was ensured. Cases were recruited prospectively. However, our study had a few limitations. As majority (two-thirds) of the EOC present as stage III and IV at diagnosis,[13] limited number of cases with early stage disease could be recruited. Although minimal (<1 mSv), the surgeon’s exposure to radioactivity was not assessed.[11] Another limitation of this study is its single-center design, which may limit the generalizability of the findings to other settings or populations. The progression free survival and overall survival of the study participants could not be studied as the study duration was 17 months. There were two cases with ITCs detected on ultrastaging but their correlation with recurrence and survival could not be determined owing to short study duration.

Conclusions

This study highlights the feasibility and diagnostic accuracy of Technetium-99 m Sulfur Colloid for SLN detection in early-stage ovarian cancer. The findings suggest that SLN mapping that SLN may have potential to become an alternative to full lymphadenectomy. However, larger studies are required to confirm its diagnostic accuracy and evaluate its long-term clinical implications.