Sentinel lymph node mapping with indocyanine green in cervical cancer patients undergoing open radical hysterectomy: a single-institution series

Purpose To assess the rate of bilateral sentinel lymph node (SLN) detection with indocyanine green (ICG), to evaluate the sensitivity and the negative predictive value of cervical cancer patients undergoing open radical hysterectomy; to compare open versus minimally invasive SLN biopsy performance and to assess factors related to no/unilateral SLN mapping. Methods We retrospectively reviewed consecutive patients with FIGO 2018 stage IA1 with lymph-vascular space involvement to IIB and IIIC1p cervical carcinoma who underwent SLN mapping with ICG followed by systematic pelvic lymphadenectomy between 05/2017 and 06/2020. Patients were divided according to surgical approach for statistical analysis. Results Eighty-five patients met inclusion criteria. Twenty-seven (31.8%) underwent open and 58 (68.2%) underwent minimally invasive SLN mapping. No difference in any SLN mapping (laparotomy 92.6% and minimally invasive 91.4%) or in SLN bilateral detection (laparotomy 72.0% and minimally invasive 84.9%) (p = 0.850 and p = 0.222, respectively), in median number of SLNs mapped and retrieved (2 in both groups, p = 0.165) and in site of SLN mapping per hemi-pelvis (right side, p = 0273 and left side, p = 0.618) was evident between open and minimally invasive approach. Per-patient sensitivity of SLN biopsy in laparotomy was 83.3% (95% CI 35.9–99.6%) and the negative predictive value was 95.0% (95% CI 76.0–99.1%). No difference in per-patient sensitivity was noted between two approaches (p = 0.300). None of the analyzed variables was associated with no/unilateral SLN mapping. Conclusion The use of ICG to detect SLN in cervical cancer treated with open surgery allows a bilateral detection, sensitivity and negative predictive value comparable to minimally invasive surgery with potential advantages of ICG compared to other tracers. Electronic supplementary material The online version of this article (10.1007/s00432-020-03393-6) contains supplementary material, which is available to authorized users.


Introduction
Despite the introduction of screening and vaccination has significantly reduced the incidence of cervical cancer in developed countries (Brisson 2020;Wright 2015), this tumor still represents the fourth most common cancer worldwide, with 569,847 new cases and 311,365 deaths in 2018 (Arbyn 2020). Treatment of early-stage disease (clinical International Federation of Gynecology and Obstetrics (FIGO) stage IA1-IB1 and IIA1) is represented by radical hysterectomy and pelvic lymphadenectomy with sentinel lymph node (SLN) biopsy (Cibula 2018). After the publication of a randomized controlled trial, which demonstrated an inferior survival outcome if patients with early-stage cervical cancer were treated with minimally invasive, compared to open radical hysterectomy (Ramirez 2018), multiple retrospective studies showed a survival difference between the two approaches, with consequent recommendations amendment by international societies (Koh 2019;Nitecki 2020;Querleu 2020), particularly for tumors larger than 2 cm (Pedone Anchora 2020). SLN biopsy has been largely studied in cervical cancer and multiple evidences support its use in early-stage disease treated with primary surgery (Cibula 2018(Cibula ,2019; also, few studies reported the use of SLN biopsy after neo-adjuvant chemotherapy (NACT) (Slama 2012;Robova 2014). Different studies reported the adoption of SLN in open radical surgery with blue dye or radioactive tracer (Silva 2005;Wydra 2006;Altgassen 2007;Kara 2008;Dostalek 2020), but experience on the use of indocyanine green (ICG) in open surgery, is limited to few case reports or case series (Furukawa 2010;Crane 2011;van der Vorst 2011;Schaafsma 2012;Buda 2016;Rychlik 2016). On the other hand, it seems well established that ICG has been showed to be superior to blue dye or radioactive tracer in SLN bilateral detection rate in minimally invasive surgery (Di Martino 2017).
The primary objective of the present study was to assess the rate of bilateral SLN detection with ICG in cervical cancer patients undergoing open radical hysterectomy. Secondary objectives were to evaluate the sensitivity and the negative predictive value of open ICG SLN biopsy, to compare open versus minimally invasive SLN biopsy performance and to assess factors related to no/unilateral SLN mapping.

Inclusion and exclusion criteria
The present retrospective observational cohort study was approved by Institutional Review Board (Dipartimento per la salute della Donna e del Bambino e della Salute Pubblica, number DIPUSVSP-26-05-2059, date 26/05/2020). After signing consent form, consecutive patients who underwent surgery at Fondazione Policlinico A. Gemelli IRCCS (Rome, Italy), for International Federation of Gynecology and Obstetrics (FIGO) 2018 (Bhatla 2018) stage IA1 with lymph-vascular space involvement (LVSI) to IIB and IIIC1p cervical carcinoma between 05/2017 and 06/2020 and had SLN mapping attempt with systematic pelvic lymphadenectomy, were included. Data were retrieved from institution's electronic database. All patients had pre-operative histology confirmation of cervical cancer and underwent pre-operative abdominal Magnetic Resonance Imaging (MRI) scan, pelvic ultrasound scan and chest X-ray. Patients with FIGO stage > IB1 underwent whole body positron emission tomography (PET)/computed tomography (CT) scan to exclude distant metastasis. Radicality of hysterectomy was classified according to Querleu-Morrow classification (Querleu 2017). Examination under anesthesia was performed at the same time of the surgery. Only women with no evidence of enlarged (i.e., short axis > 10 mm) pelvic and para-aortic lymph nodes at pre-operative MRI-scan were submitted to SLN mapping. Patients with pre-invasive cervical disease, who did not undergo systematic pelvic lymphadenectomy, with special histology sub-types (other than squamous cell carcinoma-SCC, adenocarcinoma or adenosquamous carcinoma), allergy to iodine or patients with SLN analyzed with standard H&E only, were excluded.
Surgical approach was performed by minimally invasive surgery (laparoscopic or robotic) at surgeon's discretion until October 2018 (Ramirez 2018) and for tumors < 2 cm in compliance with European Society of Gynaecological Oncology (ESGO) statement and National Comprehensive Cancer Network (NCCN) guidelines, using protecting maneuvers (no uterine manipulator and vaginal cuff formation in patients with tumor completely excised at preoperative conization), after that date (Koh 2019;Querleu 2020). Laparotomy approach was performed at surgeon's discretion until October 2018 (Ramirez 2018) and for tumors ≥ 2 cm after that date. Uterine surgery involved radical hysterectomy in patients who did not wish to retain fertility; and cervical conization or cervical biopsy followed by NACT [as part of a previously described clinical trial, NCT02323841-in press Fertility and Sterility (ClinicalTrials.gov Identifier: NCT02323841) (Clinicaltrial 2014)], in patients who desired to preserve fertility with tumor diameter ≤ 2 cm and > 2 cm, respectively. Patients with clinical diagnosis of FIGO stage IIB, underwent NACT followed by radical hysterectomy. All patients undergoing open surgery were treated with radical hysterectomy.

3
FIGO stage refers to pathologic stage for patients who underwent primary surgery and to clinical stage at diagnosis for patients undergoing NACT.

Sentinel lymph node assessment
Technique and method for SLN mapping was the same in all cases. SLN was detected after 1 ml superficial and deep cervical injections of ICG (diluted with sterile water at 1.25 mg/ ml) at 3 and 9 o'clock. About 15 min after cervical injection, pelvic retroperitoneal space was opened, and lymph nodes were assessed with near infra-red (NIR) camera: Visera Elite II (Olympus, Tokyo, Japan) and Pinpoint (Stryker, Kalamazoo, Michigan, US) in case of laparoscopic or Da Vinci Xi (Intuitive, Sunnyvale, California, US) in case of robotic approach; SLN in open surgery was detected with SPY Portable Handheld Imager (SPY-PHI) camber (Stryker, Kalamazoo, Michigan, US). SLN was defined as the ICG-positive pelvic node closest to the tumor; retroperitoneal spaces were explored with the following order to assess SLN presence: external iliac, interiliac, obturator, common iliac, parametrial and pre-sacral and low para-aortic area (Marnitz 2006;Balaya 2019). All mapped SLNs were removed and analyzed with ultrastaging (Cibula 2012) or with one-step nucleic acid amplification (OSNA) .
Ultrastaging procedure was performed as follows: hematoxylin and Eosin (H&E) negative nodes were entirely sectioned at 150 μm intervals, until exhaustion of the lymph node, according to the length of the long axis. Each level was examined by H&E and AE1/AE3. Low volume disease was defined by the American Joint Committee on Cancer (AJCC) (Schwartz 2002): macro-metastases were defined as cancer deposits larger than 2.0 mm; micro-metastases were defined as deposits between 0.2 and 2.0 mm; and isolated tumor cells were defined as deposits no greater than 0.2 mm, including the presence of single non-cohesive cytokeratinpositive cancer cells.
OSNA analysis of SLNs was performed as per previously described protocol.
Systematic bilateral pelvic lymphadenectomy was performed in all patients, regardless SLN mapping. Non-SLNs were examined with routine H&E staining.

Statistical analysis
Standard descriptive statistics were used to evaluate the distribution of each variable. Continuous variables were reported as median and categorical variables as frequency and percentage. Comparison of each variable between the groups of patients have been performed with t test for continuous variables and Chi-square or Fisher's exact test for categorical variables. Sensitivity, negative predictive value, and false-negative rates were calculated per patient and per hemi-pelvis. We considered findings to be false negative when lymphatic mapping showed drainage to one or more SLNs in a hemi-pelvis, biopsy of the SLN(s) revealed no metastases, and the patient had at least one metastatic non-SLN. A positive non-SLN in a hemipelvis with no SLN identified on lymphatic mapping was not considered to indicate a false-negative finding as our practice was to perform a complete pelvic lymphadenectomy in any case. All p values reported were two sided and a p value < 0.05 was considered statistically significant. Analysis was computed using SPSS version 26.0 (IBM Corporation 2018, Armonk, NY: IBM Corp.).

Comparison of open and minimally invasive approach
Comparison of characteristics of minimally invasive (58, 68.2%) and open approach (27, 31.8%) is reported in Table 2. Patients who underwent open approach reported higher rate of grade 3 (p = 0.028) and positive LVSI (p = 0.003), deeper stromal infiltration (median 8 mm vs. 5 mm, p = 0.016), larger maximum tumor diameter (median 25 mm vs. 15 mm, p = 0.003) and higher FIGO stage (p = 0.039). Despite these differences, no difference in any SLN mapping or in SLN bilateral detection was evident between open and minimally invasive approach (p = 0.850 and p = 0.222, respectively). Moreover, there was no difference in median number of SLNs mapped and retrieved between the two approaches (2 in both groups, p = 0.165) and in site of SLN mapping per hemi-pelvis (right side, p = 0273 and left side, p = 0.618). Figures 1  and 2 show two examples of SLN mapping in open and minimally invasive surgery, respectively.

Comparison of no/unilateral and bilateral SLN detection
Analysis of variables associated with bilateral SLN detection, compared with no/unilateral SLN detection within the entire cohort, is reported in Table 3. None of the analyzed characteristics was significantly different between the two groups. In particular, no surgery-related, or tumor-related variable was associated with higher bilateral SLN mapping. When we divided the study period in two parts, considering the first as "learning period" (33 patients, until 12/2018) and the second "experienced period" (52 patients, from 01/2019), no difference was observed in bilateral SLN detection rate (p = 0.450).

Discussion
The aim of this study was to compare the bilateral SLN detection rate with ICG of patients undergoing open and minimally invasive radical hysterectomy for cervical cancer. Following the publication of the LACC trial (Ramirez  (Nitecki 2020. SLN has become essential part of the operative management of early-stage cervical cancer, as it provides important information on lymph nodes at highest probability of metastasis and it allows a thorough analysis of these nodes by ultrastaging (Cibula 2012) or OSNA . Such analysis allows to detect low-volume metastases, which represents an important information, having some authors described the negative prognostic impact of micrometastases (Kocian 2020); nevertheless, not all studies support the negative impact of micrometastases in cervical cancer (Guani 2020).
Moreover, ICG has been demonstrated to be superior to other tracers alone (such as blue dye or radioactive tracers), and comparable with combination of blue and radioactive tracer in detecting SLNs in cervical cancer (Di Martino 2017). With this background, we intended to assess whether ICG provides such a high bilateral SLN detection rate in open surgery as in minimally invasive surgery, in view of the clinical practice change that many centers across the world had with regards to the surgical approach.
With our results, we showed no difference in any SLN and bilateral SLN detection between patients undergoing SLN mapping with open surgery compared to laparoscopic or robotic surgery. Moreover, with an overall and bilateral detection rate of 92.6% and 72.0%, SLN mapping with ICG in laparotomy is in line with previously reported pooled data from a diagnostic review, showing overall and bilateral detection rate of 91% and 60%, respectively (Tax 2015).
Differences in tumor characteristics between patients undergoing open and minimally invasive approach is likely to be related to the approach selection. Even though not significant, the difference in bilateral detection rate between the two approaches (72.0% in open versus 84.9% in minimally invasive), could be explained by the tumor characteristics of patients undergoing open approach. In fact, as previously described by (Dostálek 2018), bilateral SLN detection in large volume cervical cancer was lower, compared to smaller tumors.
Regarding sensitivity and negative predictive value in the per-patient analysis, our results demonstrated that there was no significant difference between patients undergoing open ICG SLN (83.3-95.0%, respectively) and minimally invasive ICG SLN (92.9-97.5%, respectively). The same applies for the per-hemipelvis analysis. When compared with other literature studies, we found sensitivity from open ICG SLN in the present series to be slightly lower compared with other sensitivities reported in the literature (ranging from 91.0 to 96.4%), with a negative predictive value concordant with other series (ranging from 91.0 to 100%) (Cibula 2012;Tax 2015;Salvo 2017). This is not the first study reporting the use of ICG to detect SLN in open surgery for cervical cancer. However, to date, only few case reports or small series were reported (Furukawa 2010;Crane 2011;van der Vorst 2011;Schaafsma 2012;Buda 2016;Rychlik 2016;Snyman 2018;Bedyńska 2019). Table 4 shows the characteristics of the studies which reported on the use of ICG to detect SLN in laparotomy. On the other hand, SLN in open surgery has been largely described with the use of other tracers such as blue dye, radioactive tracer (technetium) or combined technique (Silva 2005;Wydra 2006;Altgassen 2007;Kara 2008;Dostalek 2020). Nevertheless, ICG shows some advantages compared to other tracers: the low-risk of allergic reaction compared to blue dye (Papadia 2017); possibility of performing SLN biopsy in one-step at the time of surgery and the absence of patients exposure to ionizing radiation compared to radioactive tracer; lastly, it allows a real-time visualization of lymphatic channels.
The main limitation of the present study is the possible selection bias for patients submitted to open surgery; on the other hand, we have to acknowledge that this is the first study reporting the use of ICG in open surgery in a relatively large number of patients with cervical cancer, with a novel near infrared technology.  Funding Open access funding provided by Università Cattolica del Sacro Cuore within the CRUI-CARE Agreement. This study was not supported by funds.
Data availability All data and materials as well as software application or custom code support their published claims and comply with field standards.

Compliance with ethical standards
Conflicts of interest All authors declare no conflict of interest.
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