Articles. Funding Novadaq. Copyright 2018 Elsevier Ltd. All rights reserved.

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1 Near-infrared fluorescence for detection of sentinel lymph nodes in women with cervical and uterine cancers (FILM): a randomised, phase 3, multicentre, non-inferiority trial Michael Frumovitz, Marie Plante, Paula S Lee, Samith Sandadi, James F Lilja, Pedro F Escobar, Lilian T Gien, Diana L Urbauer, Nadeem R Abu-Rustum Summary Background Accurate identification of sentinel lymph nodes in patients with cancer improves detection of metastatic disease and decreases surgical morbidity. We sought to establish whether indocyanine green fluorescent dye is non-inferior to isosulfan blue dye in detecting sentinel lymph nodes in women with cervical and uterine cancers. Methods In this non-inferiority, within-patient comparison study, patients aged 18 years or older with clinical stage I endometrial or cervical cancer undergoing curative surgery were randomly assigned 1:1 to lymphatic mapping with isosulfan blue dye (visualised by white light) followed by indocyanine green (visualised by near-infrared imaging), or indocyanine green followed by isosulfan blue dye. Permuted block randomisation with stratification by study site was done with a computerised random number generator. All participants were masked to their randomisation assignment until after the procedure; however, investigators were not masked to the procedure used. Laparoscopic surgery with the PINPOINT near-infrared fluorescence imaging system (Stryker, Kalamazoo, MI, USA) was used in all cases. The primary outcome was efficacy of intraoperative indocyanine green with near-infrared fluorescence imaging versus that of isosulfan blue dye in the identification of lymph nodes, defined as the number of lymph nodes identified by indocyanine green and isosulfan blue dye, respectively (and confirmed as lymphoid tissue by histology), divided by the number of lymph nodes identified intraoperatively and excised. The study had a 5% non-inferiority margin needed to show non-inferiority of the frequency of lymph node detection with indocyanine green to that with isosulfan blue dye with 80% power at a 5% two-sided significance level. Analyses were done in both per-protocol and modified intention-to-treat populations. The trial was registered with ClinicalTrials.gov, number NCT , and is completed and closed. Findings Between Dec 21, 2015, and June 19, 2017, 180 patients were enrolled and randomly assigned to the two groups (90 to each group); 176 patients received the intervention and were evaluable (modified intention-totreat population). 13 patients with major protocol violations were subsequently excluded from the per-protocol population. 517 sentinel nodes were identified in the per-protocol population (n=163), of which 478 (92%) were confirmed to be lymph nodes on pathological processing: 219 (92%) of 238 nodes that were both blue and green, all seven nodes that were blue only, and 252 (95%) of 265 nodes that were green only (p=0 33). Seven sentinel lymph nodes were neither blue nor green but were removed for appearing suspicious or enlarged on visual examination. In total, 471 (97%) of 485 lymph nodes were identified with the green dye and 226 (47%) with the blue dye (difference 50%, 95% CI 39 62; p<0 0001). In the modified intention-to-treat population (n=176), 545 nodes were identified, of which 513 (94%) were confirmed to be lymph nodes on pathological processing: 229 (92%) of 248 nodes that were both blue and green, all nine nodes that were blue only, and 266 (95%) of 279 nodes that were green only (p=0 30). Nine sentinal lymph nodes were neither blue nor green but were removed for appearing suspicious or enlarged on visual examination. 495 (96%) of 513 nodes were identified with the green dye and 238 (46%) with the blue dye (50%, 39 61; p<0 0001). Lancet Oncol 2018 Published Online August 21, S (18) See Online/Comment S (18)30514-X University of Texas MD Anderson Cancer Center, Houston, TX, USA (Prof M Frumovitz MD, D L Urbauer MS); Laval University, CHU de Quebec, L Hotel-Dieu de Quebec, Quebec City, QC, Canada (Prof M Plante MD); Duke University Medical Center, Durham, NC, USA (P S Lee MD); Florida Gynecologic Oncology, Fort Lee, FL, USA (S Sandadi MD); Bay Area Gynecology Oncology, San Jose, CA, USA (J F Lilja MD); San Jorge Children s Hospital, San Juan, Puerto Rico (P F Escobar MD); Sunnybrook Odette Cancer Center, Toronto, ON, Canada (L T Gien MD); and Memorial Sloan-Kettering Cancer Center, New York, NY, USA (Prof N R Abu-Rustum MD) Correspondence to: Prof Michael Frumovitz, Department of Gynecologic Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA mfrumovitz@mdanderson.org Interpretation Indocyanine green dye with near-infrared fluorescence imaging identified more sentinel nodes than isosulfan blue dye in women with cervical and uterine cancers, with no difference in the pathological confirmation of nodal tissue between the two mapping substances. Funding Novadaq. Copyright 2018 Elsevier Ltd. All rights reserved. Introduction The disease status of the regional lymph nodes is one of the most important prognostic factors in all solid tumours and often drives recommendations for adjuvant chemotherapy and radiotherapy following surgical resection. The technique of lymphatic mapping and sentinel lymph node biopsy has improved surgeons ability to detect small-volume disease in lymph nodes while greatly reducing intraoperative and postoperative morbidity. This technique has been validated and is Published online August 21,

2 Research in context Evidence before this study Before development of the concept for the FILM study and the production of the subsequent protocol, we reviewed the medical literature to evaluate the off-label use of indocyanine green and near-infrared imaging for lymphatic mapping and sentinel lymph node biopsy in all solid tumours. We searched PubMed and ClinicalTrials.gov, without date or language restrictions, using the terms indocyanine green, ICG, near infrared imaging, lymphatic mapping, and sentinel node. We identified several small, single-institution retrospective reports of interstitial injection of indocyanine green for lymphatic mapping in a wide range of solid tumours including breast, uterine, cervical, vulval, lung, kidney, and colon cancers as well as cutaneous melanoma. All these studies showed that use of indocyanine green with near-infrared imaging was feasible and safe for lymphatic mapping in solid tumours and that the technique might potentially improve on substances currently approved by the US Food and Drug Administration for mapping such as patent blue dyes and radiocolloids. However, no prospective, controlled studies had compared the use of indocyanine green as a mapping substance with standard of care. Added value of this study Although the FILM study was designed as a non-inferiority comparison of the efficacy of intraoperative indocyanine green with near-infrared imaging to blue dye in the identification of lymph nodes, our results showed superiority of the experimental group over standard of care. To our knowledge, this is the first study to compare indocyanine green and isosulfan blue dye as mapping substances in a prospective, randomised controlled trial. Indocyanine green dye with near-infrared imaging identified significantly more sentinel nodes and more bilateral sentinel nodes than did isosulfan blue dye. It also identified all sentinel nodes with metastatic disease, whereas isosulfan blue dye missed a large proportion of them. Implications of all the available evidence The results of this study will serve as the basis for an application to the Food and Drug Administration for on-label use of interstitial injection of indocyanine green combined with near-infrared imaging for lymphatic mapping. Indocyanine green significantly improved sentinel lymph node identification frequency and detection of metastatic disease. If it is approved for on-label use, it will hopefully become the new standard of care for lymphatic mapping and sentinel lymph node biopsy for women with cervical and uterine cancers, and could subsequently be adopted as the mapping substance of choice across multiple subspecialties in surgical oncology. widely accepted as part of the surgical treatment of vulval and breast carcinomas and cutaneous melanoma. Gynaecological oncologists are also starting to adopt lymphatic mapping and sentinel node biopsy for women with cervical and uterine cancers. 1 3 In lymphatic mapping for cervical and uterine cancers, mapping agents are most commonly injected into the cervix, a practice that should result in drainage to bilateral sentinel nodes in the pelvis. If lymphatic mapping does not identify bilateral sentinel nodes, well accepted algorithms recommend complete pelvic lymphadenectomy on the side with no sentinel nodes detected. 4,5 Because complete pelvic lymphadenectomy increases surgical time and the risks of intraoperative vascular injury and postoperative lower extremity lymphoedema and lymphocyst formation, it is crucial that lymphatic mapping identifies bilateral sentinel nodes. Historically, patent blue dye with or without radiocolloid has been the most commonly used agent for lymphatic mapping in women with uterine cancers. However, blue dye alone identifies at least one sentinel node in only 80% of patients and bilateral sentinel nodes in as few as 50% of patients. 6 Combining blue dye with radiotracer increases the rate of detection of at least one sentinel node to 88%, but the rate of detection of bilateral sentinel nodes to only 51%. 6 With either technique, therefore, almost half of women with uterine cancer undergoing lymphatic mapping might require unilateral or bilateral pelvic lymphadenectomy, which increases the risk of surgical complications and long-term morbidity. The fluorescent dye indocyanine green has been explored as an off-label alternative to blue dye for lymphatic mapping in cervical and uterine cancers. Small series have shown the potential of interstitial indocyanine green injection to improve frequency of sentinel node detection over that observed with blue dye. 7 9 However, no prospective, randomised studies have compared interstitial blue dye with indocyanine green injections for lymphatic mapping in cervical and uterine cancers. We therefore conducted the FILM (Fluorescence Imaging for Lymphatic Mapping) trial, which was designed to compare the detection of lymph nodes after interstitial indocyanine green injection versus interstitial isosulfan blue dye injection (the standard of care) in women with cervical and uterine cancers. Methods Study design and participants The FILM trial was an international, multicentre, randomised, open-label, phase 3 study designed to assess the safety and efficacy of indocyanine green and PINPOINT near-infrared fluorescence imaging (Stryker, Kalamazoo, MI, USA) in identification of lymph nodes in women with cervical and uterine malignancies undergoing lymphatic mapping following interstitial 2 Published online August 21,

3 cervical injection of coloured dye. The study accrued patients at eight academic medical centres in the USA and Canada (appendix p 1). The trial design was a non-inferiority, within-patient comparison study. We chose this design because it would meet our primary objective and give us reliable results with fewer patients than if we had two separate randomised groups. Participating surgeons were required to have completed at least ten lymphatic mapping procedures, including at least three with the PINPOINT near-infrared fluorescence imaging system, before the initiation of enrolment. Patients were eligible for enrolment if they were 18 years of age or older, diagnosed with International Federation of Gynecology and Obstetrics clinical stage I cervical or uterine cancer (any histology), and were scheduled for curative surgery that included lymph node assessment. Any patient felt to be a safe surgical candidate was considered eligible irrespective of Eastern Cooperative Oncology Group performance status or life expectancy. Other eligibility criteria included negative nodes and absence of metastatic disease by clinical evaluation and radiological imaging. Patients were ineligible if they had previous pelvic dissection or irradiation, advanced cervical or uterine cancer, T3 or T4 lesions, cervical tumour size larger than 2 cm, hepatic dysfunction defined as a Model for End-Stage Liver Disease score of 10 or greater, renal dysfunction defined as serum creatinine of 2 0 mg/dl or greater, or a known allergy to indocyanine green, iodine, iodine dyes, isosulfan blue, or triphenylmethane. The protocol was approved by institutional review boards at each centre, and all patients enrolled gave written informed consent. The protocol is available in the appendix (p 3). Randomisation and masking Participants were prospectively enrolled into the FILM trial and underwent random assignment on the day of surgery. Randomisation was done as closely as possible in time to the mapping procedure to minimise dropouts. Participants were randomly assigned on a 1:1 basis to either lymph node mapping with isosulfan blue dye followed by lymph node mapping with indocyanine green dye and near-infrared imaging (PINPOINT), or lymph node mapping with indocyanine green dye and near-infrared imaging (PINPOINT) followed by lymph node mapping with isosulfan blue dye. Randomisation was stratified by study site, with permuted block randomisation within strata. To minimise the opportunity for the sequence to be predicted, the block size was variable and randomly chosen from small multiples of two (ie, two, four, or six). The randomisation schedules were generated in advance using a computerised random number generator. Investigational sites did not have access to the randomisation schedules. Randomisation was accomplished using a secure web-based software (REDCap). 10 Because this was a within-participant comparison study, the investigator could not be blinded to the use of blue dye or the PINPOINT device. All participants were masked to their randomisation assignment (because they were under anaesthesia) until after the procedure. Procedures Patients were removed from the study if found to not meet eligibility criteria or if they requested removal at any time before surgery (patients could request removal at any time during the study). All eligible and consenting patients underwent laparoscopic surgery performed according to the surgeon s standard practice. Indocyanine green (IC2000) injections were done with use of the PINPOINT near-infrared fluorescence imaging system (Stryker, Kalamazoo, MI, USA). After general anaesthesia was achieved, the first dye was injected in the cervix deeply and superficially at both 0300 h and 0900 h, followed by the second dye deeply and superficially at both 0300 h and 0900 h, for a total of eight injections. For the blue dye indocyanine green group, the isosulfan blue dye injections were done first, and for the indocyanine green blue dye group, the indocyanine green injections were done first. Each blue dye injection consisted of 1 ml of a 10 mg/ml isosulfan blue dye solution (1% isosulfan blue), for a total dose of 40 mg; each indocyanine green injection consisted of 1 ml of a 1 25 mg/ml indocyanine green solution (Novadaq Technologies, Mississauga, ON, Canada) for a total dose of 5 mg. The surgeon identified lymph nodes and lymphatic vessels with white light for isosulfan blue dye (blue nodes) or near-infrared imaging with PINPOINT for indocyanine green (green fluorescent nodes) depending on random isation cohort. Visibly abnormal lymph nodes were excised irrespective of whether blue dye or green fluorescence was visible. To minimise the chance that leakage of blue dye or indocyanine green would interfere with mapping, mapping was done first on one side of the pelvis and then on the other side. In both patient groups, mapping with the first dye was completed before mapping with the second dye was started, and mapping with both dyes was completed before any lymph nodes were excised. Mapping with isosulfan blue dye was considered complete when the surgeon identified all blue nodes or determined that blue nodes could not be identified. Mapping with indocyanine green was considered complete when the surgeon identified all green nodes or determined that green nodes could not be identified and the surgeon had scanned the full 360-degree area within the abdominal cavity. Once mapping with both dyes was complete and documented, all lymph nodes identified with either dye were excised. Fluorescent channels were followed in both directions to identify lymph nodes to be excised. Sentinel and nonsentinel nodes with appearance suggestive of disease underwent intraoperative frozen section analysis at the surgeon s discretion; normal-appearing nodes did not. See Online for appendix Published online August 21,

4 199 patients screened 19 excluded 180 enrolled and randomly assigned 90 assigned to blue dye indocyanine green 90 assigned to indocyanine green blue dye 3 did not receive intervention 1 due to iodine allergy 1 due to negative margins on cone biopsy 1 due to presence of intraperitoneal metastatic disease on laparoscopic exploration 1 did not receive intervention 1 had excessive bleeding and did not undergo injection of mapping substances 87 received intervention and included in mitt analysis 89 received intervention and included in mitt analysis 6 protocol violations 2 due to pregnancy test not done on day 0 1 due to incorrect blue dye used 1 due to presence of intraperitoneal metastatic disease on laparoscopic exploration 1 due to no calculation of MELD score 1 due to equipment malfunction 7 protocol violations 3 due to no calculation of MELD score 1 due to equipment malfunction 1 due to conversion to open surgery 1 due to incorrect dose of blue and green dye injections 1 due to participation in another trial 81 included in per-protocol analysis 82 included in per-protocol analysis Figure: Trial profile mitt=modified intention-to-treat. MELD=Model for End-Stage Liver Disease. Bilateral lymphatic mapping was done according to published National Comprehensive Cancer Network guidelines. 4,5 All sentinel lymph nodes resected had confirmation of nodal tissue by haematoxylin and eosin staining. Evaluation for presence of metastatic disease was performed with ultrastaging (additional sectioning and staining of the sentinel node) and immunohistochemistry per institutional protocol. There was no central review of pathological specimens. No postoperative patient follow-up was required for any primary or secondary endpoint except for adverse events and toxicities. All participants had standard-ofcare assessments throughout the study according to the hospital or institution s standard procedures, and study-specific visits to monitor occurrence of any adverse events or adverse device effects on postoperative day 1, the date of discharge, and day 30 (or within 7 days before or after this date). All participants were followed up to monitor occurrence of adverse events up to day 30 (or within 7 days before or after this date) after surgery. Adverse events were characterised as mild, moderate, or severe and assigned relationship (suspected or not suspected) to either dyes or device. Outcomes The primary endpoint was efficacy of intraoperative indocyanine green with near-infrared fluorescence imaging versus that of blue dye in the identification of lymph nodes, defined as the number of lymph nodes identified by each dye (and confirmed as lymphoid tissue by histology) divided by the number of lymph nodes identified intraoperatively and excised. Secondary end points were the rate of intraoperative detection of at least one sentinel node per patient and the rate of detection of bilateral sentinel nodes with indocyanine green compared with isosulfan blue dye, and the safety of each detection method. Two other secondary endpoints (the proportion of lymph nodes identified from following lymphatic channels and the anatomical distribution of lymph nodes) will be reported in a separate publication. Statistical analysis We hypothesised that the use of indocyanine green and near-infrared imaging would be non-inferior to isosulfan blue dye in the detection of sentinel nodes. A sample size of 525 nodes (approximately 175 evaluable participants) was required to show non-inferiority of the frequency of lymph node detection with 4 Published online August 21,

5 indocyanine green to that with isosulfan blue dye with 80% power at a 5% two-sided significance level and a 5% non-inferiority margin. Both a modified intention-to-treat (mitt) population and a per-protocol population were used for the primary analysis. All randomised and evaluable patients were included in the mitt population. Patients were excluded from the per-protocol population in cases of major deviations from the protocol (eg, incomplete or inappropriate labs drawn preoperatively, equipment or camera malfunction, presence of intraperitoneal disease on surgical exploration, incorrect dose of dye injected, use of blue dye from hospital pharmacy and not from trial kit, and allergy to iodine discovered after consent or randomisation but before injection). Analysis of the primary endpoint was done with a two-sided 95% CI for the difference in proportions using the approach described by Nam and Kwon for clustered matched-pair data (the clustering in question being lymph nodes nested within patients). 11 Formal testing for heterogeneity was not completed. We did non-inferiority testing using the per-protocol population as a conservative approach, because this population was considered the most apt to show inferiorities. If noninferiority was achieved, we planned a superiority test using the mitt population, because this population was considered the most likely to show no difference. The secondary endpoints detection of at least one lymph node per patient and detection of bilateral lymph nodes were tested using a two-sided 5% significance level with the step-down multiplicity adjustment described by Benjamini and Liu. 12 Difference in node identification in both the mitt and per-protocol populations was also assessed by site to examine whether it was appropriate to pool site data together. Study data were collected and managed using REDCap. 10 All data were analysed with SAS version 9.4 by biostatisticians in the Department of Biostatistics at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). This study is registered with ClinicalTrials.gov, number NCT Role of the funding source The funder of the study participated in the study design but did not participate in the collection, analysis, or interpretation of the data, or in the writing of this report. MF and DLU were the only authors with access to all the raw data. The corresponding author had full access to all the data and had final responsibility for the decision to submit for publication. Results Between Dec 21, 2015, and June 19, 2017, 180 patients were enrolled and randomly assigned to the two groups (90 to each group), of whom 176 received the intervention and were evaluable (figure). Baseline characteristics Blue dye indocyanine green group (n=87) were similar in the two groups (table 1). 169 (96%) of 176 patients had uterine cancer and seven (4%) had cervical cancer. After exclusion of 13 patients with major protocol violations (six in the blue dye indocyanine green group and seven in the indocyanine green blue dye group), there were 163 in the per-protocol population and primary analytical cohort. In this population, 517 nodes were identified intraoperatively and excised, of which 478 (92%) were confirmed to be lymph nodes: 219 (92%) of 238 nodes that were both blue and green, all seven nodes that were blue only, and 252 (95%) of 265 nodes that were green only (p=0 33; table 2). Seven sentinel lymph nodes were neither blue nor green but were removed for appearing suspicious or enlarged on visual examination. In total, 471 (97%) of 485 lymph nodes were identified with the green dye and 226 (47%) with the blue dye (difference 50%, 95% CI 39 62; p<0 0001). Thus, we were able to conclude that indocyanine green is not inferior to isosulfan blue dye in the identification of nodes. The mean number of nodes per patient in the per-protocol population was 3 2 (SD 1 6). At least one node was identified in 159 (98%) of 163 patients Indocyanine green blue dye group (n=89) Age, years 63 (58 71) 63 (56 68) Body-mass index, kg/m² 32 7 ( ) 31 7 ( ) Race White 66 (76%) 73 (82%) Hispanic 10 (11%) 12 (14%) African American 5 (6%) 2 (2%) Asian 4 (5%) 2 (2%) Other 2 (2%) 0 (0%) ASA classification I 10 (11%) 11 (12%) II 38 (44%) 39 (44%) III 34 (39%) 32 (36%) Unknown 5 (6%) 7 (8%) Type of cancer Uterine 84 (97%) 85 (96%) Cervical 3 (3%) 4 (4%) Enrolment site MD Anderson (TX, USA) 19 (22%) 19 (21%) Centre Hospitalier Universitaire de Quebec (QB, Canada) 17 (19%) 17 (19%) Duke Cancer Institute (NC, USA) 12 (14%) 12 (14%) Lee Memorial (FL, USA) 12 (14%) 12 (14%) Memorial Sloan-Kettering (NY, USA) 11 (13%) 11 (12%) O Connor Hospital (CA, USA) 7 (8%) 7 (8%) HIMA San Pablo (Puerto Rico) 5 (6%) 7 (8%) Sunnybrook Health Sciences Centre (ON, Canada) 4 (4%) 4 (4%) Data are median (IQR) or n (%). All patients who received the procedure are shown (modified intention-to-treat population). ASA=American Society of Anesthesiology. Table 1: Baseline characteristics Published online August 21,

6 Per-protocol population Proportion detected n/n p value Proportion detected Modified intention-to-treat population with indocyanine green and in 124 (76%) of 163 patients with isosulfan blue dye (difference 22%, 95% CI 17 32; p<0 0001). Bilateral sentinel nodes were identified in 134 (82%) patients, 52 (32%) with isosulfan blue dye and 132 (81%) with indocyanine green (49%, 41 57; p<0 0001). 176 evaluable patients received at least one injection of indocyanine green or blue dye and constituted the mitt population. In this cohort, at least one sentinel node was identified in 172 (98%) women. 545 nodes were identified in total, of which 513 (94%) were confirmed to be lymph nodes: 229 (92%) of 248 nodes that were both blue and green, all nine nodes that were blue only, and 266 (95%) of 279 nodes that were green only (p=0 30; table 2). Nine sentinel lymph nodes were neither blue nor green but were removed for appearing suspicious or enlarged on visual examination. In total, 495 (96%) of 513 nodes were identified with the green dye and 238 (46%) with the blue dye (difference 50%, 95% CI 39 61; p<0 0001). Thus, we were able to conclude that indo cyanine green is superior to isosulfan blue dye in the identification of nodes. In the mitt population, at least one node was identified in 168 (95%) of 176 patients with indocyanine green and 131 (74%) patients with isosulfan blue dye (difference 21%, 95% CI 14 28; p<0 0001). Bilateral sentinel nodes were identified in 141 (80%) of 176 patients in the mitt population. Bilateral sentinel nodes were identified in 54 (31%) of 176 patients with isosulfan blue dye and 138 (78%) of 176 patients with indocyanine green (47%, 30 56; p<0 0001). In the 545 nodes (mean 3 1 nodes [SD 1 7] per patient) identified in the mitt population, isosulfan blue dye identified 257 (47%) sentinel nodes (mean 1 5 [1 3] per patient) and indocyanine green identified 527 (97%) sentinel nodes (mean 3 0 [1 7] per patient; difference 50%; 95% CI 39 61; p<0 0001). n/n p value Confirmation of nodal tissue in sentinel lymph node Blue dye plus indocyanine 92% 219/238 92% 229/248 green Indocyanine green only 95% 252/265 95% 266/279 Blue dye only 100% 7/7 100% 9/9 Identification of one or more < < sentinel lymph nodes Indocyanine green only 98% 159/163 96% 168/176 Blue dye only 76% 124/163 74% 131/176 Identification of bilateral < < sentinel lymph nodes Indocyanine green only 81% 132/163 78% 138/176 Blue dye only 32% 54/163 31% 54/176 Table 2: Sentinel lymph node identification by indocyanine green and isosulfan blue dye Difference in node identification in both the mitt and per-protocol populations was also assessed by site to examine whether it was appropriate to pool site data together. Difference estimates and 95% CIs indicated that estimates of superiority were consistent throughout all sites (data not shown). Randomisation group did not affect the ability of isosulfan blue dye or indocyanine green to detect any or bilateral sentinel nodes in the mitt population. In the 87 patients in the blue dye indocyanine green group, at least one sentinel node was identified in 63 (72%) patients with isosulfan blue dye and in 83 (95%) patients with indocyanine green. In the 89 patients in the indocyanine green blue dye group, at least one sentinel node was identified in 68 (76%) patients with isosulfan blue dye and in 85 (96%) patients with indocyanine green. In the blue dye indocyanine green group, indocyanine green detected a sentinel node in 22 (25%) patients without a sentinel node detected by isosulfan blue dye. In the indocyanine green blue dye group, isosulfan blue dye detected a sentinel node in two (2%) patients without a sentinel node detected by indocyanine green. In the blue dye indocyanine green group, isosulfan blue dye identified bilateral sentinel nodes in 28 (32%) of 87 patients, indocyanine green identified bilateral sentinel nodes in 67 (77%) patients, and indocyanine green identified bilateral sentinel nodes in 40 (46%) patients without bilateral sentinel nodes identified by isosulfan blue dye. In the indocyanine green blue dye group, isosulfan blue dye identified bilateral sentinel nodes in 26 (29%) of 89 patients, indocyanine green identified bilateral sentinel nodes in 71 patients (80%), and isosulfan blue dye identified bilateral sentinel nodes in two patients (2%) without bilateral sentinel nodes identified by indocyanine green. As a post-hoc analysis, we assessed metastatic disease. 16 (9%) of 176 patients had metastatic disease in 21 sentinel nodes 13 (62%) both blue and green and eight (38%) green only. No metastatic sentinel nodes would have been missed with use of indocyanine green alone, whereas eight would have been missed with use of blue dye alone. Macrometastatic disease was found in eight (38%) of 21 sentinel nodes, micro metastatic disease in five (24%), and isolated tumour cells in eight (38%). There were no allergic reactions or adverse events attributable to either isosulfan blue dye or indocyanine green. Most adverse events were related to surgery (eg, postoperative pain or ileus). Discussion Although this study was designed as a non-inferiority trial, our findings suggest that indocyanine green was superior to isosulfan blue dye in identifying sentinel lymph nodes in women with cervical and uterine cancer because indocyanine green identified sentinel lymph nodes in a much larger proportion of patients than isosulfan blue dye did. Indocyanine green was also 6 Published online August 21,

7 significantly superior to isosulfan blue dye in detecting at least one sentinel node and in detecting bilateral sentinel nodes. The use of indocyanine green and isosulfan blue dye together does not seem to be necessary, because the addition of isosulfan blue dye to indocyanine green identified few sentinel nodes beyond those identified with indocyanine green alone. All metastatic sentinel nodes were detected with indocyanine green, but more than a third would have been missed had isosulfan blue dye alone been used. Finally, interstitial injection of indocyanine green seems to be safe, as we recorded no adverse events related to the compound. Our findings that isosulfan blue dye detected at least one sentinel node in 131 (74%) of 176 patients and indocyanine green detected at least one sentinel node in 168 (96%) of 176 patients in the mitt population are similar to what has been previously reported in the literature. 6 However, blue dye alone detected bilateral lymph nodes in only 54 (31%) patients in the mitt population, which is lower than the 55 60% reported in previous studies. 13,14 All surgeons participating in our study had attained proficiency in the mapping procedure, so it is difficult to attribute the low rate of detection of bilateral nodes with isosulfan blue dye alone to poor technique or learning curve. The use of isosulfan blue dye alone instead of combined blue dye and radiocolloid might have been responsible for the low detection of bilateral sentinel nodes. Retrospective studies have compared indocyanine green to blue dye plus radiocolloid with mixed results. Multiple single-institution studies have found indocyanine green to be superior to blue dye and radiocolloid in detecting bilateral sentinel nodes after a cervical injection, 15,16 but a large, multi-institutional retrospective study did not show a difference between the two techniques. 17 Systematic reviews and meta-analyses also present conflicting results, with some showing combination blue dye and radiocolloid equivalent to indocyanine green in detecting bilateral sentinel nodes 18 whereas others show that compared with use of blue dye alone, combined tracers might improve detection of at least one sentinel node but not detection of bilateral nodes. 6 Although the combination of blue dye and radiocolloid might be better than blue dye alone and equivalent to indocyanine green in detecting sentinel nodes, no studies either prospective or retrospective have compared the combination of blue dye and radiocolloid with indocyanine green. Finally, the relatively high median body-mass index (BMI) of our patients could have affected detection of bilateral nodes. The median BMI was 32 kg/m². In one retrospective study, high BMI adversely affected detection of bilateral sentinel nodes with indocyanine green or blue dye. However, the effect was much more pronounced with blue dye alone than with indocyanine green alone. 8 Because of the high rate of obesity in women with uterine cancer, many gynaecological oncologists have adopted the robotic system to assist in performing hysterectomy and staging, citing as their reason that the robotic system makes it easier to perform the lymphadenectomy portion of the surgery in women with a high BMI. 19,20 The most commonly used robotic platform, the da Vinci Surgical System (Intuitive Surgical, Sunnyvale, CA, USA), includes an option for intraoperative near-infrared imaging called the Firefly (originally developed by Novadaq technologies). The FIRES trial, 21 a prospective cohort study, showed that indocyanine green and near-infrared imaging in the da Vinci system had a sensitivity of 97% in detecting metastatic disease in sentinel nodes. The FILM protocol did not require completion of lymphadenectomy after sentinel node detection, so sensitivity and negative predictive values for the PINPOINT system cannot be calculated in our study. Moreover, the PINPOINT technology and image display used in our study differed from that of the Firefly, so the results of our study cannot be extrapolated to robotic surgery. In the FIRES study, at least one sentinel node was identified in 293 (86%) of 340 patients, which is lower than the proportion of patients shown to have at least one sentinal node with indocyanine green and the PINPOINT system in our study. Additionally, the indocyanine green used in this study (IC2000) might differ from the indocyanine green used in the FIRES trial (no details given in the trial). However, we believe lymphatic mapping and sentinel node detection with indocyanine green and near-infrared imaging is probably similar in the two systems, with potential applications of the technique in other cancers, including breast cancer and melanoma. Breast cancer, like uterine cancer, has highly predictable lymphatic drainage. In uterine cancer, more than 95% of sentinel nodes are found in the pelvis, and in breast cancer virtually all sentinel nodes are found in the axilla. Consequently, preoperative lymphatic mapping with radiocolloid and dynamic imaging (eg, lymphoscintigraphy or single photon emission CT CT [SPECT/CT]) to establish site of incision is unnecessary for lymphatic mapping in breast cancer. Improving the detection of sentinel nodes intraoperatively, therefore, is a major focus of research and innovation for breast cancer surgeons. The use of indocyanine green for detecting sentinel nodes in women with breast cancer might represent an improvement over current methods. For example, a direct comparison showed that indo cyanine green detected a sentinel node in % of patients with breast cancer whereas blue dyes detected a sentinel node in 88 89%. 22,23 Additionally, indocyanine green detects more sentinel nodes per patient than does blue dye. When compared with radiocolloid, indocyanine green does not appear to detect more sentinel nodes overall but might detect more metastatic sentinel nodes. 24 For melanoma, interest in indocyanine green as an alternative to radiocolloid for lymphatic mapping and sentinel node biopsy has been somewhat more tempered. Published online August 21,

8 Lymphatic drainage of melanoma can often be ambiguous, especially for lesions located on the trunk, head, or neck. 25 Preoperative lymphatic mapping with dynamic imaging (lymphoscintigraphy or SPECT/CT) is often necessary to identify draining nodal basins and to determine sites for surgical incision and exploration. The penetrance of near-infrared cameras to detect sentinel nodes through the skin is only 2 3 mm, so the use of indocyanine green as a substitute for radiocolloid is largely ineffective, especially for lesions located in anatomical areas with ambiguous drainage. 26 Lymphatic mapping and sentinel node biopsy is an image-guided, precision surgical procedure that is increasingly being adopted for the surgical staging of apparent cervical-confined and uterine-confined malignancies. As use of indocyanine green for lymphatic mapping in cervical and uterine malignancies becomes more routine, we should also be aware of the potential pitfalls of this new exciting technology. One major potential pitfall is removal of a presumed mapped lymph node that on final pathology is determined to be only lymphatic trunks or adipose tissue without nodal tissue. This occurrence seems to be more associated with increased indocyanine green use compared with blue dye. In our study, around 5 6% of presumed nodes with bright signal on near-infrared imaging were not confirmed to be nodes on pathology. This result is likely due to the bright green effect of the imaging with a laser-based near-infrared system. Occasionally, dilated lymphatic channels can lead to a very bright signal that leads to the channels being mistaken for nodes and excised. Mistaken excision of lymphatic trunks or adipose tissue instead of a node is potentially a major pitfall as sentinel node biopsy without complete lymphadenectomy becomes more common; failure to excise a true node could leave the disease status of an entire hemipelvis unknown. If the surgeon has any doubt, it is reasonable to send the presumed nodal tissue for an intraoperative pathological consultation to confirm nodal content. A second major potential pitfall of lymphatic mapping and sentinel node biopsy for cervical and uterine malignancies is the removal of multiple bright green nodes that appear to be true sentinel nodes but are in fact second-echelon and third-echelon nodes. This pitfall also relates to the bright signal from a laser-based, nearinfrared imaging platform. Unlike isosulfan blue dye, which rarely can be seen beyond the first or true sentinel node to secondary-echelon nodes, indocyanine green often produces bright green signal in not only the true sentinel node but also secondary, tertiary, and more distant nodal basins. The surgeon should be aware of this potential problem and could choose to remove secondary and tertiary nodes, but should not call them sentinel. Further, the enhanced pathology protocol evaluation should be limited to the true sentinel nodes, which in most studies average about three per patient with uterine malignancy. Although the initial capital expenditures needed to incorporate the use of indocyanine green and near-infrared imaging for lymphatic mapping in patients with cervical and uterine cancers might be prohibitively high for some institutions, the near-infrared imaging system could also be used for other purposes, such as evaluating perfusion of bowel or oesophageal anastomoses or delineating anatomy during cholecystectomy Moreover, the alternative to this approach is to continue to use the combination of blue dye and radiocolloid in these patients (since we have shown that blue dye alone for lymphatic mapping is suboptimal). Although blue dye is quite inexpensive, the use of radiocolloid is not, because its administration requires additional nuclear medicine technicians, nurses, and physicians, and the use of nuclear medicine facilities. Additionally, capital expenditures for open and laparoscopic gamma probes and their maintenance are also required. Finally, incorporation of sentinel lymph node biopsy with indocyanine green and near-infrared imaging might be the most cost-effective approach in treating women with gynaecological cancers when compared with complete lymphadenectomy in all patients or lymphatic mapping and sentinel node biopsy with radiocolloid or blue dye. 30 This study is limited in its inability to determine the sensitivity, negative predictive value, and oncological outcomes for lymphatic mapping and sentinel node biopsy in women with cervical and uterine cancers. The protocol was designed only to compare indocyanine green and isosulfan blue dye as mapping substances in these patients, and not as a trial to determine the validity of the sentinel node concept. Another potential limitation is the use of isosulfan blue dye alone as the comparator as opposed to combination blue dye and radiocolloid. However, although the combination of blue dye and radiocolloid might have improved detection rates in the standard group, on the basis of the retrospective data in the literature, 31 we believe indocyanine green would still be superior in detecting sentinel nodes in women with cervical and uterine cancers. Moreover, the results presented in this report reflect the outcomes from surgeons experienced with the procedure and the technology. Surgeons who are new to lymphatic mapping and sentinel lymph node biopsy should perform the procedure followed by complete lymphadenectomy until they are confident that they have achieved proficiency in accurately detecting sentinel nodes. Finally, as mentioned, the results can only be assumed valid for the specific compound (IC2000) and near-infrared system (PINPOINT) used in the study. However, we believe that studies using other formulations of indocyanine green and other nearinfrared imaging systems will probably yield similar results to our study. The US Food and Drug Administration indications for indocyanine green include only intravenous injection to determine cardiac output, to evaluate perfusion of solid 8 Published online August 21,

9 organs, and to perform ophthalmic angiography. Interstitial injection of indocyanine green for lymphatic mapping is currently an off-label use. The manufacturer of IC2000 indocyanine green dye has submitted an application to the Food and Drug Administration on the basis of the results from this study for on-label use of interstitial injection of indocyanine green combined with near-infrared imaging for lymphatic mapping. For women with cervical and uterine cancers, increasing evidence suggests that lymphatic mapping and sentinel node biopsy not only improves detection of disease in regional nodes but also decreases operative morbidity. 32,33 As lymphatic mapping and sentinel node biopsy become the standard of care in the surgical treatment of cervical and uterine cancers, improving sentinel node detection, particularly detection of bilateral sentinel nodes, will become the focus of research. As shown in this study, the incorporation of indocyanine green cervical injection and near-infrared imaging into the mapping procedure could represent an improvement over existing approaches (ie, blue dye, radiocolloid, or both) and might in the future become a standard method in these and other solid tumours. Contributors MF and NRA-R participated in study conception and design, acquisition of data, analysis and interpretation of data, drafting of the report, and critical revision. MP, PSL, SS, JFL, PFE, and LTG participated in acquisition of data and critical revision of the report. DLU participated in study conception and design, analysis and interpretation of data, and critical revision of the report. Declaration of interests MF reports grants from Novadaq/Stryker, during the conduct of the study, and personal fees from Novadaq/Stryker, grants from Navidea, personal fees from Johnson and Johnson, and personal fees from Genentech, outside the submitted work. The other authors declare no competing interests. Acknowledgments MF and DLU are supported by the National Institutes of Health/National Cancer Institute under award number P30CA References 1 Holloway RW, Abu-Rustum NR, Backes FJ, et al. Sentinel lymph node mapping and staging in endometrial cancer: a Society of Gynecologic Oncology literature review with consensus recommendations. Gynecol Oncol 2017; 146: Khoury-Collado F, St Clair C, Abu-Rustum NR. Sentinel lymph node mapping in endometrial cancer: an update. Oncologist 2016; 21: Salvo G, Ramirez PT, Levenback CF, et al. Sensitivity and negative predictive value for sentinel lymph node biopsy in women with early-stage cervical cancer. Gynecol Oncol 2017; 145: National Comprehensive Cancer Network. Cervical cancer (version ) 2017 (updated October 25, 2017). nccn.org/professionals/physician_gls/pdf/cervical.pdf (accessed March 1, 2018). 5 National Comprehensive Cancer Network. 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