Objectives
Near-infrared fluorescence imaging has the potential to improve sentinel lymph node mapping in vulvar cancer, which was assessed in the current study. Furthermore, dose optimization of indocyanine green adsorbed to human serum albumin was performed.
Study Design
Nine vulvar cancer patients underwent the standard sentinel lymph node procedure using 99m technetium-nancolloid and patent blue. In addition, intraoperative imaging was performed after peritumoral injection of 1.6 mL of 500, 750, or 1000 μM of indocyanine green adsorbed to human serum albumin.
Results
Near-infrared fluorescence sentinel lymph node mapping was successful in all patients. A total of 14 sentinel lymph nodes (average, 1.6; range, 1–4) were detected: 14 radioactive (100%), 11 blue (79%), and 14 near-infrared fluorescent (100%).
Conclusion
This study demonstrates feasibility and accuracy of sentinel lymph node mapping using indocyanine green adsorbed to human serum albumin. Considering safety, cost, and pharmacy preferences, an indocyanine green adsorbed to human serum albumin concentration of 500 μM appears optimal for sentinel lymph node mapping in vulvar cancer.
Vulvar cancer is a relatively rare disease with an annual incidence of approximately 4000 cases in the United States, resulting in 900 deaths per year. Tumor size and invasion into adjacent tissues are important factors for staging vulvar cancers, but nodal status remains the single most important prognosticator. Radical vulvectomy with en bloc inguinofemoral lymphadenectomy has been replaced in the surgical treatment of vulvar cancer by radical wide local excision or radical vulvectomy with inguinofemoral lymphadenectomy using separate groin incisions. The latter modification has significantly decreased surgery-related morbidity. However, 30% to 70% of patients treated with full inguinofemoral lymphadenectomy still suffer from lymphedema. Only 27% of patients with clinically stage I or II vulvar cancer have tumor positive lymph nodes; therefore, approximately 70% of patients undergo unnecessary lymphadenectomy.
Click Supplementary Content under the article title in the online Table of Contents
The sentinel lymph node (SLN) biopsy, as introduced in the management of cutaneous melanoma by Morton et al, was first described in vulvar cancer by Levenback in 1994. The SLN procedure in vulvar cancer patients has been validated in multicenter trials and its introduction in regular clinical practice has marked a significant reduction in lymphedema, wound infection, and wound dehiscence. Currently, the procedure usually involves a combination of a radioactive colloid and a blue dye. However, the use of radiotracers requires complex logistics including the involvement of a nuclear medicine physician and the transport of radioactivity, and is therefore not available in all clinics. Moreover, blue dyes cannot be visualized when the lymph nodes and lymphatic channels are covered by tissue, such as skin or fat.
The use of invisible near-infrared (NIR) light (700-900 nm) has several characteristics that can be advantageous in the SLN procedure, which include relatively high penetration into living tissue (millimeters to centimeters), when compared with blue dyes, and the lack of ionizing radiation. Indocyanine green (ICG) is one of only 2 clinically available NIR fluorescent agents and is currently the most optimal agent for SLN mapping. In several studies, intraoperative imaging systems in combination with ICG have been used for the SLN procedure for various types of cancer. The lymphatic channels and SLNs in vulvar cancer are often located in a relatively superficial location in the groin when compared with other tumors; therefore NIR fluorescence imaging could be particularly useful for this indication. Indeed, Crane et al reported the successful use of ICG alone at a concentration of 645 μM, in conjunction with an intraoperative imaging system for the SLN procedure in vulvar cancer. In that study involving 10 patients, 26 of 29 SLNs (90%) were detected in vivo by NIR fluorescence. Furthermore, lymphatic channels could be visualized in 5 of 16 groins (31%) containing SLNs.
Preclinical evidence demonstrated that premixing of ICG with human serum albumin (HSA, complex is ICG:HSA) increases the fluorescence intensity and hydrodynamic diameter of ICG, resulting in better retention in the SLN. The aims of the current study were to assess the use of NIR fluorescence imaging using ICG:HSA and the Mini-FLARE intraoperative imaging system for the SLN procedure in vulvar cancer and to optimize ICG:HSA dose.
Materials and Methods
Preparation of ICG:HSA
ICG (25 mg vials) was purchased from Pulsion Medical Systems (Munich, Germany) and resuspended in 10 mL of sterile water for injection for the 500 μM group, or in 5 mL of sterile water for injection for the 750 μM and 1000 μM groups, to yield stock solutions of 3.2 mM and 6.4 mM, respectively. Various amounts of this stock solution were transferred to a 50 mL vial of Cealb (20% HSA solution; Sanquin, Amsterdam, The Netherlands) to yield ICG in HSA (ICG:HSA) at a final concentration of 500 μM, 750 μM, or 1000 μM.
Intraoperative NIR fluorescence imaging
SLN mapping was performed using the Mini-FLARE image-guided surgery system as described in detail previously. Briefly, the system consists of 2 wavelength separated light sources: a “white” LED light source, generating 26,600 l× of 400 to 650 nm light to illuminate the surgical field and an NIR LED light source, generating 7.7 mW/cm 2 of fluorescence excitation light. White light and NIR fluorescence images are acquired simultaneously and displayed in real time, using custom designed optics and software. A pseudocolored (lime green) image of NIR fluorescence superimposed over the white light image is also displayed, to provide the NIR fluorescence signal in proper anatomic context.
Clinical trial
The current dose escalation clinical trial was approved by the Medical Ethics Committee of the Leiden University Medical Center and was performed in concordance with the ethical standards of the Helsinki Declaration of 1975. Nine consecutive patients that planned to undergo a SLN procedure for squamous cell vulvar carcinoma were included in this study between June 2010 and January 2011. All patients had clinically FIGO stage I vulvar cancer with a unifocal carcinoma measuring less than 4 cm in diameter, not encroaching the vagina, anus, or urethra and with negative inguinofemoral nodes as determined by palpation and ultrasonography. Exclusion criteria were pregnancy, lactation or an allergy to iodine, shellfish, or indocyanine green.
All patients gave informed consent and were anonymized. Patients received the standard-of-care SLN procedure. For our institution, this implies peritumoral injections of 60-100 MBq 99m technetium-nanocolloid on the afternoon of the day before, or the morning before surgery. Before the start of the operation, 1 mL total of patent blue V (Guerbet, France) was injected at 4 sites peritumorally. Immediately after injection of patent blue, 1.6 mL total of ICG:HSA was injected as 4 injections at the same location as the patent blue injections. After surgical scrub, the Mini-FLARE imaging head was positioned at approximately 30 cm above the surgical field. The NIR fluorescence signal was measured percutaneously, before skin incision, and continuously during the surgical procedure. Throughout the procedure, the surgeon was continuously provided with real-time NIR fluorescence image guidance. When the SLN could not be found easily by NIR fluorescence, the handheld gamma probe could be used for the localization of SLNs. Relative brightness of the SLNs was determined by measuring signal-to-background ratios (SBRs), that is the NIR fluorescence signal of the SLN divided by a directly adjacent region. Excised SNLs were analyzed ex vivo for NIR fluorescence and radioactivity and were routinely analyzed by histopathologic frozen section analysis. SLNs were fixed in formalin and embedded in paraffin for hematoxylin, eosin, and immunohistopathologic staining for AE1/AE3 at multiple levels, with an interval of 250 μm, according to the GROningen INternational Study on Sentinel nodes in Vulvar cancer (GROINSS-V) study protocol.
Statistical analysis
For statistical analysis, SPSS statistical software package (version 17.0; SPSS Inc, Chicago, IL) was used. Graphs were generated using GraphPad Prism Software (version 5.01; GraphPad, La Jolla, CA). To compare the SBR between concentration groups, a 1-way analysis of variance (ANOVA) was performed with pairwise comparison with least square difference (LSD) adjustment for multiple comparisons. Assumption of homogeneity of variances was assessed using Levene’s test. All statistical tests were 2-tailed and P < .05 was considered significant.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
