Feasibility in the Detection of Nodal Metastatic Melanoma Using Intravital Microscopy

Feasibility in the Detection of Nodal Metastatic Melanoma Using Intravital Microscopy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Feasibility in the Detection of Nodal Metastatic Melanoma Using Intravital Microscopy Emmanuel M Gabriel, Daniel T. Fisher, Minhyung Kim, Kristopher Attwood, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5161333/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While the clinical focus on the sentinel lymph node biopsy (SLNB) is the presence of intra- or extra-nodal metastases, preclinical studies suggest that tumor-draining SLNB-associated vascular architecture and adhesion properties are altered regardless of SLNB positivity. Human intravital microscopy (HIVM) has defined blood vessel abnormalities that may impact lymphocyte adhesion and systemic drug delivery at primary melanoma sites. In this pilot study of HIVM during melanoma SLNB, we sought to determine the feasibility of obtaining HIVM observations of SLNB-associated vessels. We successfully performed HIVM in all 20 SLNB patients, and 7 were found to have nodal micrometastases by standard pathology. HIVM was capable of identifying both functional and non-functional SLNB-associated vessels based on the presence or absence of fluorescent dye uptake, respectively. Comparing vessel characteristics as a secondary objective, no statistically significant differences were noted in the diameter, flow rate, functionality, or shear stress of SLNB-associated blood vessels between positive and negative SLNBs, which may likely have been a reflection of the minimal disease burden. Nonetheless, these initial observations provide the framework to optimize future trials of HIVM in cancer patients. Biological sciences/Cancer/Skin cancer/Melanoma Biological sciences/Cancer/Cancer imaging Figures Figure 1 Figure 2 Introduction Sentinel lymph node biopsy (SLNB) was originally described by Donald Morton for the staging and treatment of melanoma [ 1 ]. The multicenter selective lymphadenectomy trial (MSLT1) confirmed the prognostic value of SLNB for melanoma and its function as a staging procedure, with some clinical benefit in overall survival (OS) in select patients with intermediate-thickness melanoma [ 2 ]. The results of MSLT1 formed the basis of the MSLT2 trial that examined the outcomes for completion lymphadenectomy versus observation for positive sentinel nodes. MSLT2 confirmed that immediate completion lymphadenectomy for a positive sentinel node could be omitted with no impact on OS for the majority of patients [ 3 ]. Together, these trials raised questions regarding the clinical benefit of SLNB. However, in the absence of SLNB and/or completion lymphadenectomy, full staging may be incomplete and prognostic information that can influence decisions regarding frequency of surveillance and adjuvant therapies is lacking. By virtue of draining a primary tumor site, the sentinel lymph node represents more than just accurate staging and prognosis. On a cellular level, the sentinel node is the direct interface between the host immune system and metastatic tumor in the setting of secondary lymphoid tissue. Preclinical investigations into tumor draining lymph nodes have demonstrated how major immunologic functions such as recruitment of lymphocytes through specialized high endothelial venules (HEV) are impaired by downregulation of CCL21, a chemokine necessary for nodal entry of lymphocytes, even when actual nodal metastasis has yet to occur [ 4 ]. Soluble mediators including VEGF-D generated by primary tumors may precondition the draining node to support eventual tumor metastases by inducing changes in HEV from normally plump, cuboidal endothelium to a dilated and flat vessel architecture [ 5 ]. Rather than de novo generation of tumor vessels in SLNB, tumor-induced remodeling of HEVs not only sets the stage for tumor deposition, but also to become the nutrient tumor vasculature [ 6 , 7 ]. Primary tumors also appear to have the ability to induce stromal remodeling in tumor draining lymph nodes that downregulates CCL21 and IL-7 to further impede lymphocyte recruitment and activation, respectively [ 8 ]. Importantly, micrometastases that have gained a foothold in the tumor draining node appear to quickly co-opt the altered vasculature as a portal for intravasation and hematologic dissemination [ 7 , 9 , 10 ]. Therefore, microscopic imaging methods that could identify potential vascular alterations or micrometastasis in a sentinel lymph node in vivo might generate opportunities to preserve the sentinel node/tumor interface and create new treatment paradigms. Intravital microscopy (IVM) is the real-time evaluation of living tissue at the microscopic level (≥ 100 X magnification), which is capable of recording physiological processes such as blood flow and cell-cell interactions [ 4 , 11 , 12 ]. In oncology, preclinical IVM has elucidated mechanisms of metastasis, development of neoangiogenesis, and immune cell-tumor interactions primarily in animal models [ 13 – 16 ]. Independent of transillumination, IVM can be performed on exposed tissue of interest using an epifluorescent light source. IVM was only recently translated to the clinical arena in which melanoma tumors were interrogated by IVM in the operating room setting [ 17 ]. Clinical trials of human IVM (HIVM) proved feasible in the context of standard oncologic surgeries and generated real-time data of blood flow characteristics that was not previously possible [ 18 – 20 ]. Specifically, dynamic features such as blood flow, velocity, and real-time measurements of vessel diameters in living tissue showed differences that could not have been predicted from standard histologic evaluation of the same excised tissue [ 17 ]. The success of these first in-human trials have raised the possibility of a broader application to other oncologic procedures that could also produce relevant information of in situ human tissue prior to surgical resection [ 18 , 20 ]. Herein, we describe a completed clinical trial of HIVM of SLNB at the time of standard melanoma surgery. The primary goal of the study was to determine the feasibility of measuring vascular parameters of human lymph nodes after exposing the lymph node just prior to surgical excision. A successful observation included the ability to identify lymph node vessels, measure vessel diameters, and visualize fluorescein and/or indocyanine green (ICG) within the observed vessels. As these were SLNBs taken for melanoma, exploratory secondary objectives included comparisons between positive and negative lymph nodes and correlation to IVM measurements. Materials and Methods Patient selection Patients scheduled to undergo SLNB for melanoma were evaluated for entry into our pilot clinical trial I-284116 “Intravital Microscopy in Identifying Tumor Vessels in Patients with Melanoma Undergoing Sentinel Lymph Node Biopsy” at the Roswell Park Comprehensive Cancer Center, Buffalo, NY (ClinicalTrials.gov identifier: NCT02857374, first posted 05/08/2016). The study was approved by the Institute Review Board of Roswell Park Comprehensive Cancer Center, and all methods were performed in accordance with the Declaration of Helsinki. As groin lymph nodes were readily accessible to IVM observation, patients whose melanoma mapped to a non-groin sentinel lymph node were excluded. A full list of the inclusion and exclusion criteria is noted in Table 1 . Eligible patients who gave informed consent were enrolled in the protocol. The following data was recorded: patient demographics (age, sex, height, and weight), tumor-specific information (tumor type, location, and stage) and intravital microscopic measurements obtained from the surgery. Table 1 Study inclusion and exclusion criteria Inclusion Criteria Have an Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2 Melanoma tumor that meets indications for a groin SLN biopsy with a ≥ 10% risk of having metastasis to the draining lymph node ( i.e. , stage IB to stage IIIC melanoma of the lower body below the umbilicus) Participant must be eligible for a groin sentinel lymph node (SLN) biopsy Participant or legal representative must understand the investigational nature of this study and sign an Independent Ethics Committee/Institutional Review Board approved written informed consent form prior to receiving any study related procedure Exclusion Criteria Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements Sentinel lymph node is deemed inaccessible to microscopic observation during the operative procedure ( i.e. , sentinel node maps to a deep location or area outside of the groin) Renal dysfunction as defined as creatinine clearance < 70 mL/min by Cockroft-Gault equation Any known allergy or prior reaction to fluorescein, indocyanine green, iodine, or shellfish Unwilling or unable to follow protocol requirements Any condition which in the Investigator's opinion deems the participant an unsuitable candidate to undergo observational study (may also include preoperative testing results including electrocardiogram [EKG], chest x-ray, or pulmonary function tests) Any condition that excludes SLN biopsy as the standard of care ( e.g. , lymphadenectomy indicated) Of note, no control population was included in our pilot trial. A control population would consist of healthy volunteers without melanoma and with presumed normal lymph nodes. Because there is a 5–15% risk of lymphedema in the upper extremity and a 10–30% risk in the lower extremity with SLNB,[ 2 , 21 , 22 ] we did not include a control population due to risk and ethical considerations. Microscope design Sentinel lymph nodes were observed at 100X magnification using a highly modified Olympus microscopy system as previously described by our group [ 17 ]. The microscope was attached to a cantilevered arm to extend over the patient. Given the high magnification required to observe individual vessels and flow, vibrations in the microscope were minimized using a weighted marble base of over 360 kg. Intravenously administered fluorescein was illuminated using a 467–498 nm excitation, 513–556 nm emission dichronic filter set (Spectra Services, Rochester, NY) and ICG was illuminated using a 740–800 nm excitation, 800–860 nm emission dichronic filter set (Spectra Services, Rochester, NY) with an X-Cite 120 Led light source (Lumen Dynamics, Ontario, Canada). Images were captured using a Luca EMCCD camera (Andor Technology Ltd., Belfast, Northern Ireland) controlled through the Solis acquisition and analysis program. Images were acquired with a minimum of a 0.05-s exposure at 20 frames a second. Offline post-surgical quantification of vessel diameter and blood velocity was performed using the ImageJ software suite. Surgical technique All patients underwent standard lymphoscintigraphy with filtered technetium-99m sulfur colloid injected at the site of the primary melanoma. The SLNB location was confirmed by hand-held gamma probe (Neoprobe, Devicor Medical Products Inc., Cincinnati, OH). The skin overlying the sentinel lymph node was prepped with ChloraPrep (CareFusion Corp. San Diego, CA), and sterile towels and drapes were applied for exposure of the area. An incision was created with a scalpel in line with the longitudinal axis of the patient. With care to keep the lymph node capsule and perinodal adipose intact, the skin was elevated bluntly and retracted laterally with a Weitlaner retractor and the use of 3 − 0 Vicryl sutures (Ethicon, Raritan, NJ). Electrocautery was avoided as much as possible to prevent underlying tissue damage. In all instances, a saline soaked gauze was placed over top of the tissue to prevent desiccation before microscopic interface. After exposure of the confirmed SLNB was completed, the microscope was positioned for observation. Again, we intentionally preserved the peri-nodal tissues as to not disturb node capsule or blood supply going to the nodal hilum. At the completion of the observation period, the microscope was withdrawn, and the lymph node was removed and sent to pathology for evaluation. Standard pathologic analyses consisted of Hematoxylin and Eosin (H&E) staining as well as melanoma-specific stains including S-100, HMB-45, and Melan A. Intravital microscopic observations in patients The microscope was grossly moved into position by the surgeon over the field of interest and locked into place (as depicted in Fig. 1 A). Fine adjustments in the x and y axis were performed by using the built-in motor controls. Once the microscope was directly overlying the exposed SLNB surface, the fluorescent light source and image acquisition software were activated (Fig. 1 B). Vertical control of the microscope was performed manually by the surgeon until nodal vessels came into view on the digital monitor at 100X magnification. Lymph node vessels were clearly recognized by their well-defined architecture and branching patterns with observations focused on these areas. When present, melanoma micrometastases are typically found in the subcapsular sinus of lymph nodes (Fig. 1 B, 1 C) allowing for potential detection by IVM. To facilitate stabilization of the images, respirations were temporarily held by anesthesia for a maximum of 30 seconds. Patient respirations can lead to suboptimal imaging, but additional anesthesia or temporary holding respirations can often restore in focus, high resolution images. Therefore, clear communication with our anesthesia colleagues was key to ensure adequate HIVM observations. When a stable view of the vessels was achieved, 2.0 ml of 25% (250 mg/ml) fluorescein (Akorn Pharmaceuticals Inc., Lake Forest, Illinois) diluted in 10 ml of sterile saline was injected via a peripheral intravenous catheter. An observation was completed when fluorescein was noted to have extravasated into the nodal tissue and the vessels no longer manifested a detectable fluorescent signal. The microscope was then re-positioned with fine motor controls to observe several areas of the node. The microscopic observations were finalized when the 10 ml of diluted fluorescein was exhausted. Observations were repeated with ICG (IC-Green, Akorn Pharmaceuticals Inc., Lake Forest, Illinois) using 2.0 ml of 25% (250 mg/ml) using the same approach as with fluorescein. Of note, ICG observation produced less vascular detail than fluorescein and was omitted after the first 3 patient observations and was not included for analyses. Post-hoc analyses Post-hoc analyses were performed as previously described by our group.[ 17 ] Vessels were defined morphologically, and a single vessel was described as beginning at a branch point continuing to the next branch point. To be measured, vessels had to be 100 mm in length with no interposing branch points. Vessel density was established by enumerating vessels within a field of observation. ImageJ software was used to measure vessel diameter ( D ) and radius ( r ) at the vessel’s largest width. Blood flow velocity ( v ) was evaluated by determining the time ΔT distinct features in the fluorescent dye would take to travel a known distance ΔS and then averaging ΔS/ΔT for at least 10 points per vessel. Functionality of a given vessel was defined as a vessel that demonstrated fluorescent dye uptake/signal (Fig. 2 ) or kinetic blood flow of red blood cells. Non-functional vessels neither demonstrated dye uptake or kinetic blood flow. Percent functionality was determined as mean ± s.e.m. on a per field basis. Wall shear stress was computed as τ = 32 ηQ / πD 3 , where η is blood viscosity (assumed to be 2.2 centipoise) and Q is the blood flow rate ( Q = v * πr 2 with r being the radius of the tumor vessel). Wall shear rate was determined using the formula w = v *8/ D . The ability to discriminate between HEV and regular vessels and orders of HEV as previously described preclinically [ 23 ] was not possible due to limited fields of observation and lack of definitive anatomic references. Table 4 Comparison of vessel characteristics between negative and positive SLNB during intravital microscopic observations. Variable SLNB status Total Cohort (N = 20) Mean (Std) p value Number of observed fields negative 2.5 (0.4) 0.187 positive 3.3 (0.5) % Functional vessels negative 25.9 (11.2) 0.641 positive 30.7 (7.6) Diameter of observed vessels (µm) negative 21.3 (2.4) 0.759 positive 20.1 (1.9) Velocity of functional vessels (µm/s) negative 248.3 (53.8) 0.314 positive 321.2 (61.8) Wall Shear Stress (dyn/cm 2 ) negative 2.6 (0.7) 0.404 positive 3.9 (1.2) Wall Shear Rate (s − 1 ) negative 117.5 (33.3) 0.404 positive 175.8 (52.8) Statistical analyses Demographic and clinical characteristics were summarized using the mean and standard deviation (std) for continuous variables and using frequencies for categorical variables. No statistical method was used to predetermine sample size. Comparison of vessel characteristics between negative and positive SLNBs is presented as means with standard deviation and compared using two sample t-test. Statistical significance for all comparisons was accepted at p = 0.05. Data analyzed using R software (version 4.2.3). Results Patient demographics, SLNB-specific characteristics, and treatment outcomes Between December 2016 to August 2018, a total of 20 patients underwent SLNB with HIVM. Patient characteristics are summarized in Table 2 . Of the study cohort, 7 patients (35%) had a positive SLNB(s). Characteristics of patients who had a positive SLNB are shown in Table 2 . Of the 7 patients with a positive SLNB, 5 underwent completion lymph node dissection, and 2 of these patients had additional positive lymph nodes. There were no adverse events related to the administration of fluorescent dye or the HIVM procedure, similar to our previous HIVM studies. The overall disease burden within positive SLNBs was low with 6/7 having micrometastases < 2 mm or isolated tumor cells only as depicted in Fig. 1 C. Recurrence outcomes for each patient are also shown in Table 3 . At a median follow-up of 65 months, only 1 patient with a negative SLNB was found to have a recurrence during the study period. This patient developed both lower extremity in-transit disease and nodal recurrence 15 months after surgery. Conversely, 4/7 patients (57%) with a positive SLNB developed nodal and/or distant metastases with most occurring in the first 2 years of surveillance. Table 2 Patient and tumor variables. Variable N (%) Age (years) median (range) 58 (32–78) Sex female male 11 (55.0) 9 (45.0) Body mass index (kg/m 2 ) median (range) 28 (20–24) Primary melanoma Breslow depth (mm) mean (range) 2.2(0.5–4.7) Primary melanoma ulceration 7(35) Primary melanoma mitotic index median (range) 3(1–22) SLNB Positive Negative 7(35) 13(65) Table 3 Treatment and outcomes of patients with positive SLNB. Patient 1 2 3 4 5 6 7 SLNB disease burden (mm) 1.5 4 10 Isolated tumor cells 0.3 Isolated tumor cells 0.2 Completion lymph node dissection Yes Yes Yes No Yes Yes No Additional positive lymph nodes 0 1 0 N/A 1 N/A 0 Recurrence No Yes Yes No Yes No Yes Recurrence location N/A Soft tissue, Lung, Nodes Lung, Nodes N/A Soft tissue N/A Soft tissue, Spleen Time to recurrence (months) N/A 42 17 N/A 17 N/A 16 HIVM SLNB vessel characteristics All 20 patients were able to successfully undergo HIVM with observations of SLNB-associated blood vessels. Figure 2 depicts representative examples of HIVM observations using fluorescein at the time of SLNB, which align with our previous HIVM observations in other cancer histologies.[ 18 , 20 , 24 , 25 ] Vessel architecture in positive nodes were suggestive of the disorganized architecture seen in primary melanoma tumors [ 17 ]; however, unlike preclinical models of murine lymph nodes [ 23 ], HEV could not be reliably differentiated from non-HEV vessels in post-hoc analyses. Similar to our prior trials of HIVM in primary melanoma, primary sarcoma, or peritoneal carcinomatosis [ 17 , 18 , 20 ], functional vessels were defined as those blood vessels that took up the fluorescein dye and non-functional vessels were defined as those which did not take up the dye. Thus, functional vessels (labeled with blue arrows) were portrayed by a bright white appearance, whereas non-functional vessels (labeled with yellow arrows) were portrayed by a dark black appearance, which indicated the absence of fluorescein within the blood vessel. A comparison of vessel characteristics between negative and positive SLNBs is shown in Table 4 . Functional vessels were detected in only 26% and 30% of negative and positive SLNB, respectively. There were no statistically significant differences among any of the vessel characteristics, including density of functional or non-functional vessels, mean diameter of functional or non-functional vessels, or blood flow velocity within functional vessels for either negative or positive SLNB. The lack of differences may have been related to the limited number of functional vessels seen across all groups as well as the minimal disease burden of micrometastatic nodal melanoma. Correlations between SLNB-associated vessel characteristics and recurrence was not permissible given the small number of recurrence events. The IVM observed areas of the lymph node were not able to be localized the with standard pathologic evaluation and direct histologic comparisons to IVM observations were therefore not possible. Discussion To our knowledge and based on an extensive review of the current literature, this was the first study to demonstrate the feasibility of HIVM of tumor-draining lymph nodes. Using standard surgical techniques and our customized intravital microscope, we were able to obtain successful observations for each patient in our cohort. While our primary goal to determine the feasibility of HIVM for the SLNB was successful, our secondary goals of comparing SLNB-associated vessel characteristics and either SLNB positivity or recurrence did not yield any statistically significant differences. Yet, the findings of this clinical trial were informative to guide potential future applications of HIVM. IVM offers certain advantages over other imaging modalities, which make it a useful tool to investigate tumor-associated vessels in this pilot study and our prior HIVM trials. IVM allows for the real-time, high resolution (< 1 µm) observation of tumor-associated vessels and potential immune cell interactions.[ 11 ] While whole body imaging, including CT scan, PET-CT, and MRI, obtains a global visualization of large and medium sized blood vessels, these imaging modalities cannot determine vessel functionality at the capillary (microscopic) level. Lymphoscintigraphy with technetium injection can identify the draining SLNBs, but does not have a role for identifying tumor-associated or SLNB-associated blood vessels. Other high resolution imaging devices such as high frequency US, ocular coherence tomography (OCT), and photoacoustic microscopy (PAM) have been used to investigate tumor-associated microvessels.[ 11 ] Of these other imaging modalities, IVM offers the highest resolution (< 1 µm), whereby the others are generally limited to 10–15 µm. The tissue penetration of IVM is 200 µm, which is deeper than other modalities, such as high frequency US and OCT, which are generally limited to 10–50 µm. IVM therefore provides for a deeper assessment of blood vessels within the tissue targets. In addition, the image acquisition rate for IVM (30–100 ms) is also sufficient for tracking blood flow in real time down to individual flowing red blood cells.[ 26 ] While there is no current “gold standard” for assessing the tumor vasculature at microscopic resolution in real time in humans, for our purposes of tumor vessel imaging, IVM served as the most optimal technique. The limited correlative findings in SLNB are in contradistinction to our previous studies of HIVM of tumor tissue. The first report of HIVM in human cancer was published by our group in 2016 for primary melanoma that was surgically resected [ 17 ]. In this first-in-human study, patient melanoma-associated vessels were characterized as disorganized and tortuous compared to normal (control) blood vessels, and up to 50% of melanoma-associated vessels did not support blood flow. In addition, human melanoma-associated vessel diameters were larger than predicted from IHC or preclinical IVM. Since our initial study, we have continued HIVM observation for peritoneal carcinomatosis derived from several different histologies (including appendix, colorectal, ovarian, and mesothelioma cancers) [ 18 , 24 ]. Most recently, we have reported our HIVM findings for retroperitoneal sarcomas and brain tumors [ 20 , 25 ]. In each of these earlier clinical trials, we demonstrated that tumor areas had on average a higher density and proportion of non-functional vessels compared to control areas. Unlike our current study in melanoma-associated SLNB, our previous trial in ovarian carcinomatosis did show statistically significant correlations between tumor-associated vessels and outcomes, including disease-specific survival and response to neoadjuvant chemotherapy.[ 24 ] Several factors may have contributed to differences in our current HIVM observations in SLNB versus prior findings in tumors. All SLNB observations required surgical exposure of the tissue with the attendant potential to alter blood flow characteristics which may have accounted for the observation of only ~ 30% of all SLNB vessels manifesting blood flow. Thus, functional vessel assessments including flow rates were limited as compared to the majority of tumor studies that did not require perturbations of the tissue prior to HIVM observation. The inability to observe normal lymph nodes that are anatomically distant from tumor limits comparisons to a “true” normal control. As all SLNBs by definition are tumor-draining lymph nodes and based upon the observation that vascular changes occur within the node even prior to metastases [ 4 – 7 ], it is possible that HIVM measurements of SLNB without a normal control for comparison would be unable to detect any potential differences. Also, the alterations in vascular anatomy described preclinically may be minimal and below the threshold of detection for HIVM ( e.g. , flattening of normally cuboidal HEV) [ 5 ] and functional changes such as loss of adhesion molecules would not be detected [ 8 ]. Lastly, there is the possibility that pathologic analyses of the SLNB could have yielded false positive or false negatives. It is estimated that up to 10% of SLNB could be false positives.[ 27 ] If this were the case for any of the subjects with positive SLNB in our small cohort, then we would not expect to identify vascular differences in truly negative SLNB that were interpreted as being positive during intraoperative frozen section or final pathology (i.e., false positives). Many studies across different tumor types have shown that primary tumor-associated vessels are structurally aberrant and behave abnormally compared to normal vessels [ 28 – 30 ]. Importantly, these tumor vessel abnormalities have been shown to translate to poor responses to systemic therapies, both in preclinical animal models and in humans [ 26 , 31 – 33 ]. Our previous HIVM studies focused on known tumor-associated vessels compared to the surrounding grossly normal (control) tissue. However, in our current trial, it was unknown whether the SLNB would have metastatic disease or not. This study only included patients with clinically node negative melanoma ( i.e. , not palpable, not visible on imaging). Additionally in our cohort, the burden of disease in patients with a positive SLNB(s) was quite low, with the majority of positive SLNBs having only micrometastases or isolated tumor cells. Due to this low extent of disease, it is conceivable that HIVM measurement of differences between positive and negative SLNBs would be minimal even when probing for characteristically disorganized tumor vessel architecture. In each of our prior HIVM studies, it was readily obvious where gross tumor was present, and the tumor burden was quite high in the majority of patients (particularly those with carcinomatosis and large retroperitoneal sarcomas). Because of this limitation of low disease burden, it cannot be reliably concluded that there was no true significant difference in the SLNB-associated blood vessels of negative versus positive nodes. Additionally, we recognize that our patient cohort was small, consisting of only 20 patients. The number of SLNB positive events was also small, with only 7 cases having nodal metastasis. Thus, our trial that was primarily designed for feasibility was not sufficiently powered to analyze any associations between vessels associated with either negative or positive SLNB. Similarly, our study was underpowered to analyze any associations between SLNB-associated vessels and long-term outcomes such as melanoma recurrence. It is also possible that the cellular and molecular processes that alter SLNB-associated vessels may be different from the processes occurring with the primary tumor microenvironment which may account for the lack of positive associations between SLNB-associated vessels and oncologic outcomes. Lack of sufficient power also limited our ability to detect any true significant differences in the SLNB-associated blood vessels of negative versus positive nodes. Both the small sample size (which limited statistical power to detect differences in vessel characteristics or melanoma-associated outcomes) and the minimal disease burden within the SLNB were significant limitations of our study to make any conclusions beyond feasibility. Despite these limitations, our primary aim was accomplished as we demonstrated HIVM feasibility for the first time in SLNB. Troubleshooting the HIVM technique with clear communication with our anesthesia colleagues facilitated the acquisition of high resolution HIVM images. While the HIVM apparatus used in this study was patented and proprietary to the Roswell Park Comprehensive Cancer Center, we have also used a commercial HIVM device that is publicly available for purchase from Mauna Kea Technologies in some of our previous studies.[ 12 , 18 , 20 , 25 , 26 ] We have shown that the Mauna Kea Cellvizio fluorescent confocal microscope provided similar high resolution, tumor microvasculature images in patients with peritoneal carcinomatosis, ovarian cancer, brain tumors, sarcoma, and melanoma in-transit disease. To date, the cost of performing the HIVM observations has been covered through various research grants. As we continue to explore further applications of HIVM to the multidisciplinary treatment of various cancers, we intend to perform a cost analysis to further define the feasibility of this technique. To examine differences between positive and negative SLNB, future studies of HIVM will need to account for potential disruption of microvessels from tissue exposure either with altered surgical technique, a larger patient cohort for comparisons, or inclusion of patients with clinically positive nodal disease. As imaging technologies like HIVM continue to be refined in clinical trials, we look forward to potential novel applications of HIVM to not only characterize structural and functional differences in SLNB vessels, but also observe how immunotherapies including adoptive cell therapies (e.g., Lifileucel) and checkpoint inhibition may act at the level of the tumor draining lymph node. These IVM applications are the subject of our group’s ongoing preclinical animal studies, which still require significant optimization prior to translation to human treatment, but are nonetheless important scientific endeavors that warrant further study. Declarations Author Contribution EG, DF, and JS designed the clinical trial. VF, JK, and JS performed the SLNB surgeries. DT, MK, SE and JS performed the HIVM observations and performed the post-acquisitional analyses. AM performed the statistical analyses. EM and DF drafted the manuscript. EG, SE, and JS edited the manuscript. All authors reviewed and approved the final version of the manuscript. Data Availability All data supporting the findings of this study are available within the paper and by request to the corresponding author. References Essner, R. et al. Prognostic implications of thick (> or = 4-mm) melanoma in the era of intraoperative lymphatic mapping and sentinel lymphadenectomy. Ann. Surg. Oncol. 9 , 754–761 (2002). 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El Sharouni, M. A. et al. High discordance rate in assessing sentinel node positivity in cutaneous melanoma: Expert review may reduce unjustified adjuvant treatment. Eur. J. Cancer . 149 , 105–113 (2021). He, B. & Ganss, R. Modulation of the Vascular-Immune Environment in Metastatic Cancer. Cancers (Basel) ; 13. (2021). Katsuta, E. et al. Pancreatic adenocarcinomas with mature blood vessels have better overall survival. Sci. Rep. 9 , 1310 (2019). Mangala, L. S. et al. Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy. JCI Insight . 1 , e87754 (2016). Herrera, M. et al. Prognostic Interactions between FAP + Fibroblasts and CD8a + T Cells in Colon Cancer. Cancers (Basel) ; 12 . (2020). Mezheyeuski, A. et al. Stroma-normalised vessel density predicts benefit from adjuvant fluorouracil-based chemotherapy in patients with stage II/III colon cancer. Br. J. Cancer . 121 , 303–311 (2019). Visioni, A. et al. Intra-arterial Versus Intravenous Adoptive Cell Therapy in a Mouse Tumor Model. J. Immunother . 41 , 313–318 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5161333","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":553571900,"identity":"7e0886d0-3082-4446-85de-b94ac2b923d2","order_by":0,"name":"Emmanuel M Gabriel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFACHgaGBAYLOFeGH8pgbMCvRQLBlWwgRgsDshaDAwS0yLufPfbhQYUEA7/Y6cTPhW02PMbXDj+T+MBgI7vhAHYthmfykmcknJFgkJydu1l6Zlsaj9ntNDPJGQxpxji1NOQYMyS2STAY3M7dIM3bdhioJcFMmofhcCJOLf1vgFr+STDY387d/Ju37T+P8ez0b0At/3FqkZcA2dIAtEU6dxvQlgM8BtI5IFsO4NRiIPEumSHhmASPxO3cbdY855KBjJxiyxkGycYzcdnSn3uY8UeNjRw/0Pu3ecrsgIz0jTc+VNjJ9uGyBSrOgyzIAnQnduVgWxqwCDJ/wK1hFIyCUTAKRiAAAJ2uWH2ZQOlVAAAAAElFTkSuQmCC","orcid":"","institution":"Mayo Clinic Florida","correspondingAuthor":true,"prefix":"","firstName":"Emmanuel","middleName":"M","lastName":"Gabriel","suffix":""},{"id":553571901,"identity":"6dad960f-b1b0-436f-9a82-7c70af514ca9","order_by":1,"name":"Daniel T. 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11:31:05","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102877,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-5161333/v1/622e685d357c8cbf45decba4.html"},{"id":97342534,"identity":"b69ab219-bbdc-43ac-8012-518544adedc3","added_by":"auto","created_at":"2025-12-03 11:31:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":705982,"visible":true,"origin":"","legend":"\u003cp\u003eA) Illustration depicting the interface between the HIVM objective (100X magnification) and the dissected and exposed groin SLNB. B) Illustration of the typical location of melanoma micrometastasis in the subcapsular sinus of the lymph node. C) H\u0026amp;E staining of a representative microscopically positive SLNB obtained from one of the patients in the study cohort (100X magnification). Black asterisks denote adipose tissue and white asterisks denote melanoma micrometastasis.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5161333/v1/35fb1637e11c222107a66f07.jpg"},{"id":97342530,"identity":"038e5304-7bb4-43a4-a4d9-31bebeff8aeb","added_by":"auto","created_at":"2025-12-03 11:31:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106999,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative HIVM observations from either negative SLNBs (top panel) or positive SLNBs (bottom panel). Blue arrows denote functional SLNB-associated blood vessels as indicated by the presence of fluorescent dye, and yellow arrows denote non-functional vessels as indicated by the absence of fluorescent dye. No statistically significant differences were identified between blood vessels surrounding negative or positive SLNBs (as reported in Table 4). Bar is 100 µm.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5161333/v1/bf8582c518c16e8fa7fa69cd.jpg"},{"id":97664777,"identity":"3c6db400-0ff7-4148-a24c-b49dfbacd83b","added_by":"auto","created_at":"2025-12-08 09:14:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1502039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5161333/v1/76feb8c7-12be-493f-907a-11016ff9c696.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFeasibility in the Detection of Nodal Metastatic Melanoma Using Intravital Microscopy \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSentinel lymph node biopsy (SLNB) was originally described by Donald Morton for the staging and treatment of melanoma [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The multicenter selective lymphadenectomy trial (MSLT1) confirmed the prognostic value of SLNB for melanoma and its function as a staging procedure, with some clinical benefit in overall survival (OS) in select patients with intermediate-thickness melanoma [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The results of MSLT1 formed the basis of the MSLT2 trial that examined the outcomes for completion lymphadenectomy versus observation for positive sentinel nodes. MSLT2 confirmed that immediate completion lymphadenectomy for a positive sentinel node could be omitted with no impact on OS for the majority of patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Together, these trials raised questions regarding the clinical benefit of SLNB. However, in the absence of SLNB and/or completion lymphadenectomy, full staging may be incomplete and prognostic information that can influence decisions regarding frequency of surveillance and adjuvant therapies is lacking.\u003c/p\u003e\u003cp\u003eBy virtue of draining a primary tumor site, the sentinel lymph node represents more than just accurate staging and prognosis. On a cellular level, the sentinel node is the direct interface between the host immune system and metastatic tumor in the setting of secondary lymphoid tissue. Preclinical investigations into tumor draining lymph nodes have demonstrated how major immunologic functions such as recruitment of lymphocytes through specialized high endothelial venules (HEV) are impaired by downregulation of CCL21, a chemokine necessary for nodal entry of lymphocytes, even when actual nodal metastasis has yet to occur [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Soluble mediators including VEGF-D generated by primary tumors may precondition the draining node to support eventual tumor metastases by inducing changes in HEV from normally plump, cuboidal endothelium to a dilated and flat vessel architecture [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Rather than de novo generation of tumor vessels in SLNB, tumor-induced remodeling of HEVs not only sets the stage for tumor deposition, but also to become the nutrient tumor vasculature [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Primary tumors also appear to have the ability to induce stromal remodeling in tumor draining lymph nodes that downregulates CCL21 and IL-7 to further impede lymphocyte recruitment and activation, respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Importantly, micrometastases that have gained a foothold in the tumor draining node appear to quickly co-opt the altered vasculature as a portal for intravasation and hematologic dissemination [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, microscopic imaging methods that could identify potential vascular alterations or micrometastasis in a sentinel lymph node \u003cem\u003ein vivo\u003c/em\u003e might generate opportunities to preserve the sentinel node/tumor interface and create new treatment paradigms.\u003c/p\u003e\u003cp\u003eIntravital microscopy (IVM) is the real-time evaluation of living tissue at the microscopic level (\u0026ge;\u0026thinsp;100 X magnification), which is capable of recording physiological processes such as blood flow and cell-cell interactions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In oncology, preclinical IVM has elucidated mechanisms of metastasis, development of neoangiogenesis, and immune cell-tumor interactions primarily in animal models [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Independent of transillumination, IVM can be performed on exposed tissue of interest using an epifluorescent light source. IVM was only recently translated to the clinical arena in which melanoma tumors were interrogated by IVM in the operating room setting [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Clinical trials of human IVM (HIVM) proved feasible in the context of standard oncologic surgeries and generated real-time data of blood flow characteristics that was not previously possible [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Specifically, dynamic features such as blood flow, velocity, and real-time measurements of vessel diameters in living tissue showed differences that could not have been predicted from standard histologic evaluation of the same excised tissue [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The success of these first in-human trials have raised the possibility of a broader application to other oncologic procedures that could also produce relevant information of \u003cem\u003ein situ\u003c/em\u003e human tissue prior to surgical resection [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHerein, we describe a completed clinical trial of HIVM of SLNB at the time of standard melanoma surgery. The primary goal of the study was to determine the feasibility of measuring vascular parameters of human lymph nodes after exposing the lymph node just prior to surgical excision. A successful observation included the ability to identify lymph node vessels, measure vessel diameters, and visualize fluorescein and/or indocyanine green (ICG) within the observed vessels. As these were SLNBs taken for melanoma, exploratory secondary objectives included comparisons between positive and negative lymph nodes and correlation to IVM measurements.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient selection\u003c/h2\u003e\u003cp\u003ePatients scheduled to undergo SLNB for melanoma were evaluated for entry into our pilot clinical trial I-284116 \u0026ldquo;Intravital Microscopy in Identifying Tumor Vessels in Patients with Melanoma Undergoing Sentinel Lymph Node Biopsy\u0026rdquo; at the Roswell Park Comprehensive Cancer Center, Buffalo, NY (ClinicalTrials.gov identifier: NCT02857374, first posted 05/08/2016). The study was approved by the Institute Review Board of Roswell Park Comprehensive Cancer Center, and all methods were performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\u003cp\u003eAs groin lymph nodes were readily accessible to IVM observation, patients whose melanoma mapped to a non-groin sentinel lymph node were excluded. A full list of the inclusion and exclusion criteria is noted in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Eligible patients who gave informed consent were enrolled in the protocol. The following data was recorded: patient demographics (age, sex, height, and weight), tumor-specific information (tumor type, location, and stage) and intravital microscopic measurements obtained from the surgery.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStudy inclusion and exclusion criteria\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"1\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInclusion Criteria\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHave an Eastern Cooperative Oncology Group (ECOG) performance status of \u0026le;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMelanoma tumor that meets indications for a groin SLN biopsy with a\u0026thinsp;\u0026ge;\u0026thinsp;10% risk of having metastasis to the draining lymph node (\u003cem\u003ei.e.\u003c/em\u003e, stage IB to stage IIIC melanoma of the lower body below the umbilicus)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticipant must be eligible for a groin sentinel lymph node (SLN) biopsy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticipant or legal representative must understand the investigational nature of this study and sign an Independent Ethics Committee/Institutional Review Board approved written informed consent form prior to receiving any study related procedure\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExclusion Criteria\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSentinel lymph node is deemed inaccessible to microscopic observation during the operative procedure (\u003cem\u003ei.e.\u003c/em\u003e, sentinel node maps to a deep location or area outside of the groin)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRenal dysfunction as defined as creatinine clearance\u0026thinsp;\u0026lt;\u0026thinsp;70 mL/min by Cockroft-Gault equation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAny known allergy or prior reaction to fluorescein, indocyanine green, iodine, or shellfish\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnwilling or unable to follow protocol requirements\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAny condition which in the Investigator's opinion deems the participant an unsuitable candidate to undergo observational study (may also include preoperative testing results including electrocardiogram [EKG], chest x-ray, or pulmonary function tests)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAny condition that excludes SLN biopsy as the standard of care (\u003cem\u003ee.g.\u003c/em\u003e, lymphadenectomy indicated)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOf note, no control population was included in our pilot trial. A control population would consist of healthy volunteers without melanoma and with presumed normal lymph nodes. Because there is a 5\u0026ndash;15% risk of lymphedema in the upper extremity and a 10\u0026ndash;30% risk in the lower extremity with SLNB,[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] we did not include a control population due to risk and ethical considerations.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMicroscope design\u003c/h3\u003e\n\u003cp\u003eSentinel lymph nodes were observed at 100X magnification using a highly modified Olympus microscopy system as previously described by our group [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The microscope was attached to a cantilevered arm to extend over the patient. Given the high magnification required to observe individual vessels and flow, vibrations in the microscope were minimized using a weighted marble base of over 360 kg. Intravenously administered fluorescein was illuminated using a 467\u0026ndash;498 nm excitation, 513\u0026ndash;556 nm emission dichronic filter set (Spectra Services, Rochester, NY) and ICG was illuminated using a 740\u0026ndash;800 nm excitation, 800\u0026ndash;860 nm emission dichronic filter set (Spectra Services, Rochester, NY) with an X-Cite 120 Led light source (Lumen Dynamics, Ontario, Canada). Images were captured using a Luca EMCCD camera (Andor Technology Ltd., Belfast, Northern Ireland) controlled through the Solis acquisition and analysis program. Images were acquired with a minimum of a 0.05-s exposure at 20 frames a second.\u003c/p\u003e\u003cp\u003eOffline post-surgical quantification of vessel diameter and blood velocity was performed using the ImageJ software suite.\u003c/p\u003e\n\u003ch3\u003eSurgical technique\u003c/h3\u003e\n\u003cp\u003eAll patients underwent standard lymphoscintigraphy with filtered technetium-99m sulfur colloid injected at the site of the primary melanoma. The SLNB location was confirmed by hand-held gamma probe (Neoprobe, Devicor Medical Products Inc., Cincinnati, OH). The skin overlying the sentinel lymph node was prepped with ChloraPrep (CareFusion Corp. San Diego, CA), and sterile towels and drapes were applied for exposure of the area. An incision was created with a scalpel in line with the longitudinal axis of the patient. With care to keep the lymph node capsule and perinodal adipose intact, the skin was elevated bluntly and retracted laterally with a Weitlaner retractor and the use of 3\u0026thinsp;\u0026minus;\u0026thinsp;0 Vicryl sutures (Ethicon, Raritan, NJ). Electrocautery was avoided as much as possible to prevent underlying tissue damage. In all instances, a saline soaked gauze was placed over top of the tissue to prevent desiccation before microscopic interface. After exposure of the confirmed SLNB was completed, the microscope was positioned for observation. Again, we intentionally preserved the peri-nodal tissues as to not disturb node capsule or blood supply going to the nodal hilum. At the completion of the observation period, the microscope was withdrawn, and the lymph node was removed and sent to pathology for evaluation. Standard pathologic analyses consisted of Hematoxylin and Eosin (H\u0026amp;E) staining as well as melanoma-specific stains including S-100, HMB-45, and Melan A.\u003c/p\u003e\n\u003ch3\u003eIntravital microscopic observations in patients\u003c/h3\u003e\n\u003cp\u003eThe microscope was grossly moved into position by the surgeon over the field of interest and locked into place (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Fine adjustments in the x and y axis were performed by using the built-in motor controls. Once the microscope was directly overlying the exposed SLNB surface, the fluorescent light source and image acquisition software were activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Vertical control of the microscope was performed manually by the surgeon until nodal vessels came into view on the digital monitor at 100X magnification. Lymph node vessels were clearly recognized by their well-defined architecture and branching patterns with observations focused on these areas. When present, melanoma micrometastases are typically found in the subcapsular sinus of lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) allowing for potential detection by IVM. To facilitate stabilization of the images, respirations were temporarily held by anesthesia for a maximum of 30 seconds. Patient respirations can lead to suboptimal imaging, but additional anesthesia or temporary holding respirations can often restore in focus, high resolution images. Therefore, clear communication with our anesthesia colleagues was key to ensure adequate HIVM observations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen a stable view of the vessels was achieved, 2.0 ml of 25% (250 mg/ml) fluorescein (Akorn Pharmaceuticals Inc., Lake Forest, Illinois) diluted in 10 ml of sterile saline was injected via a peripheral intravenous catheter. An observation was completed when fluorescein was noted to have extravasated into the nodal tissue and the vessels no longer manifested a detectable fluorescent signal. The microscope was then re-positioned with fine motor controls to observe several areas of the node. The microscopic observations were finalized when the 10 ml of diluted fluorescein was exhausted. Observations were repeated with ICG (IC-Green, Akorn Pharmaceuticals Inc., Lake Forest, Illinois) using 2.0 ml of 25% (250 mg/ml) using the same approach as with fluorescein. Of note, ICG observation produced less vascular detail than fluorescein and was omitted after the first 3 patient observations and was not included for analyses.\u003c/p\u003e\n\u003ch3\u003ePost-hoc analyses\u003c/h3\u003e\n\u003cp\u003ePost-hoc analyses were performed as previously described by our group.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Vessels were defined morphologically, and a single vessel was described as beginning at a branch point continuing to the next branch point. To be measured, vessels had to be 100 mm in length with no interposing branch points. Vessel density was established by enumerating vessels within a field of observation. ImageJ software was used to measure vessel diameter (\u003cem\u003eD\u003c/em\u003e) and radius (\u003cem\u003er\u003c/em\u003e) at the vessel\u0026rsquo;s largest width. Blood flow velocity (\u003cem\u003ev\u003c/em\u003e) was evaluated by determining the time ΔT distinct features in the fluorescent dye would take to travel a known distance ΔS and then averaging ΔS/ΔT for at least 10 points per vessel. Functionality of a given vessel was defined as a vessel that demonstrated fluorescent dye uptake/signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) or kinetic blood flow of red blood cells. Non-functional vessels neither demonstrated dye uptake or kinetic blood flow. Percent functionality was determined as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m. on a per field basis. Wall shear stress was computed as \u003cem\u003eτ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;32\u003cem\u003eηQ\u003c/em\u003e/\u003cem\u003eπD\u003c/em\u003e\u003csup\u003e3\u003c/sup\u003e, where \u003cem\u003eη\u003c/em\u003e is blood viscosity (assumed to be 2.2 centipoise) and \u003cem\u003eQ\u003c/em\u003e is the blood flow rate (\u003cem\u003eQ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ev\u003c/em\u003e*\u003cem\u003eπr\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e with r being the radius of the tumor vessel). Wall shear rate was determined using the formula \u003cem\u003ew\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ev\u003c/em\u003e*8/\u003cem\u003eD\u003c/em\u003e. The ability to discriminate between HEV and regular vessels and orders of HEV as previously described preclinically [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] was not possible due to limited fields of observation and lack of definitive anatomic references.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of vessel characteristics between negative and positive SLNB during intravital microscopic observations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSLNB\u003c/p\u003e\u003cp\u003estatus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eTotal Cohort\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean (Std)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of observed fields\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.5 (0.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.187\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.3 (0.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% Functional vessels\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25.9 (11.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.641\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.7 (7.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiameter of observed vessels (\u0026micro;m)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.3 (2.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.759\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.1 (1.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVelocity of functional vessels (\u0026micro;m/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e248.3 (53.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.314\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e321.2 (61.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWall Shear Stress (dyn/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.6 (0.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.404\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.9 (1.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWall Shear Rate (s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e117.5 (33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.404\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e175.8 (52.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eDemographic and clinical characteristics were summarized using the mean and standard deviation (std) for continuous variables and using frequencies for categorical variables. No statistical method was used to predetermine sample size. Comparison of vessel characteristics between negative and positive SLNBs is presented as means with standard deviation and compared using two sample t-test. Statistical significance for all comparisons was accepted at p\u0026thinsp;=\u0026thinsp;0.05. Data analyzed using R software (version 4.2.3).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003ePatient demographics, SLNB-specific characteristics, and treatment outcomes\u003c/h2\u003e\u003cp\u003eBetween December 2016 to August 2018, a total of 20 patients underwent SLNB with HIVM. Patient characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Of the study cohort, 7 patients (35%) had a positive SLNB(s). Characteristics of patients who had a positive SLNB are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Of the 7 patients with a positive SLNB, 5 underwent completion lymph node dissection, and 2 of these patients had additional positive lymph nodes. There were no adverse events related to the administration of fluorescent dye or the HIVM procedure, similar to our previous HIVM studies. The overall disease burden within positive SLNBs was low with 6/7 having micrometastases\u0026thinsp;\u0026lt;\u0026thinsp;2 mm or isolated tumor cells only as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. Recurrence outcomes for each patient are also shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. At a median follow-up of 65 months, only 1 patient with a negative SLNB was found to have a recurrence during the study period. This patient developed both lower extremity in-transit disease and nodal recurrence 15 months after surgery. Conversely, 4/7 patients (57%) with a positive SLNB developed nodal and/or distant metastases with most occurring in the first 2 years of surveillance.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient and tumor variables.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emedian (range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58 (32\u0026ndash;78)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003efemale\u003c/p\u003e\u003cp\u003emale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (55.0)\u003c/p\u003e\u003cp\u003e9 (45.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody mass index (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emedian (range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28 (20\u0026ndash;24)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimary melanoma Breslow depth (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emean (range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.2(0.5\u0026ndash;4.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePrimary melanoma ulceration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7(35)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimary melanoma mitotic index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emedian (range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(1\u0026ndash;22)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSLNB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7(35)\u003c/p\u003e\u003cp\u003e13(65)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTreatment and outcomes of patients with positive SLNB.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSLNB disease burden (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIsolated tumor cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIsolated tumor cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompletion lymph node dissection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdditional positive lymph nodes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence location\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoft tissue,\u003c/p\u003e\u003cp\u003eLung,\u003c/p\u003e\u003cp\u003eNodes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLung,\u003c/p\u003e\u003cp\u003eNodes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSoft tissue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSoft tissue, Spleen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime to recurrence (months)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHIVM SLNB vessel characteristics\u003c/h2\u003e\u003cp\u003eAll 20 patients were able to successfully undergo HIVM with observations of SLNB-associated blood vessels. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts representative examples of HIVM observations using fluorescein at the time of SLNB, which align with our previous HIVM observations in other cancer histologies.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Vessel architecture in positive nodes were suggestive of the disorganized architecture seen in primary melanoma tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; however, unlike preclinical models of murine lymph nodes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], HEV could not be reliably differentiated from non-HEV vessels in post-hoc analyses. Similar to our prior trials of HIVM in primary melanoma, primary sarcoma, or peritoneal carcinomatosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], functional vessels were defined as those blood vessels that took up the fluorescein dye and non-functional vessels were defined as those which did not take up the dye. Thus, functional vessels (labeled with blue arrows) were portrayed by a bright white appearance, whereas non-functional vessels (labeled with yellow arrows) were portrayed by a dark black appearance, which indicated the absence of fluorescein within the blood vessel.\u003c/p\u003e\u003cp\u003eA comparison of vessel characteristics between negative and positive SLNBs is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Functional vessels were detected in only 26% and 30% of negative and positive SLNB, respectively. There were no statistically significant differences among any of the vessel characteristics, including density of functional or non-functional vessels, mean diameter of functional or non-functional vessels, or blood flow velocity within functional vessels for either negative or positive SLNB. The lack of differences may have been related to the limited number of functional vessels seen across all groups as well as the minimal disease burden of micrometastatic nodal melanoma. Correlations between SLNB-associated vessel characteristics and recurrence was not permissible given the small number of recurrence events. The IVM observed areas of the lymph node were not able to be localized the with standard pathologic evaluation and direct histologic comparisons to IVM observations were therefore not possible.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo our knowledge and based on an extensive review of the current literature, this was the first study to demonstrate the feasibility of HIVM of tumor-draining lymph nodes. Using standard surgical techniques and our customized intravital microscope, we were able to obtain successful observations for each patient in our cohort. While our primary goal to determine the feasibility of HIVM for the SLNB was successful, our secondary goals of comparing SLNB-associated vessel characteristics and either SLNB positivity or recurrence did not yield any statistically significant differences. Yet, the findings of this clinical trial were informative to guide potential future applications of HIVM.\u003c/p\u003e\u003cp\u003eIVM offers certain advantages over other imaging modalities, which make it a useful tool to investigate tumor-associated vessels in this pilot study and our prior HIVM trials. IVM allows for the real-time, high resolution (\u0026lt;\u0026thinsp;1 \u0026micro;m) observation of tumor-associated vessels and potential immune cell interactions.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] While whole body imaging, including CT scan, PET-CT, and MRI, obtains a global visualization of large and medium sized blood vessels, these imaging modalities cannot determine vessel functionality at the capillary (microscopic) level. Lymphoscintigraphy with technetium injection can identify the draining SLNBs, but does not have a role for identifying tumor-associated or SLNB-associated blood vessels. Other high resolution imaging devices such as high frequency US, ocular coherence tomography (OCT), and photoacoustic microscopy (PAM) have been used to investigate tumor-associated microvessels.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Of these other imaging modalities, IVM offers the highest resolution (\u0026lt;\u0026thinsp;1 \u0026micro;m), whereby the others are generally limited to 10\u0026ndash;15 \u0026micro;m. The tissue penetration of IVM is 200 \u0026micro;m, which is deeper than other modalities, such as high frequency US and OCT, which are generally limited to 10\u0026ndash;50 \u0026micro;m. IVM therefore provides for a deeper assessment of blood vessels within the tissue targets. In addition, the image acquisition rate for IVM (30\u0026ndash;100 ms) is also sufficient for tracking blood flow in real time down to individual flowing red blood cells.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] While there is no current \u0026ldquo;gold standard\u0026rdquo; for assessing the tumor vasculature at microscopic resolution in real time in humans, for our purposes of tumor vessel imaging, IVM served as the most optimal technique.\u003c/p\u003e\u003cp\u003eThe limited correlative findings in SLNB are in contradistinction to our previous studies of HIVM of tumor tissue. The first report of HIVM in human cancer was published by our group in 2016 for primary melanoma that was surgically resected [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this first-in-human study, patient melanoma-associated vessels were characterized as disorganized and tortuous compared to normal (control) blood vessels, and up to 50% of melanoma-associated vessels did not support blood flow. In addition, human melanoma-associated vessel diameters were larger than predicted from IHC or preclinical IVM. Since our initial study, we have continued HIVM observation for peritoneal carcinomatosis derived from several different histologies (including appendix, colorectal, ovarian, and mesothelioma cancers) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Most recently, we have reported our HIVM findings for retroperitoneal sarcomas and brain tumors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In each of these earlier clinical trials, we demonstrated that tumor areas had on average a higher density and proportion of non-functional vessels compared to control areas. Unlike our current study in melanoma-associated SLNB, our previous trial in ovarian carcinomatosis did show statistically significant correlations between tumor-associated vessels and outcomes, including disease-specific survival and response to neoadjuvant chemotherapy.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eSeveral factors may have contributed to differences in our current HIVM observations in SLNB versus prior findings in tumors. All SLNB observations required surgical exposure of the tissue with the attendant potential to alter blood flow characteristics which may have accounted for the observation of only\u0026thinsp;~\u0026thinsp;30% of all SLNB vessels manifesting blood flow. Thus, functional vessel assessments including flow rates were limited as compared to the majority of tumor studies that did not require perturbations of the tissue prior to HIVM observation. The inability to observe normal lymph nodes that are anatomically distant from tumor limits comparisons to a \u0026ldquo;true\u0026rdquo; normal control. As all SLNBs by definition are tumor-draining lymph nodes and based upon the observation that vascular changes occur within the node even prior to metastases [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], it is possible that HIVM measurements of SLNB without a normal control for comparison would be unable to detect any potential differences. Also, the alterations in vascular anatomy described preclinically may be minimal and below the threshold of detection for HIVM (\u003cem\u003ee.g.\u003c/em\u003e, flattening of normally cuboidal HEV) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and functional changes such as loss of adhesion molecules would not be detected [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lastly, there is the possibility that pathologic analyses of the SLNB could have yielded false positive or false negatives. It is estimated that up to 10% of SLNB could be false positives.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] If this were the case for any of the subjects with positive SLNB in our small cohort, then we would not expect to identify vascular differences in truly negative SLNB that were interpreted as being positive during intraoperative frozen section or final pathology (i.e., false positives).\u003c/p\u003e\u003cp\u003eMany studies across different tumor types have shown that primary tumor-associated vessels are structurally aberrant and behave abnormally compared to normal vessels [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Importantly, these tumor vessel abnormalities have been shown to translate to poor responses to systemic therapies, both in preclinical animal models and in humans [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our previous HIVM studies focused on known tumor-associated vessels compared to the surrounding grossly normal (control) tissue. However, in our current trial, it was unknown whether the SLNB would have metastatic disease or not. This study only included patients with clinically node negative melanoma (\u003cem\u003ei.e.\u003c/em\u003e, not palpable, not visible on imaging). Additionally in our cohort, the burden of disease in patients with a positive SLNB(s) was quite low, with the majority of positive SLNBs having only micrometastases or isolated tumor cells. Due to this low extent of disease, it is conceivable that HIVM measurement of differences between positive and negative SLNBs would be minimal even when probing for characteristically disorganized tumor vessel architecture. In each of our prior HIVM studies, it was readily obvious where gross tumor was present, and the tumor burden was quite high in the majority of patients (particularly those with carcinomatosis and large retroperitoneal sarcomas). Because of this limitation of low disease burden, it cannot be reliably concluded that there was no true significant difference in the SLNB-associated blood vessels of negative versus positive nodes.\u003c/p\u003e\u003cp\u003eAdditionally, we recognize that our patient cohort was small, consisting of only 20 patients. The number of SLNB positive events was also small, with only 7 cases having nodal metastasis. Thus, our trial that was primarily designed for feasibility was not sufficiently powered to analyze any associations between vessels associated with either negative or positive SLNB. Similarly, our study was underpowered to analyze any associations between SLNB-associated vessels and long-term outcomes such as melanoma recurrence. It is also possible that the cellular and molecular processes that alter SLNB-associated vessels may be different from the processes occurring with the primary tumor microenvironment which may account for the lack of positive associations between SLNB-associated vessels and oncologic outcomes. Lack of sufficient power also limited our ability to detect any true significant differences in the SLNB-associated blood vessels of negative versus positive nodes. Both the small sample size (which limited statistical power to detect differences in vessel characteristics or melanoma-associated outcomes) and the minimal disease burden within the SLNB were significant limitations of our study to make any conclusions beyond feasibility.\u003c/p\u003e\u003cp\u003eDespite these limitations, our primary aim was accomplished as we demonstrated HIVM feasibility for the first time in SLNB. Troubleshooting the HIVM technique with clear communication with our anesthesia colleagues facilitated the acquisition of high resolution HIVM images. While the HIVM apparatus used in this study was patented and proprietary to the Roswell Park Comprehensive Cancer Center, we have also used a commercial HIVM device that is publicly available for purchase from Mauna Kea Technologies in some of our previous studies.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] We have shown that the Mauna Kea Cellvizio fluorescent confocal microscope provided similar high resolution, tumor microvasculature images in patients with peritoneal carcinomatosis, ovarian cancer, brain tumors, sarcoma, and melanoma in-transit disease. To date, the cost of performing the HIVM observations has been covered through various research grants. As we continue to explore further applications of HIVM to the multidisciplinary treatment of various cancers, we intend to perform a cost analysis to further define the feasibility of this technique.\u003c/p\u003e\u003cp\u003eTo examine differences between positive and negative SLNB, future studies of HIVM will need to account for potential disruption of microvessels from tissue exposure either with altered surgical technique, a larger patient cohort for comparisons, or inclusion of patients with clinically positive nodal disease. As imaging technologies like HIVM continue to be refined in clinical trials, we look forward to potential novel applications of HIVM to not only characterize structural and functional differences in SLNB vessels, but also observe how immunotherapies including adoptive cell therapies (e.g., Lifileucel) and checkpoint inhibition may act at the level of the tumor draining lymph node. These IVM applications are the subject of our group\u0026rsquo;s ongoing preclinical animal studies, which still require significant optimization prior to translation to human treatment, but are nonetheless important scientific endeavors that warrant further study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEG, DF, and JS designed the clinical trial. VF, JK, and JS performed the SLNB surgeries. DT, MK, SE and JS performed the HIVM observations and performed the post-acquisitional analyses. AM performed the statistical analyses. EM and DF drafted the manuscript. EG, SE, and JS edited the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and by request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEssner, R. et al. Prognostic implications of thick (\u0026gt;\u0026thinsp;or =\u0026thinsp;4-mm) melanoma in the era of intraoperative lymphatic mapping and sentinel lymphadenectomy. \u003cem\u003eAnn. Surg. Oncol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 754\u0026ndash;761 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorton, D. L. et al. Final trial report of sentinel-node biopsy versus nodal observation in melanoma. \u003cem\u003eN Engl. J. 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Immunother\u003c/em\u003e. \u003cb\u003e41\u003c/b\u003e, 313\u0026ndash;318 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5161333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5161333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile the clinical focus on the sentinel lymph node biopsy (SLNB) is the presence of intra- or extra-nodal metastases, preclinical studies suggest that tumor-draining SLNB-associated vascular architecture and adhesion properties are altered regardless of SLNB positivity. Human intravital microscopy (HIVM) has defined blood vessel abnormalities that may impact lymphocyte adhesion and systemic drug delivery at primary melanoma sites. In this pilot study of HIVM during melanoma SLNB, we sought to determine the feasibility of obtaining HIVM observations of SLNB-associated vessels. We successfully performed HIVM in all 20 SLNB patients, and 7 were found to have nodal micrometastases by standard pathology. HIVM was capable of identifying both functional and non-functional SLNB-associated vessels based on the presence or absence of fluorescent dye uptake, respectively. Comparing vessel characteristics as a secondary objective, no statistically significant differences were noted in the diameter, flow rate, functionality, or shear stress of SLNB-associated blood vessels between positive and negative SLNBs, which may likely have been a reflection of the minimal disease burden. Nonetheless, these initial observations provide the framework to optimize future trials of HIVM in cancer patients.\u003c/p\u003e","manuscriptTitle":"Feasibility in the Detection of Nodal Metastatic Melanoma Using Intravital Microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 11:31:00","doi":"10.21203/rs.3.rs-5161333/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99879324-f3c9-43c0-8890-65b777c716ba","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59007019,"name":"Biological sciences/Cancer/Skin cancer/Melanoma"},{"id":59007020,"name":"Biological sciences/Cancer/Cancer imaging"}],"tags":[],"updatedAt":"2025-12-03T11:31:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 11:31:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5161333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5161333","identity":"rs-5161333","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}