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NIRF for Lymphatic Malformations

Lead Physician: Matthew Greives, MD

McGovern School of Medicine at the University of Texas Health Sciences Center at Houston and Children’s Memorial Hermann Hospital, Memorial Herman Texas Trauma Institute.

Specific Aims 

Vascular anomalies, in particular lymphatic and mixed veno-lymphatic malformations, are a common problem in the pediatric population, yet remain poorly understood from both a developmental and pathophysiological standpoint [1]. Current therapeutic interventions are limited to surgery and sclerotherapy, the latter of which targets the large lymphatic vessels and cysts within the lesions themselves, without precise knowledge of their flow patterns [2, 3]. Because accurate understanding of the anatomy and flow within these lesions is difficult to determine, multiple rounds of sclerotherapy are typically needed to treat these malformations. Current diagnostic modalities rely on radiation-based imaging techniques to generate lymphoscintigrams for assessing lymphatic drainage.  This approach does not enable real time imaging in a conscious child, fails to image flow in individuals, and remains technically difficult and non-ideal for the pediatric patients.

Previously, our collaborative engineering team developed a novel imaging modality called Near-Infrared Fluorescence Lymphatic Imaging (NIRFLI), which obviates the need for radiation and anesthesia to both diagnose AND potentially direct treatment of these vascular malformations in the pediatric and young adult population [4-6]. Preliminary studies have shown accurate, real-time assessments of lymphatic flow patterns and levels of obstruction within lymphatic malformations for patients with primary lymphedema and secondary lymphedema, from either post-surgical or post-radiation causes. In the pediatric population, we used the technology to visualize lymphatic drainage patterns in order to find the etiology of chylothorax in order to direct surgical intervention [5].

Use of NIRFLI in the pediatric vascular anomalies patient population has never been performed previously. Given UTHealth’s recent FDA and IRB approvals to safely conduct NIRFLI in the pediatric population, there is a unique clinical research opportunity to interrogate and characterize for the first time, the lymphatic function and anatomies in this population and to better inform current surgical and sclerotherapy procedures.  Specifically, we seek to:

Specific Aim 1: Use NIRFLI to evaluate the anatomy of lymphatic malformation, including the flow patterns of the lymphatic fluid, in the pediatric patient population.
Hypothesis 1: NIRFLI will accurately assess the anatomy of the lymphatic malformation in correlation with concurrent standard clinical imaging modalities in the pediatric population.
Hypothesis 2: NIRFLI will provide high resolution, real-time imaging of the flow of lymph within the lymphatic malformation of a non-anesthetized pediatric patient.

Specific Aim 2: Use NIRFLI to assess post-sclerotherapy changes to the anatomy and lymphatic flow in lymphatic malformations in the pediatric patient population.
Hypothesis 1: NIRFLI will correlate with the anatomical changes to the lymphatic malformation following sclerotherapy.
Hypothesis 2: NIRFLI will demonstrate changes in the lymphatic flow pattern following ­­

Significance

Figure 1 sml

The lymphatic system remains the least understood component of the cardiovascular system, in both its normal anatomy and pathological states. The return of lymphatic fluid from the tissues back into the circulatory system is critical to normal cardiovascular functioning. Abnormalities in lymphatic flow, both primary malformations due to abnormal genetics [4, 7-9] and secondary malformations arising from cancer [10] or trauma [11] cause significant morbidity to patients. These malformations cause soft tissue distortion, leading to physical dysfunction and pain, chylothorax [5], chylous ascites [12], and the medical abnormalities from loss of vital proteins and nutrients in this wasted fluid.

Primary lymphatic malformations (LMs) affect the pediatric population and are noted either at birth, or as ultrasound (US) technology has improved, during prenatal examinations [13, 14]. The majority of these lesions presents prior to age 2 and can occur anywhere in the patient’s body, especially in areas with high concentrations of normal lymphatic tissue [1, 15]. LMs have a predilection for the head and neck region and can cause significant airway obstructions and feeding issues for these patients (Figure 1) [16].  LMs typically grow concurrently with the growth of child but can have rapid swelling associated with localized trauma, infection, or cardiac dysfunction. Unlike hemangiomas, LMs do not undergo involution with age. Due to the bulk and poor flow in the LMs, they remain sites of physical obstruction, cosmetic deformity, and site for potential infections.

Figure 2 sml

Treatment modalities for LMs remain individualized for the patient and the specific type of LM that affects them. LMs are composed of macrocysts and/or microcysts (Figure 2). Macrocysts are typically larger (>2cm in diameter) fluid filled cavities while microcysts are smaller, more diffuse lesions. Both are highly infiltrative of the surrounding tissue and do not respect anatomical boundaries. Therapies for LMs are limited to sclerotherapy or surgical excision. Macrocystic LMs are very amenable to percutaneous US sclerotherapy [1]. The large macrocysts are drained of their lymphatic fluid and then refilled with a sclerotherapy agent (often bleomycin, doxycycline, or OK-432) to promote inflammation of the cyst walls. The treated cyst then undergoes fibrotic transition and reduces the overall size of the lymphatic malformation. Multiple rounds of sclerosis can be performed on the same lesion until all of the symptomatic or deforming macrocysts are eliminated (Figure 3). Microcystic LMs tend to be poorly responsive to percutaneous sclerotherapy as the cysts are too small to be adequately accessed for appropriate and precise sclerosant injection. As they can remain symptomatic (pain, functional or cosmetic deformation), microcystic or mixed (both macro- and microcystic LMs) are recommended for surgical excision and where appropriate newer techniques, like laser therapy.

Figure 3 sml

Lymphatic imaging in the clinical setting can be difficult, especially for children and infants.  Regardless of the type of LM encountered, anatomical understanding of the anatomical extent of the lesion is critical to developing a treatment plan. US imaging provides quick readily available imaging modality with excellent resolution and capability to determine whether macro-, microcystic or mixed LM. In addition there is no radiation involved. However US does not allow for analyses of lymphatic flow within the lesion itself nor does it fully demonstrate the extent of the lesion into deeper tissue planes. MRI provides excellent images of the extent and depth of the tissue infiltrated by the LM and gives reasonable information regarding characterization of whether macro-or microcystic (albeit inferior to US in terms of this classification). However, MRI currently cannot provide accurate lymphatic flow dynamics and pediatric patients require sedation or general anesthesia for MRI, which is not ideal as multiple rounds of MRI imaging are necessary to track the evolution of the LM over the therapeutic time course.

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Figure 4 sml

Lymphoscintigraphy is the gold-standard of lymphatic flow imaging and requires intradermal administration of 99mcTechnetium labeled colloid for uptake into the lymphatic vasculature, and subsequent imaging for several minutes under the gamma camera to obtain a grainy images that have poor spatial and temporal resolution to demonstrate lymph flow through individual lymphatic vessels. Lymphoscintigraphy also exposes the patient to radiation. Lymphangiography requires the painstaking cannulation of a lymphatic vessel, usually in the foot or intranodal injection of several milliliters of gadolinium or iodinated contrast agent for fluoroscopic, CT or MRI imaging. While fluoroscopy produces high-resolution images, the procedure is not often performed since it technically challenging requiring a lot of experience and some serious adverse events have been reported. In addition CT and fluoroscopy exposes the patient to radiation. Both lymphangiography and lymphoscintigraphy require long procedure and imaging times (for review see [1, 17]), are not “point-of-care” modalities, and necessitate sedation or occasionally anesthesia.

Herein, we propose to employ investigational NIRFLI as a rapid, “point-of-care” imaging modality to characterize the lymphatic malformations in infants and children seen in the Vascular Anomalies Clinic at UTHealth.  NIRFLI has been successfully used in adults to assess the lymphatic vasculature and its function [4, 18, 19] as well as response to manual lymphatic drainage therapy [20, 21], and recently in infants with lymphatic dysfunction [22]. 

Following an intradermal injection of trace amount of indocyanine green (ICG), the dye is rapidly taken up by the lymphatic vasculature. Tissue surfaces are illuminated with dim, near-infrared (NIR) light that penetrates tissues to excite the dye and the resultant fluorescence emanating from ICG in the lymphatics is collected by a custom camera system to provide real time visualization of lymphatic anatomy and lymphatic flow with mm and sub mm resolution at tissue depths as great as 3-4 centimeters [23] (Figure 4).  In the United States, the off-label route of intradermal administration of ICG is investigational and requires an FDA investigational new drug (IND) application. 

Figure 6 sml

In this AHA application, the PI proposes to employ investigational NIRFLI for the first time in order to characterize the anatomy and dynamics of lymph flow in children presenting with lymphatic malformations before and following sclerotherapy treatment. The study will be accomplished under an existing IND modified to accommodate the clinical studies proposed herein. Correlation between NIRFLI and the existing MRI and US anatomical information will be performed to assess the validity and potential utility of NIRFLI in the pediatric patient population. As NIRFLI does not require sedation or general anesthesia, as does MRI, it provides a potentially safe alternative for acquiring useful, real-time, anatomical images for these patients. The PI will use the flow images from the NIRFLI to assess for potential sites to optimally target in the sclerotherapy process. Sites of lymphatic fluid entry and exit from the LM, as well as sites of significant pooling within the LM will be assigned importance as targets for the subsequent sclerotherapy. In the second aim of this study, the PI will use NIRFLI to assess the changes to the LM following sclerotherapy. In particular, changes to both the anatomical extent of the lesion and flow within the remaining lesion will be assessed changes due to the sclerotherapy.

Preliminary Studies:

The Technology: The investigational NIRFLI was developed and built at UTHealth by co-I Sevick-Muraca and is unique in that it employs classified Gen III military night vision technology enabling high sensitivity and trace dose of ICG administered off-label.. The UTHealth technology has been used in studies of over 300 normal controls and subjects with lymphatic disorders, but for this application, the most pertinent studies involve its use to (i) accurately phenotype lymphatic (dys)function in families that harbor inherited lymphedema to identify causative genetic mutations [7]; (ii) demonstrate the lymphatic contribution in capillary malformation, arterio-venous malformation (CM-AVM) associated with RASA1 mutations [4]; (iii) image the drainage of peripheral lymph into lymphangiomas [6]; and (iv) evaluate the dynamic lymphatic drainage in an infant with chylothorax after congenital heart defect surgery to determine most efficacious surgical management [5].   There are key technological features that make the UTHealth technology suitable for visualizing, for the first time, the dynamics of lymph flow in lymphatic malformations in infants and children:

Figure 5 sml

  1. Imaging is rapid (with image acquisition rates between 10 Hz and more recently, 33 Hz) enabling real-time video output and visualization of afferent and efferent lymphatic drainage sites.  Once administered, ICG is rapidly taken up providing immediate visualization of lymphatic drainage.
  2. Due to focusing capabilities of the camera system, fields of view can range from 0.02 to 0.6 m2 in area to demark (i) individual, initial dysfunctional lymphatic vessels with resolution of ~300 mm as seen in Figure 5 showing abnormal lymphatic anatomy in the leg of a 35 y.o. woman with primary lymphedema.  NIRFLI movies show abnormal contractile lymphatic function pushing lymph flow distally towards the foot.
  3. Lymphatics can be visualized in vivo with as little as 2 mg and up to 25 mg of ICG administered for periods of time lasting four hours or longer with an individual imaging protocol requiring less than 15-20 minutes.
  4. The combination of sensitivity to detect small doses of ICG when combined with rapid image acquisition enables visualization of the lymphatic contractile activity that governs lymph transport.

 Figure 6 sml

 Currently all UTHealth investigational units used in the research clinics are based upon intensified back-illumination charge coupled device (CCD) sensors (termed IBCCD devices).  As shown in Figure 6, the BICCD devices have significantly better signal-to-noise and contrast performance than marketed or emerging commercial devices (such as NOVADAQ) that are not intensified with Gen III technology and were developed for detecting blood perfusion after intravenous administration of 25 mg of ICG [24].  Most recently, we have improved the investigational NIRFLI system by replacing the charge coupled device (CCD) sensor chip with a scientific complementary metal-oxide semiconductor (sCMOS) sensor chip for more rapid imaging to subtract background light enabling ambient light operation while retaining sensitivity and further improving resolution.  The clinical deployment of this new advanced IsCMOS based device is funded independently under R21 HD085691 (Sevick) but will be available to the Vascular Anomalies Clinic for the start of this AHA project.

Unpublished lymphatic imaging study by collaborators (Greives, Zvavanjanja, and Sevick)

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Figure 7 sml

Since establishing the Vascular Anomalies Clinic, one child and one young adult with lymphatic malformations or edema suggestive of lymphatic involvement have been or are underway for recruitment for investigational lymphatic imaging using the BICCD system.  In the latter case, a 20 y.o. male diagnosed with Klippel-Trenauny Syndrome (KTS) with presentation of right lower extremity overgrowth and portwine stain as a child, presented  as an adult with varicosities, and edema. NIRFLI was conducted to understand the extent of lymphatic involvement prior to sclerotherapy of venous varicosities since prior NIRF studies of similar presentations in a subject diagnosed with Parkes-Weber Syndrome (PWS) [4] and in a non-syndromic Mays-Thurner subject [6] showed overt lymphatic contributions to edema/lymphedema that were not alleviated by sclerotherapy of hemovascular abnormalities.  Figure 7 shows NIRFLI as the subject stands and lymphatic contractile motion pushes ICG-laden lymph “packets” (denoted by arrows) against gravity.  Figure 7 shows that there is a reduced number of lymphatic vessels with less contractile activity in the affected right limb as compared to the contralateral, unaffected left limb. 

Figure 8 sml

NIRFLI showed that the lymphatic anatomy in this subject appears to be comparatively intact in contrast to our prior PWS and Mays-Thurner subjects (Figure 8).  Follow-up NIRFLI after treatment could be used to determine whether sclerotherapy of venous varicosities in the affected limb reduces fluid load and results in improved lymphatic contractile function and improved number of functional vessels in the affected limb. 

In this AHA program, we will perform NIRFLI in children at the Vascular Anomalies Clinic to characterize, for the first time, lymphatic anatomy and function of lymphatic malformations as well as to visualize changes in response to sclerotherapy. 

Research Design and Methods:

Current Clinic and Treatment

Patients will be recruited from the established Vascular Anomalies Clinic at the University of Texas Health Sciences Center at Houston. This multidisciplinary clinic oversees all patients with hemangioma, lymphatic, venous, arterial, and mixed malformations and is one of only two clinics in the entire metropolitan Houston area. Currently, the clinic sees about 15 new diagnoses of lymphatic or mixed veno-lymphatic malformations annually.  At the first visit, the entire team sees all patients: plastic surgeon (Greives), interventional radiologist (Zvavanjanja), nurse coordinator (Doringo), general pediatrician, dermatologist, and pediatric surgeon. After each team member evaluates the patient, a post clinic meeting occurs to discuss the clinical plan established for each patient, which is then conveyed to the patient’s family as well as their primary pediatrician.

The current clinical pathway for patients with lymphatic malformations is as follows (Figure 9, below): all patients with LM who present to the clinic undergo US and MRI (with and without contrast) imaging to delineate the presence of macrocysts within the lesion and the extent of the LM spread in the surrounding tissue. Patients with cysts larger than 2 cm are deemed amenable to sclerotherapy and proceed with percutaneous catheter based sclerotherapy by a single radiologist (Zvavanjanja). Following each round of sclerotherapy, the patient returns to clinic for a repeat US to assess the presence of residual macrocysts, and if found, an additional round of sclerotherapy is performed. If no macrocysts are noted 6 weeks after the final round of sclerotherapy, a repeat MRI is performed and the patient is returned to the Vascular Anomalies team clinic for consideration for surgical resection of residual disease. Surgical resection, if performed, would be a team process with the extirpative surgery performed by the pediatric surgeon and the reconstructive surgery performed by the plastic surgery team (Greives).   As described in Figure 9, we will incorporate NIRFLI within the standard-of-care prior to treatment (specific aim #1) to assess information content of NIRFLI of LMs, and then after treatment (specific aim #2) in order to assess whether NIRFLI can be used to monitor changes in regional lymphatics and, in the future, effectively direct management of LMs.  We plan to recruit approximately half of the patients with new diagnoses of LM for two pre- and post- sclerotherapy sessions of NIRFLI.

Specific Aim #1: Use NIRF imaging to evaluate the anatomy of lymphatic malformation in the pediatric population.

For this study, patients between the ages of 6 months and 18 years who present with a lymphatic malformation or mixed venous lymphatic malformation will be considered for inclusion. Previous sclerotherapy or treatment by other teams or physicians will not be excluded. Contraindications for the study include patients with a documented allergy to iodine and patients with a current skin infection, eczema, or rash at the proposed injection sites. Patients whose guardians do not consent to the study will also be excluded.

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Figure 9 sml

All enrolled patients will undergo NIRFLI at the commencement of their therapeutic pathway and as described below, a second time at the completion of the sclerotherapy component of their treatment. For both imaging procedures, the patients will report to the Pediatric Surgery clinic where they will undergo a complete physical exam with vitals by the PI (Greives).

The investigational imaging will be conducted under an FDA approved protocol modified to reflect the study using the off-label use of ICG as the fluorescent contrast agent. ICG will be secured from the hospital pharmacy and diluted into final concentration of 0.25 mg/ml.  Trace dose of ICG, which provides 5 – 25 μg ICG in each injection of 0.02 - 0.1 cc, will be administered intradermally at locations of known lymphatic bundles [25] that may drain to the lesion.  Continuous pulse oximetry monitoring will be used during administration of the ICG. An emergency crash cart is immediately available in the clinic to be used to assist in securing the airway of the patient during treatment or imaging should any rare allergic reactions occur.  As few as two and as many as eight injections will be performed by physician-investigators and NIRFLI will be performed after each set of 1-2 injections to assess the drainage of ICG-laden lymph from the injection site toward the lesion and from the lesion to the regional lymph node basins in order to define the afferent and efferent lymphatics of the malformation.  Injection sites will be covered with a sterile band-aid to prevent camera oversaturation and sites of injection be noted through documentation and photography. For persons with a unilateral malformation in the upper extremity or cervical area, contralateral NIRFLI will provide a comparison between diseased and contralateral regions.  When possible, symmetrical injections made on affected and contralateral regions will be performed simultaneously in order to understand the temporal “normal” drainage pathways and how the LM impacts those drainage pathways on the affected side.   In a past study, we have found that a single 25 mg intradermal dose in an infant caused oversaturation of the NIRFLI system and obviated lymphatic imaging.  For this reason, we will begin injections with the lowest dose of ICG shown to provide lymphatic imaging (5 mg) and escalate dose from there if needed in subsequent injections.   Once the trace amounts of ICG-laden lymph empties into the hemovascular system, ICG is further diluted and rapidly cleared form the body with in 2 minutes. In the blood, the excitation light and ICG fluorescence is absorbed by hemoglobin so that at the low doses administered, there is no ICG signal from the blood vessels.  There is also no confounding signal from the liver in any of our studies.

During the study, the sequence of injections and imaging will be conducted with the child lying as still as possible for periods of 1 – 10 minutes within the caregiver’s lap and without significant restraint.   Because NIRFLI has been conducted to visualize lymphatic vessels and function in standing and moving study subjects, we do not foresee any difficulties with a squirming child.   

Bluetooth video streaming of images to an LCD screen will permit the team to assess the adequacy of the injection site and the real-time filling of the LM.  The NIRFLI results will also be superimposed overtop a white light image to provide anatomical referencing in real time.   In addition, all imaging results will be stored on a secure server for later review and data analysis by the investigation team.   The entire imaging procedure is not expected to require more than an hour of time.   

Data analysis

Because we have never visualized lymphatic anatomy and function in LMs, it is difficult to speculate which quantitatively analysis will be meaningful.  However, we will quantitate:

  • Pathways including the density, tortuosity, and branching of lymphatic vessels feeding in and draining from LMs.  We will use Fiji plugin to ImageJ (NIH) for density and branching analysis of ICG filled vessels (as described in ref [26, 27]). Lymphatic channels that quickly drain into the LM will be noted for future sclerotherapy sessions; and drainage out of the LM and back to the blood vasculature will also be noted.
  • Contractile or “pumping” function of LM associated lymphatic vessels in terms of lymph “pulses/min” as described in ref [21]; and
  • Rate of ICG filling/emptying in regions of LM as found from the rate of ICG fluorescence intensity increase/decrease in MR-identified regions.

We will use ALFIA (Automated Lymphatic Function Imaging Analysis Software) co-developed with Siemens Corporate for use with NIRFLI human data [28], in order to co-register white light and images across time to remove motion and provide quantitation of the above parameters.   We will compare these NIRFLI parameters where possible with results from the contralateral, “normal” side to provide an understanding of how the LM impacts loco-regional lymphatics. Trained technicians and engineers, who are part of the NIRFLI procedure imaging team who routinely perform this in adult studies, will perform image analysis.  Their effort is reflected in a designated per imaging session charge that also accounts for ICG purchase, pharmacy dispensal, and limited miscellaneous consumables associated with the imaging session and image analysis.

In addition, all study patients will have MRI images obtained as part of their routine clinical evaluation during their enrollment into the vascular anomalies clinic. Comparison of the NIRFLI images and the MRI will be performed to assess the anatomical similarities between the two modalities by the PI and co-investigator (Zvavanjanja). Specific markers that will be used to evaluate the utility of NIRFLI will be:

  • Size of lymphatic malformation: As the NIRFLI images are 2D representations of the 3D embedded tissue structure, they can nonetheless be correlated with the location and sizes of the lesions seen on the slices of MRI studies of the same anatomical subunit. The percent of the normal structure that is occupied by the lymphatic malformation will be calculated in both the coronal and sagittal planes at the point of maximal width. Comparison of cross-sectional areas between the 2-D MRI slices and the surface NIRFLI images will then be performed. If successful, future work will involve co-registration of the tissue surface NIRFLI images on the surface of 3-D MR images in order to evaluate the utility of NIRFLI information relative to conventional MR. Although too complicated for this initial feasibility study, NIRF imaging is capable of 3-D tomographic imaging [29] and, if this study demonstrates exceptional information, future work will seek to develop 3-D imaging for direct comparison to MR assessment of LMs.
  • Presence of macrocysts within the lymphatic malformations: Clinically significant (>2cm in diameter) macrocysts and areas of microcysts will be identified on the MRI studies. Similarly, we postulate that both macrocysts and microcysts maybe identified during the NIRFLI imaging process, presumably from different rate of filling/emptying as described above. Comparison of number, size, location, and characterization of these cysts from both MR and NIRFLI will be performed to establish utility of NIRFLI for characterization of LMs.

Data on the above points of interest in the study will be collected and statistically analyzed for difference between LM and contralateral normal regions, when possible.  Successful correlation of the NIRFL imaging images with the MRI data will be met at p<0.05.

Pitfalls and Solutions:

The clinical experience using NIRFLI is primarily in adults. In our past limited studies on infants, we found that 25mg dose of ICG was too large, causing overwhelming fluorescence that prevented identification of single vessels.  Because we do not know what the optimal doses will be to visualize afferent and efferent drainage patterns, initial injections will contain 5 mg in 0.02 cc of saline.  If insufficient to visualize the lymphatic vasculature, volume of injection will be increased up to 0.1 cc.  No more than 0.200 mg of ICG will be administered, which is below the approved i.v. dosage for children and infants.

Tissue penetration is limited to 3-4 cm beneath the skin. For larger malformations, this may be inadequate to demonstrate the true depth of the LM and the flow within this portion. To combat this, multiple angles (Coronal, sagittal) will be used during the video capture to provide a better 3D image of the entire LM.

ICG may not be taken up by the lymphatic malformation. This is highly unlikely as previous images have been successful in patients with lymphedema and one adult with a LM. Adjustments of dosage will be used if the study demonstrates poor flow within the LM.

Dark skin color may restrict imaging in certain ethnicities. Previous data demonstrates that darker skinned individuals provide better subjects for NIRFL imaging due to melanin absorption and lack of excitation light backscatter that reduces contrast in persons with fair complexion.

Participants may have an allergic reaction to ICG that can include itchiness and/or swelling; there is a rare possibility of a severe reaction, including death.  Two anaphylactic deaths have resulted from IV injection of ICG at the maximal approved dose (2 mg/kg), which is more than 300 times that to be employed in adults in this study on a mg/kg basis.  The risk of severe adverse event is extremely low.  A nurse will be present during the entire imaging procedure and provide antihistamines or other interventions if needed; a physician will be nearby or in the imaging room to provide emergency medical care if needed. 

Because of the nature of this feasibility study in a pediatric population, it is important to note that we do not, nor will have NIRFLI on normal, age-matched controls.  While the contralateral “unaffected” NIRFLI may serve as a control measure to delineate the impact of the LM on collateral lymphatics, the NIRFLI imaging team has performed imaging in normal human adults allowing some correlation to this “normal” albeit different population.

Specific Aim #2: Use NIRFLI imaging to assess post-sclerotherapy changes to the anatomy and lymphatic flow in lymphatic malformations in the pediatric patient population.

As described in Figure 9, when study subjects return to the Vascular Anomalies clinic for surgical consideration and/or annual follow-up, a second NIRFLI imaging will be performed for comparison to the pre-treatment imaging.  Injection sites will be the same as those done pre-treatment.  Because ICG can remain at the injection sites in trace quantities for several months, we will first use NIRFLI to visualize these sites (they are invisible to the eye, but not to the device), and if not present, we will use study documentation to guide the placement of ICG for the second NIRFLI imaging session.  NIRFLI and its associated data analysis will be conducted as described above in Specific Aim #1.

A second MRI will be completed at the termination of the sclerotherapy portion of the treatment algorithm as part of the standard of care and will demark any continued presence of macrocysts, and if present, microcysts that are treated most efficaciously with surgery. Similarly, NIRFLI will be performed for a second time in the same patients. The similar metrics for NIRFLI analysis and metrics of vessel anatomy, contractile function, and cyst filling/emptying will be similarly computed.  Analysis of residual cystic and lymphatic tissues will again be performed to compare the MRI data with the NIRFLI.   Specifically we seek to assess:

  • Change in size of lymphatic malformation: Following sclerotherapy the percent of the normal structure that is occupied by the lymphatic malformation will again be calculated in both the coronal and sagittal planes at the point of maximal width. Comparison of cross-sectional ratios between the MRI and the NIRF images will then be performed.
  • Presence of residual macrocysts within the lymphatic malformations: At the termination of sclerotherapy, all macrocysts should be treated and therefore missing from the final images seen in the MRI. NIRFLI will independently assess for the presence of large dilated chambers that would represent additional (previously missed) targets for sclerotherapy.
  • Changes to the pathways of lymphatic drainage flowing into the malformation: Following sclerotherapy, new patterns of lymphatic flow will likely be created to bypass sites of obstruction within the LM. NIRFL imaging will assess changes to the flow patterns of lymph into the residual LM. These remaining flow sites would represent potential targets for additional sclerotherapy or sites of necessary surgical resection.
  • Changes to the pathways of lymphatic drainage flowing from the lesion back into the circulation: Following sclerotherapy, new patters of lymphatic flow out of the LM will be identified as previous channels have undergone sclerosis. These new pathways would represent new targets for future sclerotherapy or sites of necessary surgical resection.

Similar to the pre-sclerotherapy data analysis, all images will be formatted and matched to their respective post-sclerotherapy MRI images. An independent team member will perform analysis of the video images. Still images from the video will be identified which demonstrate the largest cross-sectional area of lymphatic flow. The sizes of these LMs will be expressed as a ratio of the total area of each body part. Comparison ratios will also be created from the pre-sclerotherapy MRIs that were done as routine clinical care. Data on the above points of interest in the study will be collected and statistically analyzed. Successful correlation of the NIRFLI with the MRI data will be met at p<0.05.

Pitfalls and Solutions:

Sclerotherapy may cause dysfunctional flow in the lymphatic malformation. The exact effects of sclerotherapy on the flow of lymph within a LM are unknown. We do not currently understand the flow of lymph within the LMs prior to sclerotherapy and consequently this study represents a novel study to observe the changes to the LM and the flow within over the course of the treatment protocol.

The inflammatory process that reduces the size of cysts and the entire LM takes weeks or months to reach a steady state. Macrocysts that were previously treated can return after the inflammation resolves. The majority of this inflammation and sclerosis subsides by 6 weeks following the final treatment. Therefore the time period of 6 weeks after the final sclerotherapy session for US confirmation of macrocyst ablation will be used. Once confirmed, both the MRI and NIRF imaging will be performed within the same week to reduce any discrepancy between the two imaging modalities.

Ethical Aspects of Proposed Research

This proposal details the novel use of indocyanine green (ICG) in pediatric patients with lymphatic malformation. Previous studies have demonstrated the safety of this technology in both adults and pediatric patients with other diagnoses. This study has been approved by both the IRBs of the University of Texas Health Sciences Center at Houston and Children’s Memorial Hermann Hospital (IRB# HSC-IMM-08-0415). All approvals are specific for the use in pediatrics and have fulfilled these boards’ requirements for safety in this population. Patients will be identified by the PI (Greives) during their presentation to the Vascular Anomalies team clinic. Parents or guardians of patients will be given copies of the IRB protocol and consent form at the time of their first clinic visit for evaluation with the Vascular Anomalies team. A team member will then follow up with the guardian to answer any questions that they might have regarding the study prior to their signing the forms. All consents and forms are approved by the IRB at the above institutions.


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