Characterization of Sentinel Lymph Nodes Using Molecular Imaging with Dual Targeted Microbubbles in a Swine Model

Kartikeya Puranik

doi:10.17918/etd-6698

This work examines the characterization of sentinel lymph nodes (SLNs) in a melanoma swine model using dual targeted microbubble molecular imaging. Melanoma drainage into the SLNs is an important prognostic factor for predicting patient survival. This project proposes the identification of SLNs using a reticuloendothelial specific ultrasound contrast agent (UCA) and subsequently using a molecular contrast enhanced ultrasound imaging (CEUS) with dual targeted microbubbles to characterize metastatic spread (in an animal model). The two specific aims were: 1) to determine ex vivo binding affinity of dual targeted UCA to metastatic SLNs 2) to use molecular CEUS to characterize SLNs using histology and immunohistochemistry as reference standards. The potential benefit from this study will be a development of a minimally-invasive imaging method using UCAs to precisely localize and characterize SLN's thereby reducing the need to perform lymph node resections in patients. To test the ex vivo binding of the dual targeted and IgG (control) UCAs three set of 5 metastatic and 5 benign nodes where used. Furthermore, to check the microbubble specificity 24 slides were antigen blocked. After exposure free floating or unbounded microbubbles were removed with PBS. Subsequently, slides were dried and imaged using a microscope in phase contrast and normal modes to quantify the microbubble attachment. For the in vivo characterization of SLNs using molecular CEUS, 6 swine initially received a peritumoral injection of Sonazoid, (GE Healthcare, Oslo, Norway) to identify SLNs. Next, a dual targeted UCA was administered and imaged (after a 4 minute delay to allow for attachment). Microbubble retention was compared to that of IgG injections. In ex-vivo there was a significant difference between the number of dual targeted and IgG microbubbles attached in metastatic SLNs (0.06±0.04 vs 0.05±0.03; p=0.001) visualized in phase contrast mode. However, in normal mode this relationship was reversed (0.06±0.05 vs 0.09±0.04; p=0.03). Since there should be no IgG attachment this indicates that other cell structures may be counted as microbubbles. Additionally, images from SLNs with a metastatic involvement >65% had a dark background making it difficult to differentiate microbubbles from background. In vivo 11 SLN and 13 non-SLNs were imaged. Metastatic involvement greater than 5% was detected in 7 SLNs by pathology. The mean signal intensity of the dual-targeted contrast agent in the metastatic SLNs (17.2±15.8) was significantly higher than in the IgG-targeted control LNs (1.5±1.3; p = 0.036), while there were no significant differences in benign LNs (1.5±0.8 vs 1.6±1.6; p=0.87). There was a significant difference in dual-targeted contrast retention in metastatic vs. benign LNs (p=0.04). However, IgG-targeted control UCA demonstrated no significant retention difference in metastatic vs. benign LNs (p=0.88). In conclusion, the ex vivo study encountered unexpected variability limiting the results. On the other hand, the in vivo data showed a significant retention of dual targeted microbubble in metastatic SLNs when compared to IgG controls, while there was no significance for the benign nodes. This suggests that molecular CEUS with a dual targeted UCA can be used to non-invasively characterize metastatic SLNs; at least in a melanoma swine model and maybe in the future in humans.

Characterization of Sentinel Lymph Nodes Using Molecular Imaging with Dual Targeted Microbubbles in a Swine Model

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Characterization of Sentinel Lymph Nodes Using Molecular Imaging with Dual Targeted Microbubbles in a Swine Model

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