Improved Tumor Discrimination and Shortened Administration-to-Imaging Times in Fluorescence Guided Surgery Through Paired-Agent Protocols

doi:10.21203/rs.3.rs-111011/v1

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Improved Tumor Discrimination and Shortened Administration-to-Imaging Times in Fluorescence Guided Surgery Through Paired-Agent Protocols

https://doi.org/10.21203/rs.3.rs-111011/v1

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Background: The goal of fluorescence guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissue. However, optimal discrimination between these tissues is complicated by the non-specific uptake and retention of molecular targeted agents and the heterogeneity of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and heterogeneous signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single agent imaging for in vivo FGS.

Methods: PAI using a fluorescent anti-EGFR affibody molecule (ABY-029, eIND 122681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 hours (n = 30).

Results: PAI significantly enhanced tumor discrimination, as compared to ABY-029 alone (diagnostic accuracy - 0.94 vs 0.86, ROC curve AUC - 0.991 vs. 0.925, respectively). Additionally, the AUC of the ROC curve for PAI was stable as patient cohort number was increased from n = 1 to 20, while ABY-029 decreased as n increased, indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI reduced the administration-to-imaging time from 3 hours to 30 minutes and exhibited a statistically stronger correlation to EGFR expression heterogeneity (r = 0.58 compared to r = 0.47, p = 9B10^-8).

Conclusion: The quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

Cancer Biology

Paired-agent imaging

fluorescence guided surgery

Head and neck squamous cell carcinoma

ep-idermal growth factor receptor

ABY-029

IRDye 700DX

SupplementaryInformationJECCR.docx
Supplementary information
Supplementary Figure 1: Determining doses of paired-agent imaging using auto-fluorescence. A, For each tumor type a representative example of the autofluorescence in a tongue tumor from FaDu, Detroit 562 and A431 cell lines are shown for the 700 and 800 nm channels of the Odys-sey CLx scanner and compared to standard H&E and epidermal growth factor receptor (EGFR) immunohistochemistry (IHC). B, The binding potential (BP) calculated for each naïve tongue is shown in the corresponding box plot. Supplementary Figure 2: A, Fluorescence measurements in tumor and normal tissue are shown in the 700 and 800 channels for auto-fluorescence, 1X dose of ABY-029 and IRDye 700DX (mouse equivalent to the human microdose, 30 nanomoles), and a 1X dose of ABY-029 and 10X dose of IRDye 700DX. B, The naïve and 10X dose values of IRDye 700DX are shown for each tumor type. Supplementary Figure 3: The effect of the normalization factor (NF) on image contrast. As the normalization ratio increases, the overall signal decreases. This results in a constant contrast-to-variance ratio (CVR) that is stable when NF is varied. However, tumor-to-background ratio (TBR) increases as the signal in the normal tissue (the denominator) decreases. These results indi-cate that TBR is an unstable measure for binding potential and con not be used to compare to single agent fluorescence. Supplementary Figure 4: Image overlays created using imshowpair in Matlab allow visual as-sessment of spatial alignment between IHC and BP images. In the Checkerboard overlay, IHC is in RGB (brown pixels) while the BP map is a grey scale image. Supplementary Figure 5: EGFR molecules per cell line for squamous cell carcinomas determined by quantitative flow cytometry. Cell lines used for this study are indicated in green. Supplementary Table 1: Summary of individual trial and average values of EGFR per cell deter-mined by quantitative flow cytometry
SupplementaryInformationJECCR.docx
Supplementary information
Supplementary Figure 1: Determining doses of paired-agent imaging using auto-fluorescence. A, For each tumor type a representative example of the autofluorescence in a tongue tumor from FaDu, Detroit 562 and A431 cell lines are shown for the 700 and 800 nm channels of the Odys-sey CLx scanner and compared to standard H&E and epidermal growth factor receptor (EGFR) immunohistochemistry (IHC). B, The binding potential (BP) calculated for each naïve tongue is shown in the corresponding box plot. Supplementary Figure 2: A, Fluorescence measurements in tumor and normal tissue are shown in the 700 and 800 channels for auto-fluorescence, 1X dose of ABY-029 and IRDye 700DX (mouse equivalent to the human microdose, 30 nanomoles), and a 1X dose of ABY-029 and 10X dose of IRDye 700DX. B, The naïve and 10X dose values of IRDye 700DX are shown for each tumor type. Supplementary Figure 3: The effect of the normalization factor (NF) on image contrast. As the normalization ratio increases, the overall signal decreases. This results in a constant contrast-to-variance ratio (CVR) that is stable when NF is varied. However, tumor-to-background ratio (TBR) increases as the signal in the normal tissue (the denominator) decreases. These results indi-cate that TBR is an unstable measure for binding potential and con not be used to compare to single agent fluorescence. Supplementary Figure 4: Image overlays created using imshowpair in Matlab allow visual as-sessment of spatial alignment between IHC and BP images. In the Checkerboard overlay, IHC is in RGB (brown pixels) while the BP map is a grey scale image. Supplementary Figure 5: EGFR molecules per cell line for squamous cell carcinomas determined by quantitative flow cytometry. Cell lines used for this study are indicated in green. Supplementary Table 1: Summary of individual trial and average values of EGFR per cell deter-mined by quantitative flow cytometry
SupplementaryInformationJECCR.docx
Supplementary information
Supplementary Figure 1: Determining doses of paired-agent imaging using auto-fluorescence. A, For each tumor type a representative example of the autofluorescence in a tongue tumor from FaDu, Detroit 562 and A431 cell lines are shown for the 700 and 800 nm channels of the Odys-sey CLx scanner and compared to standard H&E and epidermal growth factor receptor (EGFR) immunohistochemistry (IHC). B, The binding potential (BP) calculated for each naïve tongue is shown in the corresponding box plot. Supplementary Figure 2: A, Fluorescence measurements in tumor and normal tissue are shown in the 700 and 800 channels for auto-fluorescence, 1X dose of ABY-029 and IRDye 700DX (mouse equivalent to the human microdose, 30 nanomoles), and a 1X dose of ABY-029 and 10X dose of IRDye 700DX. B, The naïve and 10X dose values of IRDye 700DX are shown for each tumor type. Supplementary Figure 3: The effect of the normalization factor (NF) on image contrast. As the normalization ratio increases, the overall signal decreases. This results in a constant contrast-to-variance ratio (CVR) that is stable when NF is varied. However, tumor-to-background ratio (TBR) increases as the signal in the normal tissue (the denominator) decreases. These results indi-cate that TBR is an unstable measure for binding potential and con not be used to compare to single agent fluorescence. Supplementary Figure 4: Image overlays created using imshowpair in Matlab allow visual as-sessment of spatial alignment between IHC and BP images. In the Checkerboard overlay, IHC is in RGB (brown pixels) while the BP map is a grey scale image. Supplementary Figure 5: EGFR molecules per cell line for squamous cell carcinomas determined by quantitative flow cytometry. Cell lines used for this study are indicated in green. Supplementary Table 1: Summary of individual trial and average values of EGFR per cell deter-mined by quantitative flow cytometry

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Improved Tumor Discrimination and Shortened Administration-to-Imaging Times in Fluorescence Guided Surgery Through Paired-Agent Protocols

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