Implementation and benchmarking of a novel analytical framework to clinically evaluate tumor-specific fluorescent tracers

Abstract

During the last decade, the emerging field of molecular fluorescence imaging has led to the development of tumor-specific fluorescent tracers and an increase in early-phase clinical trials without having consensus on a standard methodology for evaluating an optical tracer. By combining multiple complementary state-of-the-art clinical optical imaging techniques, we propose a novel analytical framework for the clinical translation and evaluation of tumor-targeted fluorescent tracers for molecular fluorescence imaging which can be used for a range of tumor types and with different optical tracers. Here we report the implementation of this analytical framework and demonstrate the tumor-specific targeting of escalating doses of the near-infrared fluorescent tracer bevacizumab-800CW on a macroscopic and microscopic level. We subsequently demonstrate an 88% increase in the intraoperative detection rate of tumor-involved margins in primary breast cancer patients, indicating the clinical feasibility and support of future studies to evaluate the definitive clinical impact of fluorescence-guided surgery.

Conflict of interest statement

G.M.V.D. is member of the scientific advisory board of SurgVision BV. The remaining authors declare no competing interests.

Figures

Fig. 1

The clinical analytical framework enabling correlation of intraoperative fluorescence signals with histopathology, from macroscopic to microscopic levels. a Intravenous administration of bevacizumab-800CW three days prior to surgery. b, c Color image and corresponding fluorescence image obtained in vivo during surgery to determine potential clinical value. d, e Imaging of the fresh surgical specimen, followed by serially slicing. f, g Imaging of the fresh tissue slices to determine tumor-to-background ratio based on macro-segmentation, followed by paraffin embedding. h, i Imaging of formalin-fixed paraffin-embedded (FFPE) blocks to determine heterogeneity of tracer uptake within a tumor. j, k Imaging of 10-µm-thick tissue sections for micro-segmentation to reveal microscopic biodistribution and correlation with fluorescence signals from the macroscopic to microscopic level. l, m Fluorescence microscopy to determine tracer distribution on a cellular level. Scale bars represent 1 cm, in l, m the scale bar represents 25 µm

Fig. 2

Representative images per dose group and per optical imaging method for ex vivo analyses, including MDSFR/SFF spectroscopy. Columns represent the four dose groups: 4.5 mg (a–f), 10 mg (g–l), 25 mg (m–r), 50 mg (s–x). Rows represent the imaging modality, in the upper part a white light image and in the lower part the representative fluorescence image. Tumor tissue is delineated with a dashed line. Scale bars represent 1 cm. I Mean fluorescence intensity (MFI) of normal tissue (gray) and tumor tissue (black) are depicted per dose group on the left y-axis, the right y-axis shows the tumor-to-background ratio per patient per dose group for macro-segmentation analyses, in II for MDSFR/SFF spectroscopy measurements and in III for micro-segmentation analyses. Fluorescence images are scaled using the most optimal minimum and maximum displayed value. Boxplot centerline is at median, the bounds of the box at 25th to 75th percentiles, the whiskers are depicting the min–max, tumor-to-background ratio data are depicted per patient; line indicates median value per dose group. Asterisk denotes significant (P < 0.05, Kruskal–Wallis test) values. Obelisk denotes significant (P < 0.05, Mann–Whitney U-test) values. FFPE formalin-fixed, paraffin embedded, MDSFR/SFF multi-diameter single-fiber reflectance/single-fiber fluorescence. Q.μfa,x the product of the quantum efficiency across the emission spectrum, Q[-], where Q is the fluorescence quantum yield of IRDye-800CW and μaf [mm−1] is the tracer absorption coefficient at the excitation wavelength

Fig. 3

Microscopic biodistribution in breast cancer tissue of bevacizumab-800CW based on micro-segmentation analyses. The upper row shows a representative example of the region-of-interest per tissue type based on H/E staining. The lower row shows the corresponding pseudo color fluorescence intensity image of each tissue type. In a, b the whole section is depicted, and in c, d the tumor area, e, f parenchymal breast tissue including collagen, g, h fat tissue, i, j carcinoma in situ tissue, and a combination of all tissue types in k, l. Mean fluorescence intensities of all patients per dose group, per tissue type are shown in panel m. Asterisk denotes significant (P < 0.05, Kruskal–Wallis test) values. Obelisk denotes significant (P < 0.05, Mann–Whitney U-test) values. Bars are representing the median, error bars are representing 95% confidence interval. Scale bars represent 5 mm

Fig. 4

Micro-segmentation per dose group, and per tissue type. Per dose group we plotted mean fluorescence intensity per tissue type (a–d); tumor and carcinoma in situ components shown in red. The mean fluorescence intensity per tissue type was plotted in e-i. In j the tumor-to-parenchyma ratio per dose group is plotted. Bars represent the mean and the error bars the standard deviation. Boxplot centerline is at median, the bounds of the box at 25th to 75th percentiles, the whiskers are depicting the min–max

Fig. 5

Representative examples of intraoperatively detected tumor-involved surgical margin and a tumor negative surgical margin. Columns represent from left to right intraoperative imaging, fresh specimen imaging, fresh tissue slice imaging, FFPE block imaging, and imaging of 10-µm-thick sections. The two upper rows represent a patient with a tumor-positive surgical margin, a clear fluorescence signal was detected in the surgical cavity. Subsequently, the corresponding resection plane of the excised specimen was marked with an extra suture (a, b). Fluorescence imaging of the fresh surgical specimen showed high fluorescence signals at the area of the suture mark (c, d, asterisk). Corresponding fluorescence images of fresh tissue slices, FFPE blocks and 10-µm-thick sections showed high fluorescence signals at the margin (e–j, arrows). Histopathology confirmed the presence of tumor deposits in this area (i). The lower rows represent a patient with a tumor-free surgical margin Fig. 5 k–t. Deeper sectioning of the FFPE block (q, r) was performed to investigate the probable cause of the high fluorescent area within the green dashed line (t). u, v Arrow depicts the surgical positive margin. Dashed white/black circle indicates the area with the highest fluorescence signal intensities. The asterisk represents the position of the extra suture mark. The gray box represents the origin of the FFPE block in the fresh tissue slice. The dashed white/black line delineates tumor tissue. The dashed green line delineates collagen tissue with normal parenchyma. Scale bars represent 1 cm