Perspective review of what is needed for molecular-specific fluorescence-guided surgery

Abstract

Molecular image-guided surgery has the potential for translating the tools of molecular pathology to real-time guidance in surgery. As a whole, there are incredibly positive indicators of growth, including the first United States Food and Drug Administration clearance of an enzyme-biosynthetic-activated probe for surgery guidance, and a growing number of companies producing agents and imaging systems. The strengths and opportunities must be continued but are hampered by important weaknesses and threats within the field. A key issue to solve is the inability of macroscopic imaging tools to resolve microscopic biological disease heterogeneity and the limitations in microscopic systems matching surgery workflow. A related issue is that parsing out true molecular-specific uptake from simple-enhanced permeability and retention is hard and requires extensive pathologic analysis or multiple in vivo tests, comparing fluorescence accumulation with standard histopathology and immunohistochemistry. A related concern in the field is the over-reliance on a finite number of chosen preclinical models, leading to early clinical translation when the probe might not be optimized for high intertumor variation or intratumor heterogeneity. The ultimate potential may require multiple probes, as are used in molecular pathology, and a combination with ultrahigh-resolution imaging and image recognition systems, which capture the data at a finer granularity than is possible by the surgeon. Alternatively, one might choose a more generalized approach by developing the tracer based on generic hallmarks of cancer to create a more "one-size-fits-all" concept, similar to metabolic aberrations as exploited in fluorodeoxyglucose - positron emission tomography (FDG-PET) (i.e., Warburg effect) or tumor acidity. Finally, methods to approach the problem of production cost minimization and regulatory approvals in a manner consistent with the potential revenue of the field will be important. In this area, some solid steps have been demonstrated in the use of fluorescent labeling commercial antibodies and separately in microdosing studies with small molecules.

Keywords: cancer; fluorescent; resection; surgical; therapy.

Fig. 1

Differences in bulk contrast versus high microscopic contrast are shown. Bulk tissue imaging of NIR fluorescence image from LS301 (a) overlaid on white light image of a mouse tumor, showing a fluorescence to background signal ratio, reported as 1.2 across mice. (b) High-resolution fluorescence microscopy shows colocalization (yellow) of iRFP signal (green) and LS301 fluorescence (red) exhibiting the expected high microscopic heterogeneity of the cancer. (c) Histological confirmation of the same slide showing cancerous growth corresponding to the areas marked by iRFP and LS301 fluorescence. Visualization of a tumor from trastuzumab-IRDye800CW, with a color white-light images (a), fluorescence (e) and overlay fluorescence of the two (f), with a reported tumor to background ratio of 2.7. The H&E stained tissue slides (g) and (h) show the subcutaneous tumor microscopy with heterogeneity on the 10’s of microns spatial scale, [scale bars 1 mm (g) and $50\mu \mathrm{m}$ (h)]. Fluorescent images of cetuximab-IRDye800CW are shown in serially cut fresh tumor (i) for different weights with a subsequent reduced TBR at each size, relative to normal tissue. The histological image and from H&E (j) and EGFR (k) are shown with the ex vivo fluorescent images (l) of a representative section showing the microscopic heterogeneity present in the tumor, and high labeling contrast.

Fig. 2

Example of microscopic target validation: panitumumab-IRDye800 localizing in head and neck squamous cell carcinoma (yellow line) but not in surrounding stromal and inflammatory tissues. This represents positive target validation but does not rule out off-target effects or failure to target all elements of cancer distributed throughout the tumor mass.