Diagnostic Performance of Receptor-Specific Surgical Specimen Staining Correlates with Receptor Expression Level

Intraoperative margin assessment is imperative to cancer cure but is a continued challenge to successful surgery. Breast conserving surgery is a relevant example, where a cosmetically improved outcome is gained over mastectomy, but re-excision is required in \u3e25  %   of cases due to positive or closely involved margins. Clinical translation of margin assessment modalities that must directly contact the patient or required administered contrast agents are time consuming and costly to move from bench to bedside. Tumor resections provide a unique surgical opportunity to deploy margin assessment technologies including contrast agents on the resected tissues, substantially shortening the path to the clinic. However, staining of resected tissues is plagued by nonspecific uptake. A ratiometric imaging approach where matched targeted and untargeted probes are used for staining has demonstrated substantially improved biomarker quantification over staining with conventional targeted contrast agents alone. Our group has developed an antibody-based ratiometric imaging technology using fluorescently labeled, spectrally distinct targeted and untargeted antibody probes termed dual-stain difference specimen imaging (DDSI). Herein, the targeted biomarker expression level and pattern are evaluated for their effects on DDSI diagnostic potential. Epidermal growth factor receptor expression level was correlated to DDSI diagnostic potential, which was found to be robust to spatial pattern expression variation. These results highlight the utility of DDSI for accurate margin assessment of freshly resected tumor specimens

Optimizing Fresh Specimen Staining for Rapid Identification of Tumor Biomarkers During Surgery.

Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivotissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures

Noninvasive Measurement of Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence Allowing Detection of Murine Glioma In Vivo

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups

Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)- based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches

Clinically relevant dual probe difference specimen imaging (DDSI) protocol for freshly resected breast cancer specimen staining

Background: Re-excision rates following breast conserving surgery (BCS) remain as high as ~ 35%, with positive margins detected during follow-up histopathology. Additional breast cancer resection surgery is not only taxing on the patient and health care system, but also delays adjuvant therapies, increasing morbidity and reducing the likelihood of a positive outcome. The ability to precisely resect and visualize tumor margins in real time within the surgical theater would greatly benefit patients, surgeons and the health care system. Current tumor margin assessment technologies utilized during BCS involve relatively lengthy and labor-intensive protocols, which impede the surgical work flow. Methods: In previous work, we have developed and validated a fluorescence imaging method termed dual probe difference specimen imaging (DDSI) to accurately detect benign and malignant tissue with direct correlation to the targeted biomarker expression levels intraoperatively. The DDSI method is currently on par with touch prep cytology in execution time (~ 15-min). In this study, the main goal was to shorten the DDSI protocol by decreasing tissue blocking and washing times to optimize the DDSI protocol to \u3c 10-min whilst maintaining robust benign and malignant tissue differentiation. Results: We evaluated the utility of the shortened DDSI staining methodology using xenografts grown from cell lines with varied epidermal growth factor receptor (EGFR) expression levels, comparing accuracy through receiver operator characteristic (ROC) curve analyses across varied tissue blocking and washing times. An optimized 8-min DDSI methodology was developed for future clinical translation. Conclusions: Successful completion of this work resulted in substantial shortening of the DDSI methodology for use in the operating room, that provided robust, highly receptor specific, sensitive diagnostic capabilities between benign and malignant tissues

Far-Red and Near-Infrared Seminaphthofluorophores for Targeted Pancreatic Cancer Imaging

Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research

Protoporphyrin IX Fluorescence Contrast in Invasive Glioblastomas is Linearly Correlated with Gd Enhanced Magnetic Resonance Image Contrast but has Higher Diagnostic Accuracy

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p\u3c1×10−8) and H&E+ (r = 0.74, p\u3c0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity

Imaging of Glioma Tumor with Endogenous Fluorescence Tomography

Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX. Fluorescence tomography provides a tool for preclinical molecular contrast agent assessment in oncology.1, 2, 3, 4 Systems have advanced in complexity to where noncontact imaging,5 automated boundary recovery,6 and sophisticated internal tissue shapes can be included in the recovered images. The translation of this work to humans will require molecular contrast agents that are amenable to regulatory approval and maintain tumor specificity in humans, where often nonspecific uptake of molecular imaging agents can decrease their utility. In this study, a new fluorescence tomography system coupled to microCT7 was used to illustrate diagnostic detection of orthotopic glioma tumors that were not apparent in the microCT images, using endogenous fluorescent contrast from protoporphyrin IX (PpIX). Glioma tumors provide significant endogenous fluorescence from PpIX,8, 9, 10, 11 and this is enhanced when the subject imaged has been administered aminolevulinic acid (ALA). The endogenous production process of PpIX is known to stem from the administered, ALA bypassing the regulatory inhibition of ALA synthase, allowing the heme synthesis pathway to proceed uninhibited. Since there is a limited supply of iron in the body, this process produces overabundance of PpIX rather than heme, and many tumors have been shown to have high yields of PpIX. Clinical trials with PpIX fluorescence guided resection of tumors have shown significant promise,12 and yet deep tissue imaging with PpIX fluorescence has not been exploited in clinical use. Early studies have shown that detection of these tumors with PpIX is feasible,13, 14 but no tomographic imaging has been used. This limitation in development has largely been caused by problems in wavelength filtering and low signal intensity, as well as background fluorescence from the skin limiting sensitivity to deeper structures. In the system developed and used here, this feasibility is demonstrated by imaging a human xenograft glioma model. To solve the sensitivity problem and study the ability to diagnostically image PpIX in vivo, time-correlated single-photon counting was used in the fluorescence tomography system, which provides maximum sensitivity. Figure 1a shows the system designed to match up with a microCT, allowing both x-ray structural and optical functional imaging sequentially. Lens-coupled detection of signals is acquired from the mouse using five time-resolved photomultiplier tubes (H7422P-50, Hamamatsu, Japan) with single-photon counting electronics (SPC-134 modules, Becker and Hickl GmbH, Germany). The system has fan-beam transmission geometry similar to a standard CT scanner, with single source delivery of a1-mW role= presentation \u3e1-mW pulsed diode laser light at 635nm role= presentation \u3e635nm , collimated to a 1-mm role= presentation \u3e1-mm effective area on the animal. The five detection lenses were arranged in an arc, each with 22.5-deg role= presentation \u3e22.5-deg angular separation, centered directly on the opposite side of the animal with long working distance pickup,7 allowing noncontact measurement of the diffuse light through the animal. The diffuse intensity signals collected at each of the five channels were then translated via 400-μm role= presentation \u3e400-μm fibers and split using beamsplitters to be directed toward the fluorescence (95%) and transmission (5%) channel detectors. A 650-nm role= presentation \u3e650-nm long-pass filter was used in the fluorescence channels to isolate the signal, and in the transmitted intensity signals, a neutral density filter (2 OD) was used to attenuate the signals. This latter filtering was necessary to ensure that the fluorescence and transmission. Intensity signals fell within the same dynamic range, allowing a single 1s role= presentation \u3e1s acquisition for each detector. Scans were then performed by rotating the fan-beam around the specimen to 32 locations. A GE eXplore Locus SP scanner (GE Healthcare, London, Ontario, Canada) that incorporated a detector with 94-micronpixel role= presentation \u3e94-micronpixel resolution, a 80-kV role= presentation \u3e80-kV peak voltage, and a tube current of 450μAs role= presentation \u3e450μAs , was used in acquiring the microCT data, as displayed in Fig. 2 . In this example, since soft tissue was being imaged, the CT data was largely used to image the exterior of the animal, although in future studies, it could be used to isolate the cranium region as well

Charge and Hydrophobicity Effects of NIR Fluorophores on Bone-Specific Imaging

Recent advances in near-infrared (NIR) fluorescence imaging enabled real-time intraoperative detection of bone metastases, bone growth, and tissue microcalcification. Pamidronate (PAM) has been widely used for this purpose because of its high binding affinity toward bone and remarkable therapeutic effects. Herein we describe the development of a series of PAM-conjugated NIR fluorophores that varied in net charges and hydrophobicity, and compared their bone targeting efficiency, biodistribution, and blood clearance. Since the targeting moiety, PAM, is highly negatively charged but small, the overall in vivo bone targeting and biodistribution were mediated by the physicochemical properties of conjugated fluorophores

Polymeric Micelles as Carriers for Nerve-Highlighting Fluorescent Probe Delivery

ACS Editors' Choice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.Nerve damage during surgery is a common morbidity experienced by patients that leaves them with chronic pain and/or loss of function. Currently, no clinically approved imaging technique exists to enhance nerve visualization in the operating room. Fluorescence image-guided surgery has gained in popularity and clinical acceptance over the past decade with a handful of imaging systems approved for clinical use. However, contrast agent development to complement these fluorescence-imaging systems has lagged behind with all currently approved fluorescent agents providing untargeted blood pool information. Nerve-specific fluorophores are known, however translations of these agents to the clinic has been complicated by their lipophilic nature, which necessitates specialized formulation strategies for successful systemic administration. To date the known nerve-specific fluorophores have only been demonstrated preclinically due to the necessity of a dimethyl sulfoxide containing formulation for solubilization. In the current study, a polymeric micellar (PM) formulation strategy was developed for a representative nerve-specific fluorophore from the distyrylbenzene family, BMB. The PM formulation strategy was able to solubilize BMB and demonstrated improved nerve-specific accumulation and fluorescence intensity when the same fluorophore dose was administered to mice utilizing the previous formulation strategy. The success of the PM formulation strategy will be important for moving toward clinical translation of these novel nerve-specific probes as it is nontoxic and biodegradable and has the potential to decrease the necessary dose for imaging while also improving the safety profile