Characterization of Cherenkov imaging parameters and positional constraints on an O-ring linear accelerator.

Objective. With the introduction of Cherenkov imaging technology on the Halcyon O-ring linear accelerator platform, we seek to demonstrate the imaging feasibility and optimize camera placement.Approach. Imaging parameters were probed by acquiring triggering data Cherenkov image frames for simplistic beams on the Halcyon and comparing the analyzed metrics with those from the TrueBeam platform. Camera position was analyzed by performing 3D rendering of patient treatment plans for various sites and iterating over camera positions to assess treatment area visibility.Main results. Commercial Cherenkov imaging systems are compatible with the pulse timing of the Halcyon, and this platform design favorably impacts signal to noise in Cherenkov image frames. Additionally, ideal camera placement is treatment site dependent and is always within a biconical zone of visibility centered on the isocenter. Visibility data is provided for four treatment sites, with suggestions for camera placement based on room dimensions. Median visibility values were highest for right breast plans, with values of 80.33% and 68.49% for the front and rear views respectively. Head and neck plans presented with the lowest values at 26.44% and 38.18% respectively.Significance. This work presents the first formal camera positional analysis for Cherenkov imaging on any platform and serves as a template for performing similar work for other irradiation platforms. Additionally, this study confirms the Cherenkov imaging parameters do not need to be changed for optimal imaging on the Halcyon. Lastly, the presented methodology provides a framework which could be further expanded to other optical imaging systems which rely on line of sight visibility to the patient.

Evaluating Receptor-Specific Fresh Specimen Staining for Tumor Margin Detection in Clinical Breast Specimens.

PURPOSE: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens. PROCEDURES: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining. RESULTS: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor. CONCLUSIONS: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential.

A new candidate agent for fluorescence guided neurosurgery produces high, persistent tumor contrast shortly after administration.

Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.

Maternally transferred mAbs protect neonatal mice from HSV-induced mortality and morbidity.

Neonatal herpes simplex virus (nHSV) infections often result in significant mortality and neurological morbidity despite antiviral drug therapy. Maternally transferred herpes simplex virus (HSV)-specific antibodies reduce the risk of clinically overt nHSV, but this observation has not been translationally applied. Using a neonatal mouse model, we tested the hypothesis that passive transfer of HSV-specific human mAbs can prevent mortality and morbidity associated with nHSV. The mAbs were expressed in vivo via vectored immunoprophylaxis or recombinantly. Through these maternally derived routes or through direct administration to pups, diverse mAbs to HSV glycoprotein D protected against neonatal HSV-1 and HSV-2 infection. Using in vivo bioluminescent imaging, both pre- and post-exposure mAb treatment significantly reduced viral load in mouse pups. Together these studies support the notion that HSV-specific mAb-based therapies could prevent or improve HSV infection outcomes in neonates.

A method for validating depth-resolved biodistributions in topically-stained specimen with multi-channel fluorescence cryo-imaging.

Fluorescent contrast agents targeted to cancer biomarkers are increasingly being explored for cancer detection, surgical guidance, and response monitoring. Efforts have been underway to topically apply such biomarker-targeted agents to freshly excised specimen for detecting cancer cell receptors on the surface as a method for intraoperative surgical margin assessment, including dual-probe staining methods introduce a second 'non-specific' optical agent as a control to help compensate for heterogeneous uptake and normalize the imaging field. Still, such specimen staining protocols introduce multifaceted complexity with unknown variables, such as tissue-specific diffusion, cell-specific binding and disassociation rates, and other factors, affecting the interpreted cancer-biomarker distribution across the specimen surface. The ability to recover three-dimensional dual-probe biodistributions throughout whole-specimens could offer a ground-truth validation method for examining topical staining uptake behaviors. Herein, we report on a novel method for characterizing dual-probe accumulation with 3D depth-profiles observed from a dual-probe fresh-specimen staining experiment.

Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model.

Subcutaneous (s.c.) tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse in vivo flow cytometry' (DiFC) to study CTC and CTCC dissemination in an s.c. Lewis lung carcinoma (LLC) model in mice. Tumors were grown in the rear flank and we performed DiFC up to 31 days after inoculation. At the study endpoint, lungs were excised and bioluminescence imaging (BLI) was performed to determine the extent of lung metastases. We also used fluorescence macro-cryotome imaging to visualize infiltration and growth of the primary tumor. DiFC revealed significant heterogeneity in CTC and CTCC numbers amongst all mice studied, despite using clonally identical LLC cells and tumor placement. Maximum DiFC count rates corresponded to 0.1 to 14 CTCs per mL of peripheral blood. In general, CTC numbers did not necessarily increase monotonically over time and were poorly correlated with tumor volume. However, there was a good correlation between CTC and CTCC numbers in peripheral blood and lung metastases. We attribute the differences in CTC numbers primarily due to growth patterns of the primary tumor. This study is one of the few reports of CTC shedding dynamics in sub-cutaneous metastasis models and underscores the value of in vivo methods for continuous, non-invasive CTC monitoring.

The shape of breast cancer.

PURPOSE: Little is known about the three-dimensional shape of breast cancer. Implicit to approaches that localize the center of the tumor for breast-conserving surgery (BCS) of non-palpable cancers is the assumption that breast cancers are spherical about a central point, which may not be accurate. METHODS: Pre-operative supine breast MRI images were obtained of 83 breast cancer patients undergoing partial mastectomy using supine MRI-guided resection techniques. Three-dimensional (3D) tumor models were derived after radiologists outlined tumor edges on successive MRI slices. Ideal resection volumes were determined by adding 1 cm in every dimension to the actual tumor volume. Geometrically defined parameters were used to define tumor shapes and associations between clinical variables and shapes were examined. RESULTS: Seventy-five patients had invasive cancer. Breast cancers were categorized into four tumor shapes: 34% of tumors were discoidal, 29% segmental, 19% spherical, and 18% irregular. If hypothetical spherical excisions were performed, non-spherical cases would excise 143% more tissue than the ideal resection volume. When the 3D shape of each tumor was provided to the surgeon during MR-guided BCS, the percentage of tissue overexcised in non-spherical cases was significantly less (143% vs. 66%, p < 0.001). CONCLUSIONS: Information obtained from a supine MRI can be used to generate 3D tumor models and rapidly classify breast tumor shapes. The vast majority of invasive cancers and DCIS are not spherical. Knowledge of tumor shape may allow surgeons to excise breast cancer more precisely.

Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens.

PURPOSE: Rapid, intra-operative identification of tumor tissue in the margins of excised specimens has become an important focus in the pursuit of reducing re-excision rates, especially for breast conserving surgery. Dual-probe difference specimen imaging (DDSI) is an emerging approach that uses the difference in uptake/clearance kinetics between a pair of fluorescently-labeled stains, one targeted to a biomarker-of-interest and the other an untargeted isotype, to reveal receptor-specific images of the specimen. Previous studies using antibodies labeled with either enhanced Raman particles or organic fluorophores have shown promising tumor vs. normal diagnostic performance. Yet, the unique properties of quantum dot-labeled antibody complexes (QDACs), which provide spectrally-distinct fluorescence emission from a common excitation source, make them ideal candidates for this application. Herein, we evaluate the diagnostic performance of QDAC-based DDSI in excised xenografts. PROCEDURES: Excised fresh specimens of normal tissue and human tumor xenografts with elevated expression of HER2 were stained with a HER2-targeted QDAC and an untargeted QDAC isotype. Stained specimens were imaged on a custom hyperspectral imaging system capable of spectrally separating the quantum dot signatures, and images processed using the DDSI approach. The diagnostic performance of this technique under different incubation temperatures and probe concentrations was evaluated using receiver-operator characteristic analysis. RESULTS: HER2-targeted QDAC-DDSI was able to distinguish HER2(+) tumors from normal tissue with reasonably high diagnostic performance; however, this performance was sensitive to temperature during the staining procedure. Area under the curve values were 0.61 when staining at room temperature but increased to over 0.81 when staining at 37 °C. Diagnostic performance was not affected by increasing stain concentration. CONCLUSIONS: This study is the first to report dual-probe difference imaging of specimens using QDACs and hyperspectral imaging. Our results show promising diagnostic performance under certain conditions, and compel further optimization and evaluation of this intra-operative margin assessment technique.

Tracking tumor radiotherapy response in vivo with Cherenkov-excited luminescence ink imaging.

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R2 = 0.92) but not well with total tumor volume changes (R2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

Developing a novel hyperspectral imaging cryomacrotome for whole body fluorescence imaging.

The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryo-macrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence. We present examples of tissue spectra and spectral fitting in a rodent glioma xenograft.

First experience imaging short-wave infrared fluorescence in a large animal: indocyanine green angiography of a pig brain.

The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood.

Characterizing short-wave infrared fluorescence of conventional near-infrared fluorophores.

The observed behavior of short-wave infrared (SWIR) light in tissue, characterized by relatively low scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR regime requires custom molecular probes that do not yet have proven safety profiles in humans. However, recent studies have shown that two readily available near-infrared (NIR-I) fluorophores produce measurable SWIR fluorescence, implying that other conventional fluorophores produce detectable fluorescence in the SWIR window. Using SWIR spectroscopy and wide-field SWIR imaging with tissue-simulating phantoms, we characterize and compare the SWIR emission properties of eight commercially available red/NIR-I fluorophores commonly used in preclinical and clinical research, in addition to a SWIR-specific fluorophore. All fluorophores produce measurable fluorescence emission in the SWIR, including shorter wavelength dyes such as Alexa Fluor 633 and methylene blue. This study is the first to report SWIR fluorescence from six of the eight conventional fluorophores and establishes an important comparative reference for developing and evaluating SWIR imaging strategies for biomedical applications.