Pressure-enhanced sensing of tissue oxygenation via endogenous porphyrin: Implications for dynamic visualization of cancer in surgery.

Fluorescence guidance is routinely used in surgery to enhance perfusion contrast in multiple types of diseases. Pressure-enhanced sensing of tissue oxygenation (PRESTO) via fluorescence is a technique extensively analyzed here, that uses an FDA-approved human precursor molecule, 5-aminolevulinic acid (ALA), to stimulate a unique delayed fluorescence signal that is representative of tissue hypoxia. The ALA precontrast agent is metabolized in most tissues into a red fluorescent molecule, protoporphyrin IX (PpIX), which has both prompt fluorescence, indicative of the concentration, and a delayed fluorescence, that is amplified in low tissue oxygen situations. Applied pressure from palpation induces transient capillary stasis and a resulting transient PRESTO contrast, dominant when there is near hypoxia. This study examined the kinetics and behavior of this effect in both normal and tumor tissues, with a prolonged high PRESTO contrast (contrast to background of 7.3) across 5 tumor models, due to sluggish capillaries and inhibited vasodynamics. This tissue function imaging approach is a fundamentally unique tool for real-time palpation-induced tissue response in vivo, relevant for chronic hypoxia, such as vascular diseases or oncologic surgery.

Time-gated single-pixel imaging of Cherenkov emission from a medical linear accelerator.

Cherenkov imaging is an ideal tool for real-time in vivo verification of a radiation therapy dose. Given that radiation is pulsed from a medical linear accelerator (LINAC) together with weak Cherenkov emissions, time-gated high-sensitivity imaging is required for robust measurements. Instead of using an expensive camera system with limited efficiency of detection in each pixel, a single-pixel imaging (SPI) approach that maintains promising sensitivity over the entire spectral band could be used to provide a low-cost and viable alternative. A prototype SPI system was developed and demonstrated here in Cherenkov imaging of LINAC dose delivery to a water tank. Validation experiments were performed using four regular fields and an intensity-modulated radiotherapy (IMRT) delivery plan. The Cherenkov image-based projection percent depth dose curves (pPDDs) were compared to pPDDs simulated by the treatment planning system (TPS), with an overall average error of 0.48, 0.42, 0.65, and 1.08% for the 3, 5, 7, and 9 cm square beams, respectively. The composite image of the IMRT plan achieved a 85.9% pass rate using 3%/3 mm gamma index criteria, in comparing Cherenkov intensity and TPS dose. This study validates the feasibility of applying SPI to the Cherenkov imaging of radiotherapy dose for the first time to our knowledge.

Radiation-Activated Cobalamin-Kinase Inhibitors for Treatment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most dismal diagnoses that a patient can receive. PDAC is extremely difficult to treat, as drug delivery is challenging in part due to the lack of vascularization, high stromal content, and high collagen content of these tumors. We have previously demonstrated that attaching drugs to the cobalamin scaffold provides selectivity for tumors over benign cells due to a high vitamin demand in these rapidly growing cells and an overexpression of transcobalamin receptors in a variety of cancer types. Importantly, we have shown the ability to deliver cobalamin derivatives to orthotopic pancreas tumors. Tyrosine kinase inhibitors have shown promise in treating PDAC as well as other cancer types. However, some of these inhibitors suffer from drug resistance, and as such, their success has been diminished. With this in mind, we synthesized the tyrosine kinase inhibitors erlotinib (EGFR) and dasatinib (Src) that are attached to this cobalamin platform. Both of these cobalamin-drug conjugates cause visible light-induced apoptosis, and the cobalamin-erlotinib conjugate (2) causes X-ray-induced apoptosis in MIA PaCa-2 cells. Both visible light and X-rays provide spatial control of drug release; however, utilizing X-ray irradiation offers the advantage of deeper tissue penetration. Therefore, we explored the utilization of 2 as a synergistic therapy with radiation in athymic nude mice implanted with MIA PaCa-2 tumors. We discovered that the addition of 2 caused an enhanced reduction in tumor margins in comparison with radiation therapy alone. In addition, treatment with 2 in the absence of radiation caused no significant reduction in tumor size in comparison with the controls. The cobalamin technology presented here allows for the spatial release of drugs in conjunction with external beam radiation therapy, potentially allowing for more effective treatment of deep-seated tumors with less systemic side effects.

Breast-Conserving Surgery Margin Guidance Using Micro-Computed Tomography: Challenges When Imaging Radiodense Resection Specimens.

BACKGROUND: Breast-conserving surgery (BCS) is an integral component of early-stage breast cancer treatment, but costly reexcision procedures are common due to the high prevalence of cancer-positive margins on primary resections. A need exists to develop and evaluate improved methods of margin assessment to detect positive margins intraoperatively. METHODS: A prospective trial was conducted through which micro-computed tomography (micro-CT) with radiological interpretation by three independent readers was evaluated for BCS margin assessment. Results were compared to standard-of-care intraoperative margin assessment (i.e., specimen palpation and radiography [abbreviated SIA]) for detecting cancer-positive margins. RESULTS: Six hundred margins from 100 patients were analyzed. Twenty-one margins in 14 patients were pathologically positive. On analysis at the specimen-level, SIA yielded a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 42.9%, 76.7%, 23.1%, and 89.2%, respectively. SIA correctly identified six of 14 margin-positive cases with a 23.5% false positive rate (FPR). Micro-CT readers achieved sensitivity, specificity, PPV, and NPV ranges of 35.7-50.0%, 55.8-68.6%, 15.6-15.8%, and 86.8-87.3%, respectively. Micro-CT readers correctly identified five to seven of 14 margin-positive cases with an FPR range of 31.4-44.2%. If micro-CT scanning had been combined with SIA, up to three additional margin-positive specimens would have been identified. DISCUSSION: Micro-CT identified a similar proportion of margin-positive cases as standard specimen palpation and radiography, but due to difficulty distinguishing between radiodense fibroglandular tissue and cancer, resulted in a higher proportion of false positive margin assessments.

Photon-limited Cherenkov imaging of radiation therapy dose.

Cherenkov imaging is a unique verification tool that could provide both dosimetric and tissue functional information during radiation therapy. However, the number of interrogated Cherenkov photons in tissue is always limited and tangled with stray radiation photons, severely frustrating the measurement the signal-to-noise ratio (SNR). As such, here, a noise-robust photon-limited imaging technique is proposed by comprehensively exploiting the physical rationale of low-flux Cherenkov measurements together with the spatial correlations of the objects. Validation experiments confirmed that the Cherenkov signal could be promisingly recovered with high SNR by irradiating at as few as one x ray pulse from a linear accelerator (10 mGy dose), and the Cherenkov excited luminescence imaging depth can be extended by >100% on average, for most concentrations of phosphorescent probe. This approach demonstrates that improved applications in radiation oncology could be seen when signal amplitude, noise robustness, and temporal resolution are comprehensively considered in the image recovery process.

Fluorescence guidance improves the accuracy of radiological imaging-guided surgical navigation.

BACKGROUND: Imaging-based navigation technologies require static referencing between the target anatomy and the optical sensors. Imaging-based navigation is therefore well suited to operations involving bony anatomy; however, these technologies have not translated to soft-tissue surgery. We sought to determine if fluorescence imaging complement conventional, radiological imaging-based navigation to guide the dissection of soft-tissue phantom tumors. METHODS: Using a human tissue-simulating model, we created tumor phantoms with physiologically accurate optical density and contrast concentrations. Phantoms were dissected using all possible combinations of computed tomography (CT), magnetic resonance, and fluorescence imaging; controls were included. The data were margin accuracy, margin status, tumor spatial alignment, and dissection duration. RESULTS: Margin accuracy was higher for combined navigation modalities compared to individual navigation modalities, and accuracy was highest with combined CT and fluorescence navigation (p = 0.045). Margin status improved with combined CT and fluorescence imaging. CONCLUSIONS: At present, imaging-based navigation has limited application in guiding soft-tissue tumor operations due to its inability to compensate for positional changes during surgery. This study indicates that fluorescence guidance enhances the accuracy of imaging-based navigation and may be best viewed as a synergistic technology, rather than a competing one.

Education case report: CAMPEP Medical Physics PhD education program within Engineering.

Medical physics doctoral programs have large variations in organization, administration and financing. Blending a medical physics stream into an engineering graduate program has advantages of pre-existing financial and educational infrastructures. A case study of the accredited program at Dartmouth was carried out, analyzing operational, financial, educational and outcome features. The support structures provided by each institutional partner were outlined, including engineering school, graduate school, and radiation oncology. The initiatives undertaken by founding faculty were reviewed, along with allocated resources, financial model, and peripheral entrepreneurship activities, each with quantitative outcome metrics. Currently 14 PhD students are enrolled, supported by 22 faculty across both engineering and clinical departments. The total peer-reviewed publications are ≈75/year, while the conventional medical physics fraction of this is about 14/year. Following program formation, a significant rise was seen in jointly published papers between engineering and medical physics faculty, up from 5.6 to 13.3 papers/year, with students publishing an average of 11.3/person with 5.7/person as first author. Student support was predominantly via federal grants, with a stable $5.5million/year, using about $610K/year supporting student stipends and tuition. First year funding, recruiting and staff support were via engineering school. Faculty teaching effort was supported by agreement with each home department, and student services were provided by engineering and graduate schools. Student outcomes were exceptional, with high numbers of presentations, awards, and residency placements at research universities. The lack of financial and student support in medical physics can be mitigated by this hybrid design of blending medical physics doctoral students into an engineering graduate program, providing complementary strengths. Future growth in medical physics programs might consider following this pathway, strengthening research collaborations for clinical physics and engineering faculty, as long as there is vested commitment to teach by the faculty and department leadership.

A Radiation Biological Analysis of the Oxygen Effect as a Possible Mechanism in FLASH.

The delivery of radiation at an ultra-high dose rate (FLASH) is an important new approach to radiotherapy (RT) that appears to be able to improve the therapeutic ratio by diminishing damage to normal tissues. While the mechanisms by which FLASH improves outcomes have not been established, a role involving molecular oxygen (O2) is frequently mentioned. In order to effectively determine if the protective effect of FLASH RT occurs via a differential direct depletion of O2 (compared to conventional radiation), it is essential to consider the known role of O2 in modifying the response of cells and tissues to ionising radiation (known as 'the oxygen effect'). Considerations include: (1) The pertinent reaction involves an unstable intermediate of radiation-damaged DNA, which either undergoes chemical repair to restore the DNA or reacts with O2, resulting in an unrepairable lesion in the DNA, (2) These reactions occur in the nuclear DNA, which can be used to estimate the distance needed for O2 to diffuse through the cell to reach the intermediates, (3) The longest lifetime that the reactive site of the DNA is available to react with O2 is 1-10 µsec, (4) Using these lifetime estimates and known diffusion rates in different cell media, the maximal distance that O2 could travel in the cytosol to reach the site of the DNA (i.e., the nucleus) in time to react are 60-185 nm. This calculation defines the volume of oxygen that is pertinent for the direct oxygen effect, (5) Therefore, direct measurements of oxygen to determine if FLASH RT operates through differential radiochemical depletion of oxygen will require the ability to measure oxygen selectively in a sphere of <200 nm, with a time resolution of the duration of the delivery of FLASH, (6) It also is possible that alterations of oxygen levels by FLASH could occur more indirectly by affecting oxygen-dependent cell signalling and/or cellular repair.

What Is the Meaning of an Oxygen Measurement? : Analysis of Methods Purporting to Measure Oxygen in Targeted Tissues.

Clinical measurements of O2 in tissues will inevitably provide data that are at best aggregated and will not reflect the inherent heterogeneity of O2 in tissues over space and time. Additionally, the nature of all existing techniques to measure O2 results in complex sampling of the volume that is sensed by the technique. By recognizing these potential limitations of the measures, one can focus on the very important and useful information that can be obtained from these techniques, especially data about factors that can change levels of O2 and then exploit these changes diagnostically and therapeutically. The clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of O2.

Optical emission-based phantom to verify coincidence of radiotherapy and imaging isocenters on an MR-linac.

PURPOSE: Demonstrate a novel phantom design using a remote camera imaging method capable of concurrently measuring the position of the x-ray isocenter and the magnetic resonance imaging (MRI) isocenter on an MR-linac. METHODS: A conical frustum with distinct geometric features was machined out of plastic. The phantom was submerged in a small water tank, and aligned using room lasers on a MRIdian MR-linac (ViewRay Inc., Cleveland, OH). The phantom physical isocenter was visualized in the MR images and related to the DICOM coordinate isocenter. To view the x-ray isocenter, an intensified CMOS camera system (DoseOptics LLC., Hanover, NH) was placed at the foot of the treatment couch, and centered such that the optical axis of the camera was coincident with the central axis of the treatment bore. Two or four 8.3mm x 24.1cm beams irradiated the phantom from cardinal directions, producing an optical ring on the conical surface of the phantom. The diameter of the ring, measured at the peak intensity, was compared to the known diameter at the position of irradiation to determine the Z-direction offset of the beam. A star-shot method was employed on the front face of the frustum to determine X-Y alignment of the MV beam. Known shifts were applied to the phantom to establish the sensitivity of the method. RESULTS: Couch translations, demonstrative of possible isocenter misalignments, on the order of 1mm were detectable for both the radiotherapy and MRI isocenters. Data acquired on the MR-linac demonstrated an average error of 0.28mm(N=10, R2 =0.997, σ=0.37mm) in established Z displacement, and 0.10mm(N=5, σ=0.34mm) in XY directions of the radiotherapy isocenter. CONCLUSIONS: The phantom was capable of measuring both the MRI and radiotherapy treatment isocenters. This method has the potential to be of use in MR-linac commissioning, and could be streamlined to be valuable in daily constancy checks of isocenter coincidence.

Single pixel hyperspectral Cherenkov-excited fluorescence imaging with LINAC X-ray sheet scanning and spectral unmixing.

Cherenkov light induced from megavolt (MV) X-rays during external beam radiotherapy serves as an internal light source to excite phosphors or fluorophores within biological tissues for molecular imaging. The broad spectrum of Cherenkov light leads to significant spectral overlap with any luminescence emission and, to overcome this problem, a single pixel hyperspectral imaging methodology was demonstrated here by coupling the detection with light sheet scanning and filtered back projection reconstruction of hyperspectral images. Thin scanned sheets of MV X-rays produce Cherenkov light to illuminate the planes deep within the tissue-simulating media. A fluorescence probe was excited by Cherenkov light, and a complete hyperspectral sinogram of the data was obtained through translation and rotation of the beam. Hyperspectral 2D images finally were reconstructed. Through this approach of spectral unmixing, it was possible to resolve hyperspectral images of both the Cherenkov and resulting fluorescence intensity from molecular sensors.

Active line scan with spatial gating for sub-diffuse reflectance imaging of scatter microtexture.

We examine the value of an active line scan with spatial gating for imaging sub-diffuse, wide-field reflectance microtexture. Line scanning combined with spatial gating and linear translation can be used for localized detection of features in the surface layer of a turbid target. The line scan provides broadband spatial frequency modulation, and the spatial gating effectively high-pass filters the reflectance. The major benefit of this approach is that of high dynamic range (70%-90%) signal preservation and high contrast to noise when imaging at high spatial frequencies. Alternative approaches, such as spatial frequency domain imaging, are degraded by low dynamic range in demodulated images, making it nearly impossible to image over a wide field of view at frequencies over 1.5mm-1 using commercial technology. As such, active line scanning with spatial gating presents as an inherently high sensitivity and high dynamic range method of imaging microscopic scattering features in only the surface layer of a turbid medium.

Time-gated luminescence imaging for background free in vivo tracking of single circulating tumor cells.

Fluorescence imaging is severely limited by the background and autofluorescence of tissues for in vivo detection of circulating tumor cells (CTCs). Time-gated luminescence (TGL) imaging, in combination with luminescent probes that possess hundreds of microsecond emission lifetimes, can be used to effectively suppress this background, which has predominantly nanosecond lifetimes. This Letter demonstrates the feasibility of TGL imaging using luminescent probes for the in vivo real time imaging and tracking of single CTCs circulating freely in the blood vessels with higher accuracy given by substantially higher signal-to-noise ratio. The luminescent probe used in this Letter was a commercial Eu3+ chelate (EuC) nanosphere with a super-long lifetime of near 800 µs, which enabled TGL imaging to achieve background-free detection with ∼5 times higher SNR versus steady state. Phantom and in vivo mouse studies indicated that EuC labeled tumor cells moving in medium or bloodstream at the speed of 1-2 mm/s could be captured in real time.

Dual-agent fluorescent labeling of soft-tissue sarcomas improves the contrast based upon targeting both interstitial and cellular components of the tumor milieu.

BACKGROUND: Current practices for fluorescence-guided cancer surgery utilize a single fluorescent agent, but homogeneous distribution throughout the tumor is difficult to achieve. We hypothesize that administering a perfusion and a molecular-targeted agent at their optimal administration-to-imaging time will improve whole-tumor contrast. EXPERIMENTAL DESIGN: Mice bearing subcutaneous xenograft human synovial sarcomas were administered indocyanine green (ICG) (3 mg/kg) or ABY-029 (48.7 µg/kg)-an epidermal growth factor receptor-targeted Affibody molecule-alone or in combination. Fluorescence contrast and signal distribution were compared between treatment groups. Two commercial fluorescence imaging systems were tested for simultaneous imaging of ICG and ABY-029. RESULTS: ABY-029 has a moderate positive correlation with viable tumor (ρ = 0.2 ± 0.4), while ICG demonstrated a strong negative correlation (ρ = -0.6 ± 0.1). The contrast-to-variance ratio was highest in the ABY-029 +ICG (2.5 ± 0.8), compared to animals that received ABY-029 (2.3 ± 0.8) or ICG (2.0 ± 0.5) alone. Moreover, the combination of ABY-029 + ICG minimizes the correlation between viable tumor and fluorescence intensity (ρ = -0.1 ± 0.2) indicating the fluorescence signal distribution is more homogeneous throughout the tumor milieu. CONCLUSION: Dual-agent imaging utilizing a single channel in a commercial fluorescence-guided imaging system tailored for IRDye 800CW is a promising method to increase tumor contrast in a clinical setting.

Tomographic Cherenkov-excited luminescence scanned imaging with multiple pinhole beams recovered via back-projection reconstruction.

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with a clinical linear accelerator during external beam radiotherapy to map out molecular luminescence intensity or lifetime in tissue. In order to realize a deeper imaging depth with a reasonable spatial resolution in CELSI, we optimized the original scanning procedure to complete this in a way similar to x-ray computed tomography and with image reconstruction from maximum-likelihood expectation maximization and multi-pinhole irradiation for parallelization. Resolution phantom studies showed that a 0.3 mm diameter capillary tube containing 0.01 nM luminescent nanospheres could be recognized at a depth of 21 mm into tissue-like media. Small animal imaging with a 1 mm diameter cylindrical target demonstrated that fast 3D data acquisition can be achieved by this multi-pinhole collimator approach to image high-resolution luminescence through a whole animal.

Preclinical imaging of epidermal growth factor receptor with ABY-029 in soft-tissue sarcoma for fluorescence-guided surgery and tumor detection.

BACKGROUND AND OBJECTIVES: Fluorescence-guided surgery using epidermal growth factor receptor (EGFR) targeting has been performed successfully in clinical trials using a variety of fluorescent agents. We investigate ABY-029 (anti-EGFR Affibody® molecule labeled with IRDye 800CW) compared with a small-molecule perfusion agent, IRDye 700DX carboxylate, in a panel of soft-tissue sarcomas with varying levels of EGFR expression and vascularization. METHODS: Five xenograft soft-tissue sarcoma cell lines were implanted into immunosuppressed mice. ABY-029 and IRDye 700DX were each administered at 4.98 µM. Fluorescence from in vivo and ex vivo (fresh and formalin-fixed) fixed tissues were compared. The performance of three fluorescence imaging systems was assessed for ex vivo tissues. RESULTS: ABY-029 is retained longer within tumor tissue and achieves higher tumor-to-background ratios both in vivo and ex vivo than IRDye 700DX. ABY-029 fluorescence is less susceptible to formalin fixation than IRDye 700DX, but both agents have disproportional signal loss in a variety of tissues. The Pearl Impulse provides the highest contrast-to-noise ratio, but all systems have individual advantages. CONCLUSIONS: ABY-029 demonstrates promise to assist in wide local excision of soft-tissue sarcomas. Further clinical evaluation of in situ or freshly excised ex vivo tissues using fluorescence imaging systems is warranted.

Micro-computed tomography enables rapid surgical margin assessment during breast conserving surgery (BCS): correlation of whole BCS micro-CT readings to final histopathology.

BACKGROUND: Roughly 23% of breast conserving surgery (BCS) patients undergo a second re-excision procedure due to pathologically positive surgical margins. We investigated the feasibility and potential value of micro-Computed Tomography (micro-CT) as a surgical margin guidance tool during BCS. METHODS: A cohort of 32 BCS specimens was prospectively imaged with a pre-clinical micro-CT system upon arrival in the surgical pathology laboratory. Reconstructed micro-CT scans were evaluated retrospectively by an experienced breast radiologist, who provided binary determinations whether lesions extended to the specimen margin. These readings were then compared to the final pathological diagnosis and to 2D specimen radiography readings. RESULTS: Of the 32 specimens imaged, 28 had malignant and four had benign pathological diagnoses. Overall five (four malignant, one benign) of the 32 specimens had lesion tissue extending to the margin. For all 32 specimens, micro-CT reconstructions were calculated (< 4 min. acquisition + reconstruction time) and each specimen was volumetrically analyzed by a radiologist. Of the 28 malignant specimen readings, 18 matched the final pathological diagnosis [64%, 95 CI (47%-81%)], with a negative predictive value of 89% [95 CI (74%-96%)]. Micro-CT readings revealed changes in the tumor location and margin status as compared to single-projection radiography readings. CONCLUSIONS: Micro-CT scanning of BCS specimens enabled margin status assessment over the entirety of the surgical surface in a clinically relevant time frame, provided additional spatial information over single-projection radiography, and may be a potentially useful BCS guidance tool.

Observation of short wavelength infrared (SWIR) Cherenkov emission.

Cherenkov emission induced by external beam radiation from a clinical linear accelerator has been shown in preclinical molecular imaging and clinical imaging. The broad spectrum Cherenkov emission should have a short wavelength infrared (SWIR, 1000-1700 nm) component, as predicted theoretically. To the best of our knowledge, this Letter is the first experimental observation of this SWIR Cherenkov emission induced by external beam radiation. The measured spectrum of SWIR Cherenkov emission matches the theoretical prediction, with a fluence rate near one-third of the visible and near-infrared red emissions (Vis-NIR, 400-900 nm). Imaging in water-based phantoms and biological tissues indicates that there is a sufficient fluence rate for radiotherapy dosimetry applications. The spatial resolution is improved approximately 5.3 times with SWIR Cherenkov emission detection versus Vis-NIR Cherenkov emission, which provides some improvement in the potential for higher resolution Cherenkov emission dosimetry and molecular sensing during clinical radiotherapy by imaging with SWIR wavelengths.

Application of Fluorescence-Guided Surgery to Subsurface Cancers Requiring Wide Local Excision: Literature Review and Novel Developments Toward Indirect Visualization.

The excision of tumors by wide local excision is challenging because the mass must be removed entirely without ever viewing it directly. Positive margin rates in sarcoma resection remain in the range of 20% to 35% and are associated with increased recurrence and decreased survival. Fluorescence-guided surgery (FGS) may improve surgical accuracy and has been utilized in other surgical specialties. ABY-029, an anti-epidermal growth factor receptor Affibody molecule covalently bound to the near-infrared fluorophore IRDye 800CW, is an excellent candidate for future FGS applications in sarcoma resection; however, conventional methods with direct surface tumor visualization are not immediately applicable. A novel technique involving imaging through a margin of normal tissue is needed. We review the past and present applications of FGS and present a novel concept of indirect FGS for visualizing tumor through a margin of normal tissue and aiding in excising the entire lesion as a single, complete mass with tumor-free margins.