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Background: Frozen section and smear preparation are the current standard for intraoperative histopathology during cancer surgery. However, these methods are time-consuming and subject to limited sampling. Multiphoton microscopy (MPM) is a high-resolution non-destructive imaging technique capable of optical sectioning in real time with subcellular resolution. In this report, we systematically investigated the feasibility and translation potential of MPM for rapid histopathological assessment of label- and processing-free surgical specimens. Methods: We employed a customized MPM platform to capture architectural and cytological features of biological tissues based on two-photon excited NADH and FAD autofluorescence and second harmonic generation from collagen. Infiltrating glioma, an aggressive disease that requires subcellular resolution for definitive characterization during surgery, was chosen as an example for this validation study. MPM images were collected from resected brain specimens of 19 patients and correlated with histopathology. Deep learning was introduced to assist with image feature recognition. Results: MPM robustly captures diagnostic features of glioma including increased cellularity, cellular and nuclear pleomorphism, microvascular proliferation, necrosis, and collagen deposition. Preliminary application of deep learning to MPM images achieves high accuracy in distinguishing gray from white matter and cancer from non-cancer. We also demonstrate the ability to obtain such images from intact brain tissue with a multiphoton endomicroscope for intraoperative application. Conclusion: Multiphoton imaging correlates well with histopathology and is a promising tool for characterization of cancer and delineation of infiltration within seconds during brain surgery.

PURPOSE/OBJECTIVE(S) Severe lymphopenia during chemoradiation is often associated with higher risk of death, disease progression and distant metastases. Recent data has shown that head and neck cancer (HNC) patients with lymphopenia has a decreased 5-year overall survival of 70 vs 90%. While both radiation and chemotherapy have been associated with lymphopenia, the mechanism of action remains unclear. Here, we propose a spatially-defined model that evaluates the combined effects of lymphotoxic radiation doses to head and neck organs-at-risk (HN-OARs) which include the cervical bone marrow and the circulating blood pool. Our objective is to investigate the spatial dose relationships related to treatment-related lymphopenia (TRL) in HNC patients. MATERIALS/METHODS We have identified a prospectively acquired dataset of 40 HNC patients treated at our institution with 31 oropharynx, 3 oral cavity, 4 nasopharynx and 2 larynx cases. Weekly lymphocyte counts were extracted at baseline and for 8 weeks since initiation of radiotherapy. Chemotherapy timing, dosage and regimens were documented. HN-OARs were manually contoured on CT simulation scans. To generate the spatially-defined linear regression model, we included these inputs (x): internal jugular veins (IJVs), carotid arteries (CAs), capillary bed (CB) and vertebral bodies (VBs). The output (y) is defined by % decrease between baseline and nadir lymphocyte counts. Then, we segmented VBs by vertebral levels, and other HN-OARs by disease laterality (ipsilateral vs contralateral). IJVs and CAs are further segmented by anatomic zones (zone 1: C1 to hyoid, zone 2: hyoid to cricoid, and zone 3: cricoid to C7). Stepwise selection was used to identify influential HN-OARs. Finally, we proposed spatial dose parameters associated with high risk lymphopenia (defined as ≥70% decrease) and performed sensitivity/specificity (Se/Sp) analyses. RESULTS Using stepwise (forward) selection, we identified IJVs as potential targets to spare low dose bath parameters Dmin and D90 (P-values 0.028 to 0.056), and VBs as potential targets to spare point max doses Dmax and D10 (P-values < 0.001 to 0.020). To avoid overfitting, we also performed backward selection with a more liberal P-value criterion of 0.50 which showed similar results. Key anatomic zones for potential dose sparing include C1-3 VBs and contralateral zone 1 IJVs. Overall, we proposed these dose parameters to limit TRL risks: Dmax ≤ 60 Gy for VBs (Se/Sp: 76%/86%, AUC 0.84) and D90 ≤ 13 Gy for contralateral IJVs (Se/Sp: 72%/86%, AUC 0.79). CONCLUSION The risk of treatment-related lymphopenia (TRL) appears to be spatially related to radiation doses to the VBs and IJVs. Better understanding of the spatial dose relationship with HN-OARs may lead to insights in reducing TRL risks and improving oncologic outcomes, especially as we consider the benefits of recent dose de-escalation studies (to reduce point max doses) and proton treatment techniques (to reduce low dose baths) in the management of head and neck cancers.

Summary Background During laparoscopic or robotic assisted laparoscopic prostatectomy, the surgeon lacks tactile feedback which can help him tailor the size of the excision. Ultrasound elastography (USE) is an emerging imaging technology which maps the stiffness of tissue. In the paper we are evaluating USE as a palpation equivalent tool for intraoperative image guided robotic assisted laparoscopic prostatectomy. Material/Methods Two studies were performed: 1) A laparoscopic ultrasound probe was used in a comparative study of manual palpation versus USE in detecting tumor surrogates in synthetic and ex-vivo tissue phantoms; N=25 participants (students) were asked to provide the presence, size and depth of these simulated lesions, and 2) A standard ultrasound probe was used for the evaluation of USE on ex-vivo human prostate specimens (N=10 lesions in N=6 specimens) to differentiate hard versus soft lesions with pathology correlation. Results were validated by pathology findings, and also by in-vivo and ex-vivo MR imaging correlation. Results In the comparative study, USE displayed higher accuracy and specificity in tumor detection (sensitivity=84%, specificity=74%). Tumor diameters and depths were better estimated using USE versus with manual palpation. USE also proved consistent in identification of lesions in ex-vivo prostate specimens; hard and soft, malignant and benign, central and peripheral. Conclusions USE is a strong candidate for assisting surgeons by providing palpation equivalent evaluation of the tumor location, boundaries and extra-capsular extension. The results encourage us to pursue further testing in the robotic laparoscopic environment.

More complete brain cancer resection can prolong survival and delay recurrence. However, it is challenging to distinguish cancer from noncancer tissues intraoperatively, especially at the transitional, infiltrative zones. This is especially critical in eloquent regions (for example, speech and motor areas). This study tested the feasibility of label-free, quantitative optical coherence tomography (OCT) for differentiating cancer from noncancer in human brain tissues. Fresh ex vivo human brain tissues were obtained from 32 patients with grade II to IV brain cancer and 5 patients with noncancer brain pathologies. On the basis of volumetric OCT imaging data, pathologically confirmed brain cancer tissues (both high- and low-grade) had significantly lower optical attenuation values at both cancer core and infiltrated zones when compared with noncancer white matter, and OCT achieved high sensitivity and specificity at an attenuation threshold of 5.5 mm(-1) for brain cancer patients. We also used this attenuation threshold to confirm the intraoperative feasibility of performing in vivo OCT-guided surgery using a murine model harboring human brain cancer. Our OCT system was capable of processing and displaying a color-coded optical property map in real time at a rate of 110 to 215 frames per second, or 1.2 to 2.4 s for an 8- to 16-mm(3) tissue volume, thus providing direct visual cues for cancer versus noncancer areas. Our study demonstrates the translational and practical potential of OCT in differentiating cancer from noncancer tissue. Its intraoperative use may facilitate safe and extensive resection of infiltrative brain cancers and consequently lead to improved outcomes when compared with current clinical standards.

The purpose of this critical review is to explore the controversy regarding the relationship between radiation dose to the neural progenitor cell (NPC) niches and patient outcomes, in terms of both toxicity and tumor control. NPCs in the subventricular zone (SVZ) and hippocampus are paradoxically associated with long-term neurocognitive sequelae of brain irradiation, as well as resistance to therapy and tumor recurrence. The reconciliation of these somewhat opposing functions is challenging. Current literature suggests that radiation and other treatments against the NPC in the hippocampus and the SVZ may influence patient outcome. As a result, both the SVZ and the hippocampus could have important implications on radiation treatment planning strategies, and future laboratory and clinical evaluations will be critical in designing studies to optimize treatment outcome, effectiveness, and safety.

Aim: To assess the potential for injury to normal tissues in mice due to heating systemically delivered magnetic nanoparticles in an alternating magnetic field (AMF). Materials & methods: Twenty three male nude mice received intravenous injections of dextran–superparamagnetic iron oxide nanoparticles on days 1–3. On day 6, they were exposed to AMF. On day 7, blood, liver and spleen were harvested and analyzed. Results: Iron deposits were detected in the liver and spleen. Mice that had received a high-particle dose and a high AMF experienced increased mortality, elevated liver enzymes and significant liver and spleen necrosis. Mice treated with low-dose superparamagnetic iron oxide nanoparticles and a low AMF survived, but had elevated enzyme levels and local necrosis in the spleen. Conclusion: Magnetic nanoparticles producing only modest heat output can cause damage, and even death, when sequestered in sufficient concentrations. Dextran–superparamagnetic iron oxide nanoparticles are deposited in the liver and spleen, making these the sites of potential toxicity. Original submitted 16 August 2011; Revised submitted 21 March 2012; Published online 26 July 2012

Ex vivo human brain tissues from 8 glioma patients and 5 controls are imaged using SS-OCT, with higher glioma attenuation values and easily detectable and specific tumor features (microcyst, necrosis and pallisading) for intraoperative resections.

Purpose: To compare the accuracy of detecting tumor location and size in the prostate using both manual palpation and ultrasound elastography (UE). Methods: Tumors in the prostate were simulated using both synthetic and ex vivo tissue phantoms. 25 participants were asked to provide the presence, size and depth of these simulated lesions using manual palpation and UE. Ultrasound images were captured using a laparoscopic ultrasound probe, fitted with a Gore-Tetrad transducer with frequency of 7.5 MHz and a RF capture depth of 4-5 cm. A MATLAB GUI application was employed to process the RF data for ex vivo phantoms, and to generate UE images using a cross-correlation algorithm. Ultrasonix software was used to provide real time elastography during laparoscopic palpation of the synthetic phantoms. Statistical analyses were performed based on a two-tailed, student t-test with α = 0.05. Results: UE displays both a higher accuracy and specificity in tumor detection (sensitivity = 84%, specificity = 74%). Tumor diameters and depths are better estimated using ultrasound elastography when compared with manual palpation. Conclusions: Our results indicate that UE has strong potential in assisting surgeons to intra-operatively evaluate the tumor depth and size. We have also demonstrated that ultrasound elastography can be implemented in a laparoscopic environment, in which manual palpation would not be feasible. With further work, this application can provide accurate and clinically relevant information for surgeons during prostate resection.

The blood-brain barrier (BBB), with its charged, lipid-based, continuous basement membrane and specialized transporters, is optimized to exclude potentially threatening compounds from the central nervous system (CNS). In the setting of CNS diseases, the careful construction of the BBB is thought to restrict access of therapeutic agents that may otherwise be effective, contributing to poorer outcomes. However, there is controversy in the neurotherapeutics community about the degree to which this hypothesis is true; some argue that the BBB is not the critical factor because it naturally opens in the setting of brain pathology and that techniques to circumvent the BBB do not improve drug efficacy in many instances.1 Moreover, some drugs do not need direct access to brain tissue in order to have their therapeutic effect. For example, fingolimod and bevacizumab are effective in patients with multiple sclerosis and glioblastoma, respectively, although their primary mechanism of action is systemic. Additionally, in some brain tumors, achieving target drug concentrations in the brain either fails to show efficacy or, in fact, causes toxicity.2 In these instances, the factor impeding success of neurotherapeutics is clearly not the BBB.

Purpose: To improve the utility of in‐room video‐based system for continuous quantitative monitoring of patient motion independent of environmental changes in lighting and geometry. Method and Materials: A video‐based tracking system was developed using a webcam with 600 by 450 pixels and Microsoft Visual C++. It is mounted at the end of the treatment couch to constantly view a specified region on the patient's torso of 50 cm by 30 cm at 55 degrees viewing angle. Small high contrast “sticky” markers are placed within the viewing region for easy detection. Environmental disturbances such as changes in machine positions and lightings are minimized through appropriate real‐time image subtraction and processing techniques. An alarm is activated when the patient movement is deemed out‐of‐tolerance. For validation, a lead ball phantom and a flat “sticky” marker of 1 cm diameter each were used to determine detectable target displacements. Each tracking session was automatically recorded for further data analysis. Pre‐clinical evaluations were performed on two subjects. Results: Target displacements of (2, 2, 5) mm for the lead ball phantom and (4,4,7) mm for the flat phantom are readily detected in the lateral, superior‐inferior and vertical directions respectively. Most importantly, the system detects these changes in the presence of environmental disturbances which include large changes in room lighting, and couch and gantry positions. The system fails when the camera's view is obstructed. Conclusion: Our system provides an effective approach to track and monitor patient position after highly accurate setup, such as using cone‐beam CT. It is considerably lower in cost relative to existing commercial tracking systems. The use of more optimal markers and 2 cameras are under investigation to improve detection resolution. With further refinement, the system can be adapted for routine clinical use that is superior to present in‐room video‐monitoring systems.

INTRODUCTION: Tumor cells are passively carried by extracellular fluid flow to regional lymph nodes in most systemic cancers including breast cancer, melanoma and colorectal cancer. Although the brain does not contain lymphatics, cancer spread within the brain may be a function of extracellular fluid velocity. As malignant brain tumors are characterized by a disrupted blood-brain-barrier that leaks albumin from blood vessels into brain parenchyma, we hypothesize that this albumin influx will pull water in osmotically and increase flow rates and glioma cell dissemination down adjacent white matter tracts. High flow rates may increase tumor dissemination via the WMT. This study sought to determine if increasing intracranial albumin concentrations affect WMT flow rates. If true, restricting albumin extravasation (through steroids or VEGF inhibitors) has the potential to reduce glioma dissemination and may have implications for future radiation planning and steroid/VEGF inhibitor dosing. METHODS: Our study examines flow rates in white matter tracts after intracranial administration of albumin. 0.12-6 mg bovine albumin were mixed with 20 μL of 2% Evans Blue and injected stereotactically into the right frontal lobe at the gray-white matter junction over 80 minutes in 15 anesthetized Sprague-Dawley female rats. Seven hours later, the rats were sacrificed and the brains sectioned into 0.5mm coronal slices, and imaged to determine interstitial flow rates. RESULTS: Average anterior-posterior (AP) velocity for rats receiving 0.12 mg and 6.0 mg of albumin were 0.43 and 1.02 mm/hour respectively (p = 0.029). Correlation was observed between administered albumin dose and interstitial fluid velocity (R = 0.57). In vivo MRI results were consistent with ex vivo Evans blue results and demonstrate markedly increased edema (T2) in the hemisphere where albumin was injected. CONCLUSIONS: This study demonstrates higher interstitial fluid flow rates in adjacent white matter tracts in response to albumin-induced-edema. These observations provide insight into expected pathways for glioma dissemination which could be used to tailor radiation plans for individual patients. Furthermore, these findings provide a rationale for interventions to reduce glioma dissemination by limiting the extravasation of albumin through the blood-brain-barrier while local radiation is being administered. Citation Format: Carmen Kut, Vadappuram Chacko, Betty Tyler, Arvind P. Pathak, Zaver Bhujwalla, Elliot R. McVeigh, Xingde Li, Daniel A. Herzka, Stuart A. Grossman. Determining extracellular fluid velocity along white matter tracts with injections of increasing concentrations of intracranial albumin using both histological and radiological techniques. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2013-3931