Isaiah Norton

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo. Virtual Histology During brain tumor surgery, precision is key. Removing healthy tissue can cause neurologic deficits; leaving behind tumor tissue can allow cancer to spread and treatment to fail. To help the surgeon clearly see tumor versus normal tissue, Ji and colleagues developed a stimulated Raman scattering (SRS) microscopy method and demonstrated its ability to identify malignant human brain tissue. In SRS microscopy, laser beams are directed at the tissue sample to generate a series of output signals called “Raman spectra.” These spectra depend on the molecular composition of the tissue. Ji et al. implanted human brain cancer (glioblastoma) cells into mice, allowed them to infiltrate and grow into tumors, and then removed slices for SRS imaging. From the resulting spectra, the authors were able to differentiate the two major components of brain tissue—lipid-rich white matter and protein-rich cortex—as well as tumors, which are full of proteins. Intraoperatively, using an imaging window into mouse brains, the authors found that SRS microscopy could locate tumor infiltration in areas that appeared normal by eye, which suggests that this tool could be applied during surgery. Imaging fresh tissue slices ex vivo could also complement or perhaps replace standard hematoxylin and eosin (H&E) staining in the clinic because it avoids artifacts inherent in imaging frozen or fixed tissues. To this end, Ji and colleagues showed that SRS microscopy could identify hypercellular tumor regions in fresh surgical specimens from a patient with glioblastoma. Certain diagnostic features were present in these specimens and readily identified by SRS, including pseudopalisading necrosis and microvascular proliferation. The next step will be to apply SRS microscopy to a large collection of human specimens to see whether this technology may be useful in quickly distinguishing glioblastoma from healthy tissue, both outside and inside the operating room. Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Classifying Human Brain Tumors by Lipid Imaging with Mass Spectrometry

Brain tissue biopsies are required to histologically diagnose brain tumors, but current approaches are limited by tissue characterization at the time of surgery. Emerging technologies such as mass spectrometry imaging can enable a rapid direct analysis of cancerous tissue based on molecular composition. Here, we illustrate how gliomas can be rapidly classified by desorption electrospray ionization-mass spectrometry (DESI-MS) imaging, multivariate statistical analysis, and machine learning. DESI-MS imaging was carried out on 36 human glioma samples, including oligodendroglioma, astrocytoma, and oligoastrocytoma, all of different histologic grades and varied tumor cell concentration. Gray and white matter from glial tumors were readily discriminated and detailed diagnostic information could be provided. Classifiers for subtype, grade, and concentration features generated with lipidomic data showed high recognition capability with more than 97% cross-validation. Specimen classification in an independent validation set agreed with expert histopathology diagnosis for 79% of tested features. Together, our findings offer proof of concept that intraoperative examination and classification of brain tissue by mass spectrometry can provide surgeons, pathologists, and oncologists with critical and previously unavailable information to rapidly guide surgical resections that can improve management of patients with malignant brain tumors.

Ambient mass spectrometry for the intraoperative molecular diagnosis of human brain tumors

The main goal of brain tumor surgery is to maximize tumor resection while preserving brain function. However, existing imaging and surgical techniques do not offer the molecular information needed to delineate tumor boundaries. We have developed a system to rapidly analyze and classify brain tumors based on lipid information acquired by desorption electrospray ionization mass spectrometry (DESI-MS). In this study, a classifier was built to discriminate gliomas and meningiomas based on 36 glioma and 19 meningioma samples. The classifier was tested and results were validated for intraoperative use by analyzing and diagnosing tissue sections from 32 surgical specimens obtained from five research subjects who underwent brain tumor resection. The samples analyzed included oligodendroglioma, astrocytoma, and meningioma tumors of different histological grades and tumor cell concentrations. The molecular diagnosis derived from mass-spectrometry imaging corresponded to histopathology diagnosis with very few exceptions. Our work demonstrates that DESI-MS technology has the potential to identify the histology type of brain tumors. It provides information on glioma grade and, most importantly, may help define tumor margins by measuring the tumor cell concentration in a specimen. Results for stereotactically registered samples were correlated to preoperative MRI through neuronavigation, and visualized over segmented 3D MRI tumor volume reconstruction. Our findings demonstrate the potential of ambient mass spectrometry to guide brain tumor surgery by providing rapid diagnosis, and tumor margin assessment in near–real time.

Intraoperative mass spectrometry mapping of an onco-metabolite to guide brain tumor surgery

Significance The diagnosis of tumors during surgery still relies principally on an approach developed over 150 y ago: frozen section microscopy. We show that a validated molecular marker—2-hydroxyglutarate generated from isocitrate dehydrogenase 1 mutant gliomas—can be rapidly detected from tumors using a form of ambient MS that does not require sample preparation. We use the Advanced Multimodality Image Guided Operating Suite at Brigham and Women’s Hospital to demonstrate that desorption electrospray ionization MS could be used to detect residual tumor that would have been left behind in the patient. The approach paves the way for the clinical testing of MS-based intraoperative monitoring of tumor metabolites, an advance that could revolutionize the care of surgical oncology patients. For many intraoperative decisions surgeons depend on frozen section pathology, a technique developed over 150 y ago. Technical innovations that permit rapid molecular characterization of tissue samples at the time of surgery are needed. Here, using desorption electrospray ionization (DESI) MS, we rapidly detect the tumor metabolite 2-hydroxyglutarate (2-HG) from tissue sections of surgically resected gliomas, under ambient conditions and without complex or time-consuming preparation. With DESI MS, we identify isocitrate dehydrogenase 1-mutant tumors with both high sensitivity and specificity within minutes, immediately providing critical diagnostic, prognostic, and predictive information. Imaging tissue sections with DESI MS shows that the 2-HG signal overlaps with areas of tumor and that 2-HG levels correlate with tumor content, thereby indicating tumor margins. Mapping the 2-HG signal onto 3D MRI reconstructions of tumors allows the integration of molecular and radiologic information for enhanced clinical decision making. We also validate the methodology and its deployment in the operating room: We have installed a mass spectrometer in our Advanced Multimodality Image Guided Operating (AMIGO) suite and demonstrate the molecular analysis of surgical tissue during brain surgery. This work indicates that metabolite-imaging MS could transform many aspects of surgical care.

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Significance This study is the first demonstration, to our knowledge, of the application of desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for discrimination of breast cancer and delineation of tumor margins. Using DESI-MSI, it is possible to discriminate between cancerous and adjacent normal tissue on the basis of the detection and specific spatial distributions of different lipid species. This study proves the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. It will allow the surgeon to access to this information in real time so as to make accurate intraoperative decisions quickly. It will result in improved cosmesis and decrease the need for multiple operations for margin reexcision. Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.

Interactive Diffusion Tensor Tractography Visualization for Neurosurgical Planning

BACKGROUND: Diffusion tensor imaging (DTI) infers the trajectory and location of large white matter tracts by measuring the anisotropic diffusion of water. DTI data may then be analyzed and presented as tractography for visualization of the tracts in 3 dimensions. Despite the important information contained in tractography images, usefulness for neurosurgical planning has been limited by the inability to define which are critical structures within the mass of demonstrated fibers and to clarify their relationship to the tumor. OBJECTIVE: To develop a method to allow the interactive querying of tractography data sets for surgical planning and to provide a working software package for the research community. METHODS: The tool was implemented within an open source software project. Echo-planar DTI at 3 T was performed on 5 patients, followed by tensor calculation. Software was developed that allowed the placement of a dynamic seed point for local selection of fibers and for fiber display around a segmented structure, both with tunable parameters. A neurosurgeon was trained in the use of software in < 1 hour and used it to review cases. RESULTS: Tracts near tumor and critical structures were interactively visualized in 3 dimensions to determine spatial relationships to lesion. Tracts were selected using 3 methods: anatomical and functional magnetic resonance imaging-defined regions of interest, distance from the segmented tumor volume, and dynamic seed-point spheres. CONCLUSION: Interactive tractography successfully enabled inspection of white matter structures that were in proximity to lesions, critical structures, and functional cortical areas, allowing the surgeon to explore the relationships between them.

A combined fMRI and DTI examination of functional language lateralization and arcuate fasciculus structure: Effects of degree versus direction of hand preference

The present study examined the relationship between hand preference degree and direction, functional language lateralization in Brocas and Wernickes areas, and structural measures of the arcuate fasciculus. Results revealed an effect of degree of hand preference on arcuate fasciculus structure, such that consistently-handed individuals, regardless of the direction of hand preference, demonstrated the most asymmetric arcuate fasciculus, with larger left versus right arcuate, as measured by DTI. Functional language lateralization in Wernickes area, measured via fMRI, was related to arcuate fasciculus volume in consistent-left-handers only, and only in people who were not right hemisphere lateralized for language; given the small sample size for this finding, future investigation is warranted. Results suggest handedness degree may be an important variable to investigate in the context of neuroanatomical asymmetries.

Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain – insights that would be difficult or impossible to extract by use of radiolabeled compounds.

The DTI Challenge: Toward Standardized Evaluation of Diffusion Tensor Imaging Tractography for Neurosurgery

Diffusion tensor imaging (DTI) tractography reconstruction of white matter pathways can help guide brain tumor resection. However, DTI tracts are complex mathematical objects and the validity of tractography‐derived information in clinical settings has yet to be fully established. To address this issue, we initiated the DTI Challenge, an international working group of clinicians and scientists whose goal was to provide standardized evaluation of tractography methods for neurosurgery. The purpose of this empirical study was to evaluate different tractography techniques in the first DTI Challenge workshop.

Development of Stereotactic Mass Spectrometry for Brain Tumor Surgery

BACKGROUND: Surgery remains the first and most important treatment modality for the majority of solid tumors. Across a range of brain tumor types and grades, postoperative residual tumor has a great impact on prognosis. The principal challenge and objective of neurosurgical intervention is therefore to maximize tumor resection while minimizing the potential for neurological deficit by preserving critical tissue. OBJECTIVE: To introduce the integration of desorption electrospray ionization mass spectrometry into surgery for in vivo molecular tissue characterization and intraoperative definition of tumor boundaries without systemic injection of contrast agents. METHODS: Using a frameless stereotactic sampling approach and by integrating a 3-dimensional navigation system with an ultrasonic surgical probe, we obtained image-registered surgical specimens. The samples were analyzed with ambient desorption/ionization mass spectrometry and validated against standard histopathology. This new approach will enable neurosurgeons to detect tumor infiltration of the normal brain intraoperatively with mass spectrometry and to obtain spatially resolved molecular tissue characterization without any exogenous agent and with high sensitivity and specificity. RESULTS: Proof of concept is presented in using mass spectrometry intraoperatively for real-time measurement of molecular structure and using that tissue characterization method to detect tumor boundaries. Multiple sampling sites within the tumor mass were defined for a patient with a recurrent left frontal oligodendroglioma, World Health Organization grade II with chromosome 1p/19q codeletion, and mass spectrometry data indicated a correlation between lipid constitution and tumor cell prevalence. CONCLUSION: The mass spectrometry measurements reflect a complex molecular structure and are integrated with frameless stereotaxy and imaging, providing 3-dimensional molecular imaging without systemic injection of any agents, which can be implemented for surgical margins delineation of any organ and with a rapidity that allows real-time analysis.