Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

Expansion of endogenous T cells in CSF of pediatric CNS tumor patients undergoing locoregional delivery of IL13R〿2-targeting CAR T cells: an interim analysis.

Outcomes for pediatric brain tumor patients remain poor, and there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. Here, we present interim results from the first six pediatric patients treated on an ongoing phase I clinical trial (NCT04510051) of IL13BBζ-CAR T cells delivered weekly into the lateral cerebral ventricles, identifying clonal expansion of endogenous CAR-negative CD8+ T cells in the cerebrospinal fluid (CSF) over time. Additionally, of the five patients evaluable for disease response, three experienced transient radiographic and/or clinical benefit not meeting protocol criteria for response. The first three patients received CAR T cells alone; later patients received lymphodepletion before the first infusion. There were no dose limiting toxicities (DLTs). Aside from expected cytopenias in patients receiving lymphodepletion, serious adverse events possibly attributed to CAR T cell infusion were limited to one episode of headache and one of liver enzyme elevation. One patient withdrew from treatment during the DLT period due to a Grade 3 catheter-related infection and was not evaluable for disease response, although this was not attributed to CAR T cell infusion. Importantly, scRNA- and scTCR-sequence analyses provided insights into CAR T cell interaction with the endogenous immune system. In particular, clonally expanded endogenous CAR- T cells were recovered from the CSF, but not the peripheral blood, of patients who received intraventricular IL13BBζ-CAR T cell therapy. Additionally, although immune infiltrates in CSF and post-therapy tumor did not generally correlate, a fraction of expanded T cell receptors (TCRs) was seen to overlap between CSF and tumor. This has important implications for what samples are collected on these trials and how they are analyzed. These initial findings provide support for continued investigation into locoregionally-delivered IL13BBζ-CAR T cells for children with brain tumors.

Subtemporal Transcollateral Sulcus Approach for Temporal Horn Tumors: 2-Dimensional Operative Video and Description of the Technique.

INTRODUCTION: Purely temporal intraventricular tumors are uncommon. Given their deep location, access to these brain tumors can be challenging in terms of preserving brain tissue. The subtemporal approach spares the lateral temporal cortex and is a less traumatic corridor to reach intraventricular temporal tumors. OBJECTIVE: To describe and assess the feasibility of the subtemporal transcollateral approach for the removal of a temporal horn tumor. METHODS: We describe the subtemporal transcollateral sulcus operative technique detailed step-by-step and depicted through both video and illustrations to surgically resect a left intraventricular temporal mass in a 44-year-old woman who presented with worsening memory deficits. The surgery was performed under general anesthesia and with the use of a microscope and neuronavigation. RESULTS: The patient did not suffer from any postoperative complications. Her vision was intact, and her memory deficit was unchanged. A brain MRI showed complete removal of the tumor. The pathological examination revealed a World Health Organization grade I meningioma. CONCLUSION: The subtemporal transsulcal approach seems to be an efficient and safe way to access intraventricular lesions within the temporal horn while avoiding any disruption of the optic radiations and temporal language areas.

Molecular Imaging of Glucose Metabolism for Intraoperative Fluorescence Guidance During Glioma Surgery.

PURPOSE: This study evaluated the use of molecular imaging of fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as a discriminatory marker for intraoperative tumor border identification in a murine glioma model. PROCEDURES: 2-NBDG was assessed in GL261 and U251 orthotopic tumor-bearing mice. Intraoperative fluorescence of topical and intravenous 2-NBDG in normal and tumor regions was assessed with an operating microscope, handheld confocal laser scanning endomicroscope (CLE), and benchtop confocal laser scanning microscope (LSM). Additionally, 2-NBDG fluorescence in tumors was compared with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence. RESULTS: Intravenously administered 2-NBDG was detectable in brain tumor and absent in contralateral normal brain parenchyma on wide-field operating microscope imaging. Intraoperative and benchtop CLE showed preferential 2-NBDG accumulation in the cytoplasm of glioma cells (mean [SD] tumor-to-background ratio of 2.76 [0.43]). Topically administered 2-NBDG did not create sufficient tumor-background contrast for wide-field operating microscope imaging or under benchtop LSM (mean [SD] tumor-to-background ratio 1.42 [0.72]). However, topical 2-NBDG did create sufficient contrast to evaluate cellular tissue architecture and differentiate tumor cells from normal brain parenchyma. Protoporphyrin IX imaging resulted in a more specific delineation of gross tumor margins than intravenous or topical 2-NBDG and a significantly higher tumor-to-normal-brain fluorescence intensity ratio. CONCLUSION: After intravenous administration, 2-NBDG selectively accumulated in the experimental brain tumors and provided bright contrast under wide-field fluorescence imaging with a clinical-grade operating microscope. Topical 2-NBDG was able to create a sufficient contrast to differentiate tumor from normal brain cells on the basis of visualization of cellular architecture with CLE. 5-Aminolevulinic acid demonstrated superior specificity in outlining tumor margins and significantly higher tumor background contrast. Given the nontoxicity of 2-NBDG, its use as a topical molecular marker for noninvasive in vivo intraoperative microscopy is encouraging and warrants further clinical evaluation.

Cerebellar infarction requiring surgical decompression in patient with COVID 19 pathological analysis and brief review.

BACKGROUND: This report and literature review describes a case of a COVID-19 patient who suffered a cerebellar stroke requiring neurosurgical decompression. This is the first reported case of a sub-occipital craniectomy with brain biopsy in a COVID-19 patient showing leptomeningeal venous intimal inflammation. CLINICAL DESCRIPTION: The patient is a 48-year-old SARS-COV-2 positive male with multiple comorbidities, who presented with fevers and respiratory symptoms, and imaging consistent with multifocal pneumonia. On day 5 of admission, the patient had sudden change in mental status, increased C-Reactive Protein, ferritin and elevated Interleukin-6 levels. Head CT showed cerebral infarction from vertebral artery occlusion. Given subsequent rapid neurologic decline from cerebellar swelling and mass effect on his brainstem emergent neurosurgical intervention was performed. Brain biopsy found a vein with small organizing thrombus adjacent to focally proliferative intima with focal intimal neutrophils. CONCLUSION: A young man with COVID-19 and suspected immune dysregulation, complicated by a large cerebrovascular ischemic stroke secondary to vertebral artery thrombosis requiring emergent neurosurgical intervention for decompression with improved neurological outcomes. Brain biopsy was suggestive of inflammation from thrombosed vessel, and neutrophilic infiltration of cerebellar tissue.

Provision of rapid and specific ex vivo diagnosis of central nervous system lymphoma from rodent xenograft biopsies by a fluorescent aptamer.

OBJECTIVE: Differentiating central nervous system (CNS) lymphoma from other intracranial malignancies remains a clinical challenge in surgical neuro-oncology. Advances in clinical fluorescence imaging contrast agents and devices may mitigate this challenge. Aptamers are a class of nanomolecules engineered to bind cellular targets with antibody-like specificity in a fraction of the staining time. Here, the authors determine if immediate ex vivo fluorescence imaging with a lymphoma-specific aptamer can rapidly and specifically diagnose xenografted orthotopic human CNS lymphoma at the time of biopsy. METHODS: The authors synthesized a fluorescent CNS lymphoma-specific aptamer by conjugating a lymphoma-specific aptamer with Alexa Fluor 488 (TD05-488). They modified human U251 glioma cells and Ramos lymphoma cells with a lentivirus for constitutive expression of red fluorescent protein and implanted them intracranially into athymic nude mice. Three to 4 weeks postimplantation, acute slices (biopsies, n = 28) from the xenografts were collected, placed in aptamer solution, and imaged with a Zeiss fluorescence microscope. Three aptamer staining concentrations (0.3, 1.0, and 3.0 µM) and three staining times (5, 10, and 20 minutes) followed by a 1-minute wash were tested. A file of randomly selected images was distributed to neurosurgeons and neuropathologists, and their ability to distinguish CNS lymphoma from negative controls was assessed. RESULTS: The three staining times and concentrations of TD05-488 were tested to determine the diagnostic accuracy of CNS lymphoma within a frozen section time frame. An 11-minute staining protocol with 1.0-µM TD05-488 was most efficient, labeling 77% of positive control lymphoma cells and less than 1% of negative control glioma cells (p < 0.001). This protocol permitted clinicians to positively identify all positive control lymphoma images without misdiagnosing negative control images from astrocytoma and normal brain. CONCLUSIONS: Ex vivo fluorescence imaging is an emerging technique for generating rapid histopathological diagnoses. Ex vivo imaging with a novel aptamer-based fluorescent nanomolecule could provide an intraoperative tumor-specific diagnosis of CNS lymphoma within 11 minutes of biopsy. Neurosurgeons and neuropathologists interpreted images generated with this molecular probe with high sensitivity and specificity. Clinical application of TD05-488 may permit specific intraoperative diagnosis of CNS lymphoma in a fraction of the time required for antibody staining.

Evaluation of Diagnostic Accuracy Following the Coadministration of Delta-Aminolevulinic Acid and Second Window Indocyanine Green in Rodent and Human Glioblastomas.

PURPOSE: Fluorescence-guided-surgery offers intraoperative visualization of neoplastic tissue. Delta-aminolevulinic acid (5-ALA), which targets enzymatic abnormality in neoplastic cells, is the only approved agent for fluorescence-guided neurosurgery. More recently, we described Second Window Indocyanine Green (SWIG) which targets neoplastic tissue through enhanced vascular permeability. We hypothesized that SWIG would demonstrate similar clinical utility in identification of high-grade gliomas compared with 5-ALA. PROCEDURES: Female C57/BL6 and nude/athymic mice underwent intracranial implantation of 300,000 GL261 and U87 cells, respectively. Tumor-bearing mice were euthanized after administration of 5-ALA (200 mg/kg intraperitoneal) and SWIG (5 mg/kg intravenous). Brain sections were imaged for protoporphyrin-IX and ICG fluorescence. Fluorescence and H&E images were registered using semi-automatic scripts for analysis. Human subjects with HGG were administered SWIG (2.5 mg/kg intravenous) and 5-ALA (20 mg/kg oral). Intraoperatively, tumors were imaged for ICG and protoporphyrin-IX fluorescence. RESULTS: In non-necrotic tumors, 5-ALA and SWIG demonstrated 90.2 % and 89.2 % tumor accuracy (p value = 0.52) in U87 tumors and 88.1 % and 87.7 % accuracy (p value = 0.83) in GL261 tumors. The most distinct difference between 5-ALA and SWIG distribution was seen in areas of tumor-associated necrosis, which often showed weak/no protoporphyrin-IX fluorescence, but strong SWIG fluorescence. In twenty biopsy specimens from four subjects with HGG, SWIG demonstrated 100 % accuracy, while 5-ALA demonstrated 75-85 % accuracy; there was 90 % concordance between SWIG and 5-ALA fluorescence. CONCLUSION: Our results provide the first direct comparison of the diagnostic utility of SWIG vs 5-ALA in both rodent and human HGG. Given the broader clinical utility of SWIG compared with 5-ALA, our data supports the use of SWIG in tumor surgery to improve the extent of safe resections. CLINICAL TRIAL: NCT02710240 (US National Library of Medicine Registry; https://www.clinicaltrials.gov/ct2/show/NCT02710240?id=NCT02710240&draw=2&rank=1 ).

Delta-Aminolevulinic Acid-Mediated Photodiagnoses in Surgical Oncology: A Historical Review of Clinical Trials.

Fluorescence imaging is an emerging clinical technique for real-time intraoperative visualization of tumors and their boundaries. Though multiple fluorescent contrast agents are available in the basic sciences, few fluorescence agents are available for clinical use. Of the clinical fluorophores, delta aminolevulinic acid (5ALA) is unique for generating visible wavelength tumor-specific fluorescence. In 2017, 5ALA was FDA-approved for glioma surgery in the United States. Additionally, clinical studies suggest this agent may have utility in surgical subspecialties outside of neurosurgery. Data from dermatology, OB/GYN, urology, cardiothoracic surgery, and gastrointestinal surgery show 5ALA is helpful for intraoperative visualization of malignant tissues in multiple organ systems. This review summarizes data from English-language 5ALA clinical trials across surgical subspecialties. Imaging systems, routes of administration, dosing, efficacy, and related side effects are reviewed. We found that modified surgical microscopes and endoscopes are the preferred imaging devices. Systemic dosing across surgical specialties range between 5 and 30 mg/kg bodyweight. Multiple studies discussed potential for skin irritation with sun exposure, however this side effect is infrequently reported. Overall, 5ALA has shown high sensitivity for labeling malignant tissues and providing a means to visualize malignant tissue not apparent with standard operative light sources.

Pediatric spinal cord biopsy: A case series from a high-volume referral center.

Pediatric patients presenting with intramedullary spinal cord lesions often require specific diagnoses to guide their treatment plans. Though results from magnetic resonance imaging and lumbar puncture may narrow the differential diagnosis, these tests cannot always provide a definitive diagnosis. In such cases, spinal cord biopsy may be undertaken to provide a specific histopathologic diagnosis for guiding treatment. Data from the adult population show 24% of spinal cord biopsies can be nondiagnostic and the procedure may carry a 21% complication rate. Therefore, spinal cord biopsy may portend a similar high risk-to-benefit ratio in the pediatric population. Here, we review spinal cord biopsy cases scheduled for diagnosis, and not debulking, at a high volume pediatric referral center during a seventeen-year period. We report our experience with five patients who met our inclusion criteria. Due to the rarity of the procedure, statistically significant factors associated with improved diagnostic yield or peri-operative complication could not be identified. A definitive diagnosis which guided the post-operative treatment plan was obtained in four of our five patients. None of our patients developed post-operative motor deficits. However, these patients were susceptible to the same risks of open spine surgery, such as wound infections and spinal deformities. Our case series shows that intramedullary spinal cord biopsies may provide tissue for obtaining histopatholgic diagnoses. However, the potential risks of complication, and the possibility of obtaining nondiagnostic tissue, should be discussed with patients, families and their medical treatment teams.

Neurosurgical Flexible Probe Microscopy with Enhanced Architectural and Cytological Detail.

BACKGROUND: Microscopic delineation and clearance of tumor cells at neurosurgical excision margins potentially reduce tumor recurrence and increase patient survival. Probe-based in vivo fluorescence microscopy technologies are promising for neurosurgical in vivo microscopy. OBJECTIVE: We sought to demonstrate a flexible fiberoptic epifluorescence microscope capable of enhanced architectural and cytological imaging for in vivo microscopy during neurosurgical procedures. METHODS: Eighteen specimens were procured from neurosurgical procedures. These specimens were stained with acridine orange and imaged with a 3-dimensional (3D)-printed epifluorescent microscope that incorporates a flexible fiberoptic probe. Still images and video sequence frames were processed using frame alignment, signal projection, and pseudo-coloring, resulting in resolution enhancement and an increased field of view. RESULTS: Images produced displayed good nuclear contrast and architectural detail. Grade 1 meningiomas demonstrated 3D chords and whorls. Low-grade meningothelial nuclei showed streaming and displayed regularity in size, shape, and distribution. Oligodendrogliomas showed regular round nuclei and a variably staining background. Glioblastomas showed high degrees of nuclear pleomorphism and disarray. Mitoses, vascular proliferation, and necrosis were evident. CONCLUSIONS: We demonstrate the utility of a 3D-printed, flexible probe microscope for high-resolution microscopic imaging with increased architectural detail. Enhanced in vivo imaging using this device may improve our ability to detect and decrease microscopic tumor burden at excision margins during neurosurgical procedures.

Epithelioid glioblastoma presenting as aphasia in a young adult with ovarian cancer: A case report.

Our patient's clinical history and preoperative radiographic evaluation suggested central nervous system (CNS) metastatic disease. Ultimately, final pathology revealed epithelioid glioblastoma (eGBM), a newly classified CNS primary tumor. This reinforces the importance of direct tissue sampling and including eGBM on the differential for young patients with histories of systemic cancer presenting with new CNS lesions.

Fluorescence-guided stereotactic biopsy: a proof-of-concept study.

OBJECTIVEAccurate histopathological diagnoses are often necessary for treating neuro-oncology patients. However, stereotactic biopsy (STB), a common method for obtaining suspicious tissue from deep or eloquent brain regions, fails to yield diagnostic tissue in some cases. Failure to obtain diagnostic tissue can delay initiation of treatment and may result in further invasive procedures for patients. In this study, the authors sought to determine if the coupling of in vivo optical imaging with an STB system is an effective method for identification of diagnostic tissue at the time of biopsy.METHODSA minimally invasive fiber optic imaging system was developed by coupling a 0.65-mm-diameter coherent fiber optic fluorescence microendoscope to an STB system. Human U251 glioma cells were transduced for stable expression of blue fluorescent protein (BFP) to produce U251-BFP cells that were utilized for in vitro and in vivo experiments. In vitro, blue fluorescence was confirmed, and tumor cell delineation by fluorescein sodium (FNa) was quantified with fluorescence microscopy. In vivo, transgenic athymic rats implanted with U251-BFP cells (n = 4) were utilized for experiments. Five weeks postimplantation, the rats received 5-10 mg/kg intravenous FNa and underwent craniotomies overlying the tumor implantation site and contralateral normal brain. A clinical STB needle containing our 0.65-mm imaging fiber was passed through each craniotomy and images were collected. Fluorescence images from regions of interest ipsilateral and contralateral to tumor implantation were obtained and quantified.RESULTSLive-cell fluorescence imaging confirmed blue fluorescence from transduced tumor cells and revealed a strong correlation between tumor cells quantified by blue fluorescence and FNa contrast (R2 = 0.91, p < 0.001). Normalized to background, in vivo FNa-mediated fluorescence intensity was significantly greater from tumor regions, verified by blue fluorescence, compared to contralateral brain in all animals (301.7 ± 34.18 relative fluorescence units, p < 0.001). Fluorescence intensity measured from the tumor margin was not significantly greater than that from normal brain (p = 0.89). Biopsies obtained from regions of strong fluorescein contrast were histologically consistent with tumor.CONCLUSIONSThe authors found that in vivo fluorescence imaging with an STB needle containing a submillimeter-diameter fiber optic fluorescence microendoscope provided direct visualization of neoplastic tissue in an animal brain tumor model prior to biopsy. These results were confirmed in vivo with positive control cells and by post hoc histological assessment. In vivo fluorescence guidance may improve the diagnostic yield of stereotactic biopsies.

Confocal scanning microscopy provides rapid, detailed intraoperative histological assessment of brain neoplasms: Experience with 106 cases.

OBJECTIVES: Frozen section histological analysis is currently the mainstay for intraprocedural tissue diagnosis during the resection of intracranial neoplasms and for evaluating tumor margins. However, frozen sections are time-consuming and often do not reveal the histological features needed for final diagnosis when compared with permanent sections. Confocal scanning microscopy (CSM) with certain stains may be a valuable technology that can add rapid and detailed histological assessment advantage for the neurosurgical operating room. This study describes potential advantages of CSM imaging of fresh human brain tumor tissues labeled with acriflavine (AF), acridine orange (AO), cresyl violet (CV), methylene blue (MB), and indocyanine green (ICG) within the neurosurgical operating room facility. PATIENTS AND METHODS: Acute slices from orthotopic human intracranial neoplasms were incubated with AF/AO and CV solutions for 10 s and 1 min respectively. Staining was also attempted with MB and ICG. Samples were imaged using a bench-top CSM system. Histopathologic features of corresponding CSM and permanent hematoxylin and eosin images were reviewed for each case. RESULTS: Of 106 cases, 30 were meningiomas, 19 gliomas, 13 pituitary adenomas, 9 metastases, 6 schwannomas, 4 ependymomas, and 25 other pathologies. CSM using rapid fluorophores (AF, AO, CV) revealed striking microvascular, cellular and subcellular structures that correlated with conventional histology. By rapidly staining and optically sectioning freshly resected tissue, images were generated for intraoperative consultations in less than one minute. With this technique, an entire resected tissue sample was imaged and digitally stored for tele-pathology and archiving. CONCLUSION: CSM of fresh human brain tumor tissue provides clinically meaningful and rapid histopathological assessment much faster than frozen section. With appropriate stains, including specific cellular structure or antibody staining, CSM could improve the timeliness of intraoperative decision-making, and the neurosurgical-pathology workflow during resection of human brain tumors, ultimately improving patient care.

Acridine Orange as a Novel Photosensitizer for Photodynamic Therapy in Glioblastoma.

BACKGROUND: Photodynamic therapy combines the effects of a chemical agent with the physical energy from light or radiation to result in lysis of cells. Acridine orange (AO) is a molecule with fluorescence properties that has been demonstrated to possess photosensitizing properties. The objective of this study was to investigate the photodynamic effect of AO on glioblastoma cell viability and growth. METHODS: Glioblastoma cells (N = 8000 cells/well at 0 hours) were exposed to AO followed by white unfiltered light-emitting diode light. Cultures were exposed to either 10 or 30 minutes of light. The cell number per well was determined at 0, 24, 48, and 72 hours after exposure. RESULTS: A dramatic cytocidal effect of AO after exposure to 10 minutes of white light was observed. There was almost complete eradication of glioblastoma cells over a 72-hour period. Although AO or light alone exhibited some effect on cell growth, it was not as pronounced as the combination of AO and light. CONCLUSIONS: This is the first study to our knowledge to demonstrate the photodynamic effect of AO in glioblastoma cells. These data support the need for further studies to characterize and evaluate whether this striking cytotoxic effect can be achieved in vivo. The combination of AO and exposure to white unfiltered light-emitting diode light may have potential future applications in management of glioblastoma.

In Vivo Microscopy in Neurosurgical Oncology.

Intraoperative neurosurgical histopathologic diagnoses rely on evaluation of rapid tissue preparations such as frozen sections and smears with conventional light microscopy. Although useful, these techniques are time consuming and therefore cannot provide real-time intraoperative feedback. In vivo molecular imaging techniques are emerging as novel methods for generating real-time diagnostic histopathologic images of tumors and their surrounding tissues. These imaging techniques rely on contrast generated by exogenous fluorescent dyes, autofluorescence of endogenous molecules, fluorescence decay of excited molecules, or light scattering. Large molecular imaging instruments are being miniaturized for clinical in vivo use. This review discusses pertinent imaging systems that have been developed for neurosurgical use and imaging techniques currently under development for neurosurgical molecular imaging.

Immediate ex-vivo diagnosis of pituitary adenomas using confocal reflectance microscopy: a proof-of-principle study.

OBJECTIVE The objective of this study was to evaluate the feasibility of using confocal reflectance microscopy (CRM) ex vivo to differentiate adenoma from normal pituitary gland in surgical biopsy specimens. CRM allows for rapid, label-free evaluation of biopsy specimens with cellular resolution while avoiding some limitations of frozen section analysis. METHODS Biopsy specimens from 11 patients with suspected pituitary adenomas were transported directly to the pathology department. Samples were immediately positioned and visualized with CRM using a confocal microscope located in the same area of the pathology department where frozen sections are prepared. An H & E-stained slide was subsequently prepared from imaged tissue. A neuropathologist compared the histopathological characteristics of the H & E-stained slide and the matched CRM images. A second neuropathologist reviewed images in a blinded fashion and assigned diagnoses of adenoma or normal gland. RESULTS For all specimens, CRM contrasted cellularity, tissue architecture, nuclear pleomorphism, vascularity, and stroma. Pituitary adenomas demonstrated sheets and large lobules of cells, similar to the matched H & E-stained slides. CRM images of normal tissue showed scattered small lobules of pituitary epithelial cells, consistent with matched H & E-stained images of normal gland. Blinded review by a neuropathologist confirmed the diagnosis in 15 (94%) of 16 images of adenoma versus normal gland. CONCLUSIONS CRM is a simple, reliable approach for rapidly evaluating pituitary adenoma specimens ex vivo. This technique can be used to accurately differentiate between pituitary adenoma and normal gland while preserving biopsy tissue for future permanent analysis, immunohistochemical studies, and molecular studies.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.