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  • By Author: Preul, Mark C. x
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Robert Lynagh, Mark Ishak, Joseph Georges, Danielle Lopez, Hany Osman, Michael Kakareka, Brandon Boyer, H. Warren Goldman, Jennifer Eschbacher, Mark C. Preul, Peter Nakaji, Alan Turtz, Steven Yocom and Denah Appelt

OBJECTIVE

Accurate 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.

METHODS

A 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.

RESULTS

Live-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.

CONCLUSIONS

The 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.

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Michael A. Mooney, Joseph Georges, Mohammedhassan Izady Yazdanabadi, Katherine Y. Goehring, William L. White, Andrew S. Little, Mark C. Preul, Stephen W. Coons, Peter Nakaji and Jennifer M. Eschbacher

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.

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Joseph Georges, Aqib Zehri, Elizabeth Carlson, Joshua Nichols, Michael A. Mooney, Nikolay L. Martirosyan, Layla Ghaffari, M. Yashar S. Kalani, Jennifer Eschbacher, Burt Feuerstein, Trent Anderson, Mark C. Preul, Kendall Van Keuren-Jensen and Peter Nakaji

Glioblastoma is the most common primary brain tumor with a median 12- to 15-month patient survival. Improving patient survival involves better understanding the biological mechanisms of glioblastoma tumorigenesis and seeking targeted molecular therapies. Central to furthering these advances is the collection and storage of surgical biopsies (biobanking) for research. This paper addresses an imaging modality, confocal reflectance microscopy (CRM), for safely screening glioblastoma biopsy samples prior to biobanking to increase the quality of tissue provided for research and clinical trials. These data indicate that CRM can immediately identify cellularity of tissue biopsies from animal models of glioblastoma. When screening fresh human biopsy samples, CRM can differentiate a cellular glioblastoma biopsy from a necrotic biopsy without altering DNA, RNA, or protein expression of sampled tissue. These data illustrate CRM's potential for rapidly and safely screening clinical biopsy samples prior to biobanking, which demonstrates its potential as an effective screening technique that can improve the quality of tissue biobanked for patients with glioblastoma.

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Nikolay L. Martirosyan, Joseph Georges, Jennifer M. Eschbacher, Daniel D. Cavalcanti, Ali M. Elhadi, Mohammed G. Abdelwahab, Adrienne C. Scheck, Peter Nakaji, Robert F. Spetzler and Mark C. Preul

Object

The authors sought to assess the feasibility of a handheld visible-wavelength confocal endomicroscope imaging system (Optiscan 5.1, Optiscan Pty., Ltd.) using a variety of rapid-acting fluorophores to provide histological information on gliomas, tumor margins, and normal brain in animal models.

Methods

Mice (n = 25) implanted with GL261 cells were used to image fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA), acridine orange (AO), acriflavine (AF), and cresyl violet (CV). A U251 glioma xenograft model in rats (n = 5) was used to image sulforhodamine 101 (SR101). A swine (n = 3) model with AO was used to identify confocal features of normal brain. Images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope. Histological samples were acquired through biopsies from matched imaging areas. Samples were visualized with a benchtop confocal microscope. Histopathological features in corresponding confocal images and photomicrographs of H & E–stained tissues were reviewed.

Results

Fluorescence induced by FNa, 5-ALA, AO, AF, CV, and SR101 and detected with the confocal endomicroscope allowed interpretation of histological features. Confocal endomicroscopy revealed satellite tumor cells within peritumoral tissue, a definitive tumor border, and striking fluorescent cellular and subcellular structures. Fluorescence in various tumor regions correlated with standard histology and known tissue architecture. Characteristic features of different areas of normal brain were identified as well.

Conclusions

Confocal endomicroscopy provided rapid histological information precisely related to the site of microscopic imaging with imaging characteristics of cells related to the unique labeling features of the fluorophores. Although experimental with further clinical trial validation required, these data suggest that intraoperative confocal imaging can help to distinguish normal brain from tumor and tumor margin and may have application in improving intraoperative decisions during resection of brain tumors.