Clinical feasibility of miniaturized Lissajous scanning confocal laser endomicroscopy for indocyanine green-enhanced brain tumor diagnosis.

Abstract

Intraoperative real-time confocal laser endomicroscopy (CLE) is an alternative modality for frozen tissue histology that enables visualization of the cytoarchitecture of living tissues with spatial resolution at the cellular level. We developed a new CLE with a "Lissajous scanning pattern" and conducted a study to identify its feasibility for fluorescence-guided brain tumor diagnosis. Conventional hematoxylin and eosin (H&E) histological images were compared with indocyanine green (ICG)-enhanced CLE images in two settings (1): experimental study with in vitro tumor cells and ex vivo glial tumors of mice, and (2) clinical evaluation with surgically resected human brain tumors. First, CLE images were obtained from cultured U87 and GL261 glioma cells. Then, U87 and GL261 tumor cells were implanted into the mouse brain, and H&E staining was compared with CLE images of normal and tumor tissues ex vivo. To determine the invasion of the normal brain, two types of patient-derived glioma cells (CSC2 and X01) were used for orthotopic intracranial tumor formation and compared using two methods (CLE vs. H&E staining). Second, in human brain tumors, tissue specimens from 69 patients were prospectively obtained after elective surgical resection and were also compared using two methods, namely, CLE and H&E staining. The comparison was performed by an experienced neuropathologist. When ICG was incubated in vitro, U87 and GL261 cell morphologies were well-defined in the CLE images and depended on dimethyl sulfoxide. Ex vivo examination of xenograft glioma tissues revealed dense and heterogeneous glioma cell cores and peritumoral necrosis using both methods. CLE images also detected invasive tumor cell clusters in the normal brain of the patient-derived glioma xenograft model, which corresponded to H&E staining. In human tissue specimens, CLE images effectively visualized the cytoarchitecture of the normal brain and tumors. In addition, pathognomonic microstructures according to tumor subtype were also clearly observed. Interestingly, in gliomas, the cellularity of the tumor and the density of streak-like patterns were significantly associated with tumor grade in the CLE images. Finally, panoramic view reconstruction was successfully conducted for visualizing a gross tissue morphology. In conclusion, the newly developed CLE with Lissajous laser scanning can be a helpful intraoperative device for the diagnosis, detection of tumor-free margins, and maximal safe resection of brain tumors.