In vivo dual-axis confocal microscopy of 5-ALA-induced PpIX to guide low-grade glioma resections

摘要 切除范围与胶质瘤患者的预后相关，如无进展生存期。图像- 基于核磁共振和现在的荧光引导手术(FGS)的引导技术已经发展到 提高外科医生显示大体肿瘤边缘的能力。然而，有基本的限制。 广域成像方法，如MRI和FGS，如检测播散性肿瘤细胞的灵敏度较低 弥漫性胶质瘤的浸润性边缘，以及图像的非定量和主观性 释义。随着使用5-ALA的FGS的出现，以及它最近于2017年获得FDA的批准，这一差距 在低级别胶质瘤(LGG)和高级别胶质瘤(HGG)患者之间，就切除程度而言， 可能会变宽，因为LGG很少产生足够的PPIX荧光，通过广场FGS进行检测。 因此，显然有必要改进术中技术，使其具有检测和 量化肿瘤空洞边缘残留LGG以提高切除和延迟的程度 复发。我们已经证明，高分辨率共聚焦显微镜具有观察稀疏物体的灵敏度 在接受5-ALA治疗的LGG患者中，PpIX的亚细胞表达甚至超出了放射学边缘。 因此，我们将优化手持光学切片显微镜来成像5-ALA诱导的PPIX LGG患者的切除边缘，结合实时视频马赛克，以促进大的 组织区域，这将最大限度地减少成像异质脑组织时的采样偏差(目标1)。为了 促进临床对这些技术的接受，我们将建立显微之间的关系 PpIX的表达模式和公认的生物学指标，如肿瘤负荷和增殖指数 (目标2)。最后，我们将探讨PPIX的定量显微成像的假说 切缘可以预测手术切除的范围，如目前术后MRI所定义的，这将提示 它对于优化切除以最大限度地减少和/或延迟复发具有价值(目标3)。总而言之，这些结果 将为未来优化切除程序和结果的随机对照临床研究铺平道路 对于LGG患者(成人和儿童)，他们中的许多人可以有良好的生存结果和生活质量。

Summary Extent-of-resection is correlated with glioma patient outcomes such as progression-free survival. Image- guidance technologies, based on MRI and now fluorescence-guided surgery (FGS), have been developed to improve the surgeon’s ability to visualize gross tumor margins. However, there are fundamental limitations to wide-field imaging methods such as MRI and FGS, such as poor sensitivity to detect disseminated tumor cells at the infiltrative margins of diffuse gliomas, as well as the non-quantitative and subjective nature of image interpretation. With the emergence of FGS using 5-ALA, and its recent approval by the FDA in 2017, the gap between low-grade glioma (LGG) and high-grade glioma (HGG) patients, in terms of extent of resection, will likely widen since LGGs rarely generate sufficient PpIX fluorescence to be detected via wide-field FGS. Consequently, there is a clear need for improved intraoperative techniques with the sensitivity to detect and quantify residual LGGs at the margins of the tumor cavity in order to improve the extent of resection and delay recurrence. We have shown that high-resolution confocal microscopy has the sensitivity to visualize the sparse sub-cellular expression of PpIX in LGG patients treated with 5-ALA, even beyond the radiographic margins. Therefore, we will optimize a handheld optical-sectioning microscope to image 5-ALA-induced PpIX at the final resection margins in LGG patients, together with real-time video mosaicking to facilitate the imaging of large tissue areas, which will minimize sampling bias when imaging heterogeneous brain tissues (Aim 1). In order to facilitate the clinical acceptance of these techniques, we will establish a relationship between the microscopic patterns of PpIX expression and well-established biological metrics such as tumor burden and proliferative index (Aim 2). Finally, we will explore the hypothesis that quantitative microscopic imaging of PpIX of the resection margins is predictive of extent of resection, as currently defined by post-operative MRI, which would suggest that it has value for optimizing resections to minimize and/or delay recurrence (Aim 3). Collectively, these results will pave the way for future randomized controlled clinical studies to optimize resection procedures and outcomes for LGG patients (adults and children), many of whom can have good survival outcomes and quality of life.