Over the last three decades, our understanding of the molecular changes associated with cancer development and progression has advanced greatly. This has led to new cancer therapeutics targeted against specific molecular pathways; such therapies show great promise to reduce mortality, in part by enabling physicians to tailor therapy for patients based on a molecular profile of their tumor. Unfortunately, the tools for definitive cancer diagnosis – light microscopic examination of biopsied tissue stained with nonspecific dyes – remain focused on the analysis of tissue ex vivo. There is an important need for new clinical tools to support the molecular diagnosis of cancer. Optical molecular imaging is emerging as a technique to help meet this need. Targeted, optically active contrast agents can specifically label extra-and intracellular biomarkers of cancer. Optical images can be acquired in real time with high spatial resolution to image-specific molecular targets, while still providing morphologic context. This article reviews recent advances in optical molecular imaging, highlighting the advances in technology required to improve early cancer detection, guide selection of targeted therapy and rapidly evaluate therapeutic efficacy.

Abstract. In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

By mimicking the variable resolution of the human eye, a newly designed foveated endomicroscopic objective shows the potential to improve current endoscopic based techniques of identifying abnormal tissue in the esophagus and colon. The prototype miniature foveated objective is imaged with a confocal microscope to provide large field of view images combined with a high resolution central region to rapidly observe morphological structures associated with cancer development in a mouse model.

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Multiplex immunofluorescence (mIF) has become an established method for characterizing the tumor immune microenvironment. Currently, hematoxylin and eosin (H&E) staining is typically performed on a serial tissue section to provide morphologic context for the mIF data. Serial sections do not represent identical cell populations, however, and are not always possible to obtain when the amount of available tissue is limited. Consequently, a method to examine the same tissue section by both fluorescence and brightfield imaging would be advantageous. Here we present a streamlined method and system for serial mIF and H&E staining on a single tissue slide for a comprehensive analysis to support high-throughput tissue immunophenotyping. The UltiMapper I/O PD-L1 kit was used for multiplex immunofluorescence staining of CD8, CD68, PD-L1, and pan-Cytokeratin in formalin-fixed, paraffin-embedded (FFPE) samples from human tonsil and primary tumor biopsies using the Leica Biosystems BOND RX autostainer. Stained tissues were imaged in five spectrally distinct fluorescence channels (DAPI, FITC, TRITC, Cy5, Cy7) on the RareCyte CyteFinder II HT Instrument. Slides were de-coverslipped and stained with H&E, then imaged with brightfield using the CyteFinder II HT instrument. To segment the tumor and stroma tissue regions, a HALO AI classifier was created. Fluorescence images were analyzed using the HALO Highplex FL module to identify CD8+ cytotoxic T-cells, CD68+ macrophages, CD68+/PD-L1+ immuno-suppressive macrophages, pan-CK+ tumor cells, and pan-CK+/PD-L1+ immune-evading tumor cells within the tumor and stromal regions identified by the H&E stain. This novel combination of fluorescence and brightfield analysis enables deep phenotyping of immune cells as well as assessment of cellular morphology and tumor architecture in the same tissue section. Citation Format: Katir K. Patel, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Kate Lillard, Anne Hellebust, Melinda Duplessis, Kyla Teplitz, Tad George. Sequential same slide multiplex immunofluorescence and H&E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2677.