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Raman-enhanced spectroscopy (RESpect) probe, which enhances Raman spectroscopy technology through a portable fiber-optic device, characterizes tissues and cells by identifying molecular chemical composition showing distinct differences/similarities for potential tumor markers or diagnosis. In a feasibility study with the ultimate objective to translate the technology to the clinic, a panel of pediatric non-Hodgkin lymphoma tissues and non-malignant specimens had RS analyses compared between standard Raman spectroscopy microscope instrument and RESpect probe. Cryopreserved tissues were mounted on front-coated aluminum mirror slides and analyzed by standard Raman spectroscopy and RESpect probe. Principal Component Analysis revealed similarities between non-Hodgkin lymphoma subtypes but not follicular hyperplasia. Standard Raman spectroscopy and RESpect probe fingerprint comparisons demonstrated comparable primary peaks. Raman spectroscopic fingerprints and peaks of pediatric non-Hodgkin lymphoma subtypes and follicular hyperplasia provided novel avenues to pursue diagnostic approaches and identify potential new therapeutic targets. The information could inform new insights into molecular cellular pathogenesis. Translating Raman spectroscopy technology by using the RESpect probe as a potential point-of-care screening instrument has the potential to change the paradigm of screening for cancer as an initial step to determine when a definitive tissue biopsy would be necessary.

HIV-positive individuals are at increased risk for precancerous anal squamous intraepithelial lesions (SILs). Anal cytology and digital rectal examination are performed as screening tools, but extensive training and appropriate instruments are required to follow up on an abnormal anal cytology. Thus, novel approaches to SIL evaluation could improve better health care follow-up by efficient and timely diagnosis to offer treatment options. Recently, Raman-enhanced spectroscopy (RESpect) has emerged as a potential new tool for early identification of SIL. RESpect is a noninvasive, label-free, laser-based technique that identifies molecular composition of tissues and cells. HIV-serodiscordant couples had anal biopsies obtained during high-resolution anoscopy. RESpect was performed on the specimens. Principal component analysis of the data identified differences between normal and abnormal tissue as well as HIV-positive and HIV-negative individuals of each couple even with similar pathologies. RESpect has the potential to change the paradigm of anal pathology diagnosis and could provide insight into different pathways leading to SIL in HIV-serodiscordant couples.

Purpose: Raman spectroscopy (RS) is a noninvasive laser-based technique that provides a very high level of confidence in detecting various molecules through their vibrational modes. A monochromatic laser is used to excite a sample, and inelastic scattered light generated by the sample is used to measure Raman spectra of the sample. Because vibration modes of a molecule are very unique to the molecule, RS is sensitive and highly selective to the molecular composition of various chemicals including biologic tissues and cells. By using micro-Raman systems capable of measuring Raman spectra of single biologic cell, the objective of the study focused on identifying molecular differences between normal B-cells and Burkitt non-Hodgkin lymphoma (BNHL) cells, hypothesizing that unique RS fingerprints of BL could be identified with the overall goal to apply the information for novel diagnostic or treatment strategies. Methods: Normal B-cells were isolated from peripheral blood using a negative selection Robosep kit (EasySep Human B-Cell Isolation Kit, Stemcell Technologies, Cambridge, MA). BNHL cell lines (Ramos and CA46, ATCC, Manassas, VA), were cultured, washed, and resuspended in a 0.9% saline solution at a concentration of 1 x 106 cells/mL. The cells were mounted on a clean aluminum reflective slide and single cells were analyzed with a micro-Raman system utilizing a 785-nm laser excitation and an automated xyz microscope stage. Asymmetric Least Squares (AsLS) was applied to the spectral data (n=20 spectra per cell type) to obtain baseline spectra. Principal Component Analysis (PCA) followed by k-nearest neighboring (KNN) method was applied to analyze the variance in the spectral data and assess the diagnostic accuracy of the loadings from PCA. Results: The Raman fingerprints of normal B-cells and BNHL cells identified characteristic peak differences between the cell types, ranging from DNA/protein concentrations to saccharide bonds. These were further analyzed using AsLS and PCA. AsLS informed the PCA, which identified variances in the peaks that formed distinct clusters to differentiate normal B-cells from BL NHL cells. PCA effectively compiles the peaks, demonstrating that the first three components accounted for 97.45% of variance in the data. Several peaks were identified as the major contributors to the loadings, and some (e.g., 831, 1083, 1210, 1257, 1360, 1578, 1658 cm-1) were also reported as biomarkers of BNHL. The KNN on the PCs revealed that diagnostic accuracy was 100% and specificity was 100%. Conclusions: Raman fingerprints of normal B-cells versus BNHL cells demonstrated unique clustering of molecular changes that could potentially distinguish neoplastic cells from normal cells. As a novel and innovative tool, RS could contribute towards diagnostic resources and new paradigms for intervention for BNHL and other diseases. This study was supported by the St. Baldrick’s and McCabe Foundations and in part by NIH U54MD007584. Citation Format: Natalie Kamada, Robert Oda, Tiffany Shieh, Melissa Agsalda-Garcia, Tayro Acosta-Maeda, Anupam Misra, So Yung Choi, Eunjung Lim, Bruce Shiramizu. Unique Raman spectroscopic fingerprints of Burkitt non-Hodgkin lymphoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A04.

Background Anal dysplasia is a potentially chronic disease that affects HIV-seropositive and-seronegative men who have sex with men (MSM) and transgender women. Novel approaches to AIN screening could improve healthcare through access to timely care and treatment since appropriate training and equipment are currently required for screening and follow-up. To pioneer new ideas, the foundation for new technology relies on collecting data to design new tools. Recently, Raman spectroscopy (RESpect), a laser-based technology, has identified unique anal tissue fingerprints. We assessed anal tissue for RESpect phenotypes for differences in HIV-serodiscordant couples. Methods HIV-serodiscordant couples were enrolled in a clinical study to assess anal biopsy specimens as per IRB guidelines. Anal tissue was flash-frozen and mounted onto aluminum reflective slides which were then subjected to RESpect point scans accumulations. RESpect information was processed using asymmetric least squares to baseline the data and subjected to principal component analysis (PCA). Results To date, data from 2 couples showed that PCA distinguishes between HIV+ and HIV- individuals of the couples. In HIV+ individuals, PCA also distinguishes AIN from normal tissue. Conclusions RESpect has shown to identify not only AIN amongst all individuals but also suggested there may be a unique HIV effect in the RESpect data from anal tissue. Further work on RESpect could provide groundbreaking information towards the design of a RESpect monitoring instrument to diagnose and follow patients for AIN. Supported in part by R21CA216830 and U54MD007584.