Anal dysplasia screening and human papillomavirus characterization by Raman-enhanced spectroscopy in individuals infected with human immunodeficiency virus

Field: Medicine (Health Sciences)

Background: People living with human immunodeficiency virus (PLHIV) are at increased risk for developing anal cancer and anal dysplasia due to persistent infection by high-risk human papillomavirus (HPV). It is recommended they undergo anal cancer screening with anal cytology and, if abnormal cytology is reported, a more invasive high-resolution anoscopy (HRA) to obtain biopsies for definitive diagnosis. However, HRA and biopsy are invasive, and variable sensitivity and specificity have been reported for both anal cytology and HRA with biopsy. Raman-enhanced spectroscopy (RESpect), a noninvasive laser-based technology that characterizes the chemical and molecular composition of cells and tissues, has demonstrated the potential to improve current standards of anal cancer screening. The feasibility of translating RESpect clinically relies on consistent Raman-based characterization of dysplastic anal tissues and a positive control for squamous epithelial carcinoma. By comparing the spectral profiles of anal tissues biopsied from PLHIV and cervical carcinoma cell lines acquired by RESpect microscope (micro-Raman) and portable RESpect probe instruments, this study evaluates the clinical potential for RESpect probe screening as a real-time, noninvasive supplemental tool in detecting anal dysplasia. Methods: Participants being evaluated for anal cancer screening at the Hawai‘i Center for AIDS clinic were recruited following guidelines approved by the University of Hawai‘i Institutional Review Board. Anal cytology was followed by HRA and biopsy to obtain two tissue samples from the same location: one specimen for RESpect analysis and one specimen for pathology assessment. The tissues for RESpect analysis were cut into two before mounting onto reflective aluminum slides for spectra acquisition by the micro-Raman and RESpect probe instruments. HeLa and SiHa cervical carcinoma cell lines were similarly mounted and analyzed by micro-Raman and RESpect probe. Following baseline correction and smoothing, spectra were averaged to produce a representative spectrum of each cell or tissue specimen and normalized. Averaged spectra representing negative, low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL) pathologies were visually inspected to identify significant differences in intensity, as determined by a two-sample t-test. To determine the feasibility of using HeLa and SiHa cell lines as positive controls of HPV-infected squamous epithelial carcinoma, peak differences identified by visual inspection of HeLa and SiHa spectra were compared to the peaks distinguished in anal tissues. Results: Forty-three participants were enrolled to date. Overall agreement between cytology and biopsy results was 71%, with Cohen’s kappa (κ) = 0.484 (95% CI: 0.266–0.741) indicating moderate agreement. Comparison across pathologies (26 negative, 12 LSIL, and 5 HSIL) revealed spectral differences. Based on visual observation, LSIL and HSIL tissues demonstrated averaged spectra closer to each other than to negative tissue. Significant peak differences (p < 0.2) between tissue pathologies were found at three regions in both the micro-Raman and RESpect probe spectra. Between cervical carcinoma cell lines, SiHa cells generally exhibited a higher intensity than HeLa cells, and peak differences were observed at six spectral regions, with a consistent peak difference at 1554 cm–1 corresponding to amide II distinguished in both cells and tissues. Conclusion: Peak differences elucidated from micro-Raman and RESpect probe spectral fingerprints of anal tissues and cervical carcinoma cell lines demonstrate the potential of RESpect to distinguish between pathology grades and support the integration of RESpect technology into the anal cancer screening paradigm. Previous studies have demonstrated similar differences between normal and dysplastic/cancerous cervical tissues at peaks associated with amide as well as similar agreement between cytology and biopsy results. These findings lay the foundation to consider RESpect as a real-time, noninvasive screening tool for further exploration.