Diagnosis and monitoring of hepatocellular carcinoma in Hepatitis C virus patients using attenuated total reflection Fourier transform infrared spectroscopy

Abstract

Background: Current diagnostic methods for assessment of hepatitis C virus related hepatocellular carcinoma and subsequent categorization of hepatocellular carcinoma into non-angio-invasive hepatocellular carcinoma and angio-invasive hepatocellular carcinoma, to establish appropriate treatment strategies, are costly, invasive and requires multiple screening steps. This demands alternative diagnostic approaches that are cost-effective, time-efficient, and minimally invasive, while maintaining their efficacy for screening of hepatitis c virus related hepatocellular carcinoma. In this study, we propose that attenuated total reflection Fourier transform infrared in conjunction with principal component analysis - linear discriminant analysis and support vector machine multivariate algorithms holds a potential as a sensitive tool for the detection of hepatitis C virus-related hepatocellular carcinoma and the subsequent categorization of hepatocellular carcinoma into non-angio-invasive hepatocellular carcinoma and angio-invasive hepatocellular carcinoma.

Methods: Freeze-dried sera samples collected from 31 hepatitis c virus related hepatocellular carcinoma patients and 30 healthy individuals, were used to acquire mid-infrared absorbance spectra (3500-900 cm-¹) using attenuated total reflection Fourier transform infrared. Chemometric machine learning techniques were utilized to build principal component analysis - linear discriminant analysis and support vector machine discriminant models for the spectral data of hepatocellular carcinoma patients and healthy individuals. Sensitivity, specificity, and external validation on blind samples were calculated.

Results: Major variations were observed in the two spectral regions i.e., 3500-2800 and 1800-900 cm-¹. IR spectral signatures of hepatocellular carcinoma were reliably different from healthy individuals. Principal component analysis - linear discriminant analysis and support vector machine models computed 100% accuracy for diagnosing hepatocellular carcinoma. To classify the non-angio-invasive hepatocellular carcinoma/ angio-invasive hepatocellular carcinoma status, diagnostic accuracy of 86.21% was achieved for principal component analysis - linear discriminant analysis. While the support vector machine showed a training accuracy of 98.28% and a cross-validation accuracy of 82.75%. External validation for support vector machine based classification observed 100% sensitivity and specificity for accurately classifying the freeze-dried sera samples for all categories.

Conclusions: We present the specific spectral signatures for non-angio-invasive hepatocellular carcinoma and angio-invasive hepatocellular carcinoma, which were prominently differentiated from healthy individuals. This study provides an initial insight into the potential of attenuated total reflection Fourier transform infrared to diagnose hepatitis C virus related hepatocellular carcinoma but also to further categorize into non-angio-invasive and angio-invasive hepatocellular carcinoma.

Keywords: Attenuated total reflection Fourier transform infrared spectroscopy; Diagnostics; Hepatitis C virus; Hepatocellular carcinoma; Principal component analysis – linear discriminant analysis; Support vector machine.