Raman spectroscopy for the preoperative diagnosis of thyroid cancer and its subtypes: An in vitro proof-of-concept study.

OBJECTIVE: In 2016, there were an estimated 56 870 new cases of thyroid cancer (TC) in the USA. Fine needle aspiration cytology (FNAC) is the most safe, accurate and cost-effective method for the initial investigation of thyroid nodules. FNAC is limited by the inability to diagnose malignancy in follicular-patterned lesions accurately and, as a result, 20%-30% of cases under investigation for TC are classified as cytologically indeterminate, illustrating a problem with current FNAC procedure. Raman spectroscopy has shown promising results for the detection of many cancers; however, to date there has been no report on the performance of Raman spectroscopy on thyroid cytological samples. The aim of this study was to examine whether Raman spectroscopy could be used to correctly classify cell lines representing benign thyroid cells and various subtypes of TC. METHODS: A benign thyroid cell line and seven TC cell lines were prepared as ThinPrep® cytology slides and analysed with Raman spectroscopy. Principal components analysis and linear discriminant analysis were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different TC subtypes. RESULTS: The spectral differences separating benign and TC cell lines were assigned to differences in the composition of nucleic acids, lipids, carbohydrates and protein in the benign and cancer cells. Good sensitivities (74%-85%), specificities (65%-93%) and diagnostic accuracies (71%-88%) were achieved for the identification of TC. CONCLUSION: These findings suggest that Raman spectroscopy has potential for preoperative TC diagnosis on FNAC samples.

Classification of cytological samples from oral potentially malignant lesions through Raman spectroscopy: A pilot study.

The potential of Raman microspectroscopy of exfoliated cells has been demonstrated for oral cancer diagnosis. In this study, brush biopsies were collected from the buccal mucosa/tongue of healthy donors (n = 31) and from oral mucosal dysplastic lesions (n = 31 patients). Raman spectra were acquired and subjected to partial least squares-discriminant analysis (PLS-DA). The patient samples could be differentiated from healthy donor samples with 96% sensitivity and 95% specificity. Furthermore, PLS-DA models were developed based on cytopathological and histopathological assessment. Low and high grade dysplasia could be discriminated with 64% sensitivity and 65% specificity based on cytopathological assessment, while 81% sensitivity and 86% specificity could be achieved when histopathological assessment was within six months of the brush biopsy sampling. Therefore, this explorative study has successfully demonstrated that Raman spectroscopy may have a role in monitoring patients with dysplasia and may reduce the need for multiple biopsies.

Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells.

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Investigation of the influence of high-risk human papillomavirus on the biochemical composition of cervical cancer cells using vibrational spectroscopy.

The main aetiology of cervical cancer is infection with high-risk human papillomavirus (HPV). Cervical cancer is almost 100% curable if detected in the early stages. Thus, information about the presence and levels of HPV in patient samples has high clinical value. As current screening methods, such as the Pap smear test, are highly subjective and in many cases show low sensitivity and specificity, new supportive techniques are desirable to improve the quality of cervical cancer screening. In this study, vibrational spectroscopic techniques (Raman and Fourier Transform Infra Red absorption) have been applied to the investigation of four cervical cancer cell lines: HPV negative C33A, HPV-18 positive HeLa with 20-50 integrated HPV copies per cell, HPV-16 positive SiHa with 1-2 integrated HPV strands per cell and HPV-16 positive CaSki containing 60-600 integrated HPV copies per cell. Results show that vibrational spectroscopic techniques can discriminate between the cell lines and elucidate cellular differences originating from proteins, nucleic acids and lipids. Similarities between C33A and SiHa cells were exhibited in the Raman and infrared spectra and were confirmed by Principal Component Analysis (PCA). Analysis of the biochemical composition of the investigated cells, with the aid of PCA, showed a clear discrimination between the C33A-SiHa group and HeLa and CaSki cell lines indicating the potential of vibrational spectroscopic techniques as a support to current methods for cervical cancer screening.

Raman microspectroscopic study for the detection of oral field cancerisation using brush biopsy samples.

Field cancerisation (FC) is potentially an underlying cause of poor treatment outcomes of oral squamous cell carcinoma (OSCC). To explore the phenomenon using Raman microspectroscopy, brush biopsies from the buccal mucosa, tongue, gingiva and alveolus of healthy donors (n = 40) and from potentially malignant lesions (PML) of Dysplasia Clinic patients (n = 40) were examined. Contralateral normal samples (n = 38) were also collected from the patients. Raman spectra were acquired from the nucleus and cytoplasm of each cell, and subjected to partial least squares-discriminant analysis (PLS-DA). High discriminatory accuracy for donor and PML samples was achieved for both cytopalmic and nuclear data sets. Notably, contralateral normal (patient) samples were also accurately discriminated from donor samples and contralateral normal samples from patients with multiple lesions showed a similar spectral profile to PML samples, strongly indicating a FC effect. These findings support the potential of Raman microspectroscopy as a screening tool for PML using oral exfoliated cells.

A pilot study for early detection of oral premalignant diseases using oral cytology and Raman micro-spectroscopy: Assessment of confounding factors.

This study demonstrates the efficacy of Raman micro-spectroscopy of oral cytological samples for differentiating dysplastic, potentially malignant lesions from those of normal, healthy donors. Cells were collected using brush biopsy from healthy donors (n = 20) and patients attending a Dysplasia Clinic (n = 20). Donors were sampled at four different sites (buccal mucosa, tongue, alveolus, gingiva), to ensure matched normal sites for all lesions, while patient samples were taken from clinically evident, histologically verified dysplastic lesions. Spectra were acquired from the nucleus and cytoplasm of individual cells of all samples and subjected to partial least squares-discriminant analysis. Discriminative sensitivities of 94% and 86% and specificity of 85% were achieved for the cytoplasm and nucleus, respectively, largely based on lipidic contributions of dysplastic cells. Alveolar/gingival samples were differentiated from tongue/buccal samples, indicating that anatomical site is potentially a confounding factor, while age, gender, smoking and alcohol consumption were confirmed not to be.

Current Advances in the Application of Raman Spectroscopy for Molecular Diagnosis of Cervical Cancer.

Raman spectroscopy provides a unique biochemical fingerprint capable of identifying and characterizing the structure of molecules, cells, and tissues. In cervical cancer, it is acknowledged as a promising biochemical tool due to its ability to detect premalignancy and early malignancy stages. This review summarizes the key research in the area and the evidence compiled is very encouraging for ongoing and further research. In addition to the diagnostic potential, promising results for HPV detection and monitoring treatment response suggest more than just a diagnosis prospective. A greater body of evidence is however necessary before Raman spectroscopy is fully validated for clinical use and larger comprehensive studies are required to fully establish the role of Raman spectroscopy in the molecular diagnostics of cervical cancer.