Early detection of cervical neoplasia by Raman spectroscopy

Abstract

Early detection of malignant tumours, or their precursor lesions, improves patient outcome. High risk human papillomavirus (HPV), particularly HPV16, infection can lead to the development of uterine cervical neoplasia, and therefore, the identification in clinical samples of the effects of HPV infection may have clinical value. In this report, we apply Raman microspectroscopy to live and fixed cultured cells to discriminate between defined cell types. Raman spectra were acquired from primary human keratinocytes (PHK), PHK expressing the E7 gene of HPV 16 (PHK E7) and CaSki cells, an HPV16-containing cervical carcinoma-derived cell line. Averaged Raman spectra showed variations, mostly in peaks originating from DNA and proteins, consistent with HPV gene expression and cellular changes associated with neoplasia, in both live and fixed cells. Principal component analysis produced good discrimination between the cell types, with sensitivities of up to 100% for the comparison of fixed PHK and CaSki. These results demonstrate the ability of Raman spectroscopy to discriminate between cell types representing different stages of cervical neoplasia. More specifically, this technique was able to identify cells expressing the HPV 16 E7 gene accurately and objectively, suggesting that this approach may be of value in diagnosis. Moreover, the ability to detect the effects of the virus in fixed samples also demonstrates the compatibility of Raman spectroscopy with current cervical screening methods. ' 2007 Wiley-Liss, Inc.Key words: Raman spectroscopy; cervix; neoplasia; diagnosis; human papillomavirus Cervical cancer is the second most common cancer in women worldwide, and infection with oncogenic or 'high-riskÕ human papillomavirus (HPV) types is the most significant risk factor in its aetiology. 1 HPV is present in 99.7% of invasive cervical cancers, 1,2 and therefore, early detection of the effects of HPV infection, particularly when accompanied by neoplastic changes, could improve the diagnosis of HPV-associated neoplasia. The current primary screening tool for cervical neoplasia is the Pap smear, which involves the microscopic examination of exfoliated cells for morphological abnormalities. Although effective, this technique is labour intensive and results in a significant number of false positives/negatives 3 as it is based upon a subjective interpretation of the sample. This increases the need for an objective screening tool that gives an early and confident diagnosis.Optical diagnostic techniques, such as drug-assisted tumour fluorescence, 4 natural tissue fluorescence 5 or Fourier transform infrared spectroscopy 6 have attracted much interest recently as they offer the possibility of nonintrusive objective diagnostics both in vitro and in vivo. Emerging as a forerunner among these diagnostic technologies is Raman spectroscopy. This is a laser-based scattering spectroscopy and refers to scattered light, from a molecular or cellular sample, which exhibits a frequency shift that reflects the energy of specific molecula...

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

“…With respect to PC3, there is a separation of the CaSki group from the other cell lines and it is important to note that in the PC3 loading there is a distinctive protein peak at 1036cm -1 , 70 which has a clearly different contribution in the CaSki mean spectrum compared to other cell lines (figure 2B and Tyr 44 . Raman and FTIR PCs are dominated by features associated with changes of protein conformation structures; β-sheet, α-helix and disordered structure in PC1, PC2 and PC3, respectively.…”

“…In the literature, there is a wide spectrum of experiments concerning cervical 70 cancer investigation with vibrational spectroscopy techniques, demonstrating the usefulness of these methods in understanding cervical cancer progression from the molecular level, and showing its potential application in cervical cancer screening and diagnosis . Raman spectroscopy has also 75 been used to study cell lines derived from the cervix, revealing spectral variations, mostly in peaks originating from DNA and proteins, in cell lines expressing the E7 gene of HPV-16 compared to cells not affected by HPV 44 . Furthermore, a recent study has demonstrated that in low- 5 grade cytology infected with high-risk oncogenic HPV16 or HPV18, it was possible to differentiate samples from women based on whether they were aged 20-29 years vs. 30-39 years using attenuated total reflection infrared spectroscopy 45 .…”

Sensitivity of Raman spectroscopy to normal patient variability

“…14 Although Raman spectroscopy is inherently a sensitive technique, previous studies using Raman to detect cervical dysplasia both in vivo and in vitro have reported a wide range of sensitivity (70-100%) and specificity (70-100%) rates. 1,[15][16][17][18][19] Krishna et al used a benchtop Raman spectroscopy system to acquire information from cervical samples ex vivo and classified normal compared to malignant samples at sensitivity and specificity rates of 75-99.5%. 19 Because infection with certain strains of human papillomavirus (HPV) is the cause of cervical dysplasia in >99% of cases worldwide, [20][21][22] certain research groups have used Raman spectroscopy to identify differences between HPV types.…”

“…Jess et al used a confocal Raman microscope system and were able to discriminate varying HPV types in live and fixed cells with sensitivity and specificity rates of 70-100%. 15 Our research group has used a portable probe-based Raman spectroscopy system to distinguish between normal and malignant cervical samples in cell culture, in vitro and in vivo. The sensitivity and specificity rates of this work have ranged between 81 and 97%.…”

Modulated Raman spectroscopy for enhanced identification of bladder tumor cells in urine samples

“…Comparing the relative intensities of the spectral peaks for SV-HUC-1 and MGH spectra show that urothelial cells (SV-HUC-1) are characterized by relatively strong protein and carbohydrate Raman peaks, in the spectral region 1100 to 1300 cm − 1 , which suggest greater concentrations of proteins and carbohydrates in nonmalignant cells. 23 In contrast, the spectra from bladder cancer cells (MGH) show a significant increase in the peaks pertaining to ring breathing modes in DNA bases, such as 669, 727, 785, 828, 1095, and 1578 cm − 1 , suggesting an increase in the DNA concentration of bladder cancer cells. Tumor cells commonly exhibit an increase in DNA content in the nucleus.…”