flavin-adenine-dinucleotide and Uterine-Cervical-Neoplasms

While metabolic changes are considered a cancer hallmark, their assessment has not been incorporated in the detection of early or precancers, when treatment is most effective. Here, we demonstrate that metabolic changes are detected in freshly excised human cervical precancerous tissues using label-free, non-destructive imaging of the entire epithelium. The images rely on two-photon excited fluorescence from two metabolic co-enzymes, NAD(P)H and FAD, and have micron-level resolution, enabling sensitive assessments of the redox ratio and mitochondrial fragmentation, which yield metrics of metabolic function and heterogeneity. Simultaneous characterization of morphological features, such as the depth-dependent variation of the nuclear:cytoplasmic ratio, is demonstrated. Multi-parametric analysis combining several metabolic metrics with morphological ones enhances significantly the diagnostic accuracy of identifying high-grade squamous intraepithelial lesions. Our results motivate the translation of such functional metabolic imaging to

Topics: Cervix Uteri; Epithelium; Female; Flavin-Adenine Dinucleotide; Humans; Metabolic Networks and Pathways; Mitochondrial Dynamics; NAD; NADP; Optical Imaging; Precancerous Conditions; Reproducibility of Results; Sensitivity and Specificity; Staining and Labeling; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

We report the ex vivo results of an in-house fabricated portable device based on polarized fluorescence measurements in the clinical environment. This device measures the polarized fluorescence and elastic scattering spectra with 405-nm laser and white light sources, respectively. The dominating fluorophore with 405-nm excitation is flavin adenine dinucleotide (FAD) with a fluorescence peak around 510 nm. The measured spectra are highly modulated by the interplay of scattering and absorption effects. Due to this, valuable information gets masked. To reduce these effects, intrinsic fluorescence was extracted by normalizing polarized fluorescence spectra with polarized elastic scattering spectra obtained. A number of fluorophores contribute to the fluorescence spectra and need to be decoupled to understand their roles in the progression of cancer. Nelder-Mead method has been utilized to fit the spectral profile with Gaussian to decouple the different bands of contributing fluorophores (FAD and porphyrin). The change in concentration of FAD during disease progression manifests in the change in ratio of total area to FWHM of its Gaussian profile. Receiver operating characteristic (ROC) curve analysis has been used to discriminate different grades of cervical precancer by using the ratio as input parameter. The sensitivity and specificity for discrimination of normal samples from CIN I (cervical intraepithelial neoplasia) are 75% and 54%, respectively. Further, the normal samples can be discriminated from CIN II samples with 100% and 82% sensitivity and specificity, respectively, and the CIN I from CIN II samples can also be discriminated with 100% sensitivity and 90% specificity, respectively. The results show that the change in the concentration of (FAD) can be used as a marker to discriminate the different grades of the cancer and biochemical changes at an early stage of the cancer can also be monitored with this technique.

Topics: Biomarkers, Tumor; Early Detection of Cancer; Equipment Design; Female; Flavin-Adenine Dinucleotide; Humans; Optical Imaging; Sensitivity and Specificity; Spectrometry, Fluorescence; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The label-free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label-free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer.

Topics: Adult; Aged; Early Detection of Cancer; Female; Flavin-Adenine Dinucleotide; Humans; Middle Aged; NAD; Optical Imaging; Spectrometry, Fluorescence; Uterine Cervical Neoplasms; Young Adult

Intrinsic fluorescence spectra of the human normal, cervical intraepithelial neoplasia 1 (CIN1), CIN2, and cervical cancer tissue have been extracted by effectively combining the measured polarized fluorescence and polarized elastic scattering spectra. The efficacy of principal component analysis (PCA) to disentangle the collective behavior from smaller correlated clusters in a dimensionally reduced space in conjunction with the intrinsic fluorescence is examined. This combination unambiguously reveals the biochemical changes occurring with the progression of the disease. The differing activities of the dominant fluorophores, collagen, nicotinamide adenine dinucleotide, flavins, and porphyrin of different grades of precancers are clearly identified through a careful examination of the sectorial behavior of the dominant eigenvectors of PCA. To further classify the different grades, the Mahalanobis distance has been calculated using the scores of selected principal components.

Topics: Cervix Uteri; Collagen; Disease Progression; Female; Flavin-Adenine Dinucleotide; Humans; NAD; Optical Imaging; Porphyrins; Principal Component Analysis; Sensitivity and Specificity; Spectrometry, Fluorescence; Uterine Cervical Neoplasms

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.

Topics: Cell Line, Transformed; Cells, Cultured; Female; Flavin-Adenine Dinucleotide; Fluorescence; Humans; L-Lactate Dehydrogenase; Microscopy, Confocal; NAD; Spectrometry, Fluorescence; Uterine Cervical Neoplasms

Topics: Apoptosis; Cervix Uteri; Cisplatin; Female; Flavin-Adenine Dinucleotide; Fluorescence; Humans; Keratinocytes; NAD; Spectrometry, Fluorescence; Uterine Cervical Neoplasms

Topics: Carcinoma in Situ; Female; Flavin-Adenine Dinucleotide; Humans; NAD; Precancerous Conditions; Spectrometry, Fluorescence; Uterine Cervical Neoplasms

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.

Topics: Cervix Uteri; Epithelial Cells; Female; Flavin-Adenine Dinucleotide; Fluorescence; Humans; Luminescent Proteins; Microscopy, Confocal; NAD; Uterine Cervical Neoplasms