Detection and classification of gastrointestinal cancer and other pathologies through quantitative analysis of optical coherence tomography data and goniophotometry

The changes in light interaction between healthy and diseased tissues have been investigated as a potential diagnostic application. Here we attempt to differentiate between healthy and pathological gastrointestinal tissues using quantitative analysis of optical coherence tomography (OCT) data and goniophotometry. A goniophotometer was constructed and calibrated using titanium oxide and microsphere phantoms. Measurements were carried out on human gastrointestinal tissue sections collected using the methodology described below. The anisotropy factor g was extracted from the scattering curves by fitting the Henyey-Greenstein function. Measurements on human samples were in the forward scattering range with g 0.6-0.7, in agreement with the literature. Optical coherence tomography imaging was carried out on gastrointestinal tissues collected from patients undergoing elective surgery or endoscopy at St. Mary’s Hospital, London. In total 146 patients were included. Data was processed using gradient analysis of signal attenuation and morphological analysis with kNN classification. Results were correlated with histological diagnoses. Gradient analysis results were statistically significant across most categories, showing particularly good differences in the gradient distributions between healthy and diseased oesophageal tissues. Morphological analysis and kNN classification produced sensitivity and specificity values for healthy oesophagus and cancer in surgical specimens reaching 100% / 97.87% and 99.99% / 99.91% respectively and high accuracy in detecting Barrett's oesophagus in endoscopic specimens, with sensitivity and specificity values of 99.80% and 99.02%. Results in rectal tissue where also noteworthy, with detection of dysplasia reaching a sensitivity and specificity of 99.55% / 96.01%. Despite limitations in our work, we have shown that the detection of gastrointestinal pathologies using quantitative analysis of OCT data is a promising technique with good ex vivo results. Transferring the methodology to the in vivo domain holds a lot of potential as a future quick and reliable diagnostic technique.Open Acces

Risk Factors for Gastric Cancer and Risk Stratification in Singapore Chinese Population

Ph.DDOCTOR OF PHILOSOPH

An Investigation of the Diagnostic Potential of Autofluorescence Lifetime Spectroscopy and Imaging for Label-Free Contrast of Disease

The work presented in this thesis aimed to study the application of fluorescence lifetime spectroscopy (FLS) and fluorescence lifetime imaging microscopy (FLIM) to investigate their potential for diagnostic contrast of diseased tissue with a particular emphasis on autofluorescence (AF) measurements of gastrointestinal (GI) disease. Initially, an ex vivo study utilising confocal FLIM was undertaken with 420 nm excitation to characterise the fluorescence lifetime (FL) images obtained from 71 GI samples from 35 patients. A significant decrease in FL was observed between normal colon and polyps (p = 0.024), and normal colon and inflammatory bowel disease (IBD) (p = 0.015). Confocal FLIM was also performed on 23 bladder samples. A longer, although not significant, FL for cancer was observed, in paired specimens (n = 5) instilled with a photosensitizer. The first in vivo study was a clinical investigation of skin cancer using a fibre-optic FL spectrofluorometer and involved the interrogation of 27 lesions from 25 patients. A significant decrease in the FL of basal cell carcinomas compared to healthy tissue was observed (p = 0.002) with 445 nm excitation. A novel clinically viable FLS fibre-optic probe was then applied ex vivo to measure 60 samples collected from 23 patients. In a paired analysis of neoplastic polyps and normal colon obtained from the same region of the colon in the same patient (n = 12), a significant decrease in FL was observed (p = 0.021) with 435 nm excitation. In contrast, with 375 nm excitation, the mean FL of IBD specimens (n = 4) was found to be longer than that of normal tissue, although not statistically significant. Finally, the FLS system was applied in vivo in 17 patients, with initial data indicating that 435 nm excitation results in AF lifetimes that are broadly consistent with ex vivo studies, although no diagnostically significant differences were observed in the signals obtained in vivo.Open Acces

REAL-TIME FIBER OPTIC RAMAN SPECTROSCOPY FOR EARLY DIAGNOSIS OF PRECANCER AND CANCER IN VIVO IN THE UPPER GASTROINTESTINAL TRACT

Ph.DDOCTOR OF PHILOSOPH

Autosoom-retsessiivne Stargardti tõbi: fenotüübiline heterogeensus ja genotüübi-fenotüübi seosed

Väitekirja elektrooniline versioon ei sisalda publikatsiooneStargardti tõbi (STGD1) on kõige sagedasem pärilik võrkkesta kollatähni düstroofia põhjustades progresseeruvat nägemislangust sageli juba lapseeast. Haigust põhjustavad mutatsioonid ABCA4 geenis, mille tulemusena ladestuvad võrkkesta toksilised jääkproduktid põhjustades fotoretseptorite kadu. Praeguseks on teada üle 1000 haigustekkelise mutatsiooni ABCA4 geenis, mille tõttu on haiguse kliiniline pilt (fenotüüp) väga mitmekesine. Meie uuringu peamisteks eesmärkideks olid STGD1 puhuste võrkkesta struktuurimuutuste analüüs ja leida võimalikke seoseid haiguse fenotüüpide ja (ABCA4) mutatsioonide vahel. Doktoritöös leidsime, et vastupidiselt senise arusaama kohaselt esineb varases haiguse faasis noortel STGD1 haigetel lisaks fotoretseptorite kihi õhenemisele välise piirimembraani oluline paksenemine võrreldes kontrollgrupiga. Tõenäoliselt iseloomustab leid võrkkesta gliiarakkude mööduvat hüpertroofiat, olles STGD1 varajaseks kliiniliseks markeriks, mis omab kliinilist potentsiaali diagnoosimaks haigust varases faasis. Teiseks analüüsisime STGD1 korral harva esinevat fenotüüpi, mida nimetatakse kollatähni tsentri (fovea) tühimikuks (ingl.k. optical gap; foveal cavitation). Tegemist on foveas paiknevate fotoretseptorite kaoga, mille tulemusena moodustub võrkkesta väliskihtidesse struktuurne tühimik. Võrkkesta kuvamisuuringute analüüsil näitasime, kuidas fenotüüp dünaamiliselt kujuneb ja kaob ning lõime selle baasil arengustaadiumid. Vastupidiselt siiani juhtivale arusaamale, et STGD1 puhul esineb primaarselt võrkkesta pigmentepiteelirakkude (RPE) kõhetumine näitasime kuvamisuuringutel, et fovea tühimiku korral toimub esmaselt just fotoretseptorite kadu, millele järgneb RPE atroofia. Ühtlasi leidsime genotüübi-fenotüübi analüüsil, et STGD1 haigetel esineb statistiliselt tugev seos ABCA4 geenis paikneva p. G1961E mutatsiooni ja fovea tühimiku vahel. Kolmandaks näitasime, et STGD1 võib fenotüübilt sarnaneda hüdroksüklorokviin (Plaquenil) võrkkesta toksiliste muutusega. Moodustub nn „foveat säilitava“ fenotüübi variant, mille tulemusena on kollatähni teravanägemise punktis võrkkest suhteliselt hästi säilinud ning patsiendid on avastamise hetkel enamasti sümptomite vabad. Fenotüüpide detailne iseloomustamine, dünaamika hindamine ja genotüübi-fenotüübi seoste kirjeldamine võiks paremini aidata planeerida ja analüüsida ravimi, geeniteraapia ja tüvirakuteraapia uuringuid ja nende tulemusi STGD1 kontekstisStargardt disease (STGD1) is the most common form of inherited macular dystrophy causing progressive visual loss often from childhood. The disease is caused by mutations in the ABCA4 gene resulting in progressive accumulation of toxic visual cycle byproducts in the retina leading to photoreceptor loss. More than 1000 disease-causing ABCA4 mutations have been described resulting in remarkable diverse clinical (phenotypic) expression of the disease. The main aims of our study were to analyze STGD1-associated retinal structural changes as well as phenotypic expression via multimodal imaging and to detect possible genotype-phenotype associations. In the thesis studies we determined that in addition to thinning of photoreceptor-associated (ellipsoid zone) layer, there is a statistically significant thickening of external limiting membrane in young STGD1 patients compared to age-matched controls. The finding describes probably transient hypertrophy of retinal glial cells and could clinically be an important early stage disease marker, holding a diagnostic potential in early diagnosis of the STGD1. We also analyzed STGD1 patients with foveal cavitation (optical gap) phenotype. It is a rare manifestation of the STGD1 characterized by focal loss of photoreceptors in the fovea leaving a sub-foveal optically empty space detectable with spectral- domain optical coherent tomography (SD-OCT). With multimodal imaging we showed how the phenotype develops and dynamically behaves, based on which we described developmental stages of this phenotype. In contrast to the common understanding, that the first cells being affected in STGD1 are the retinal pigment epithelial (RPE) cells, we showed that, in foveal cavitation, the first cells which disappear are photoreceptors followed by atrophy of the RPE cells. We also detected a strong association between foveal cavitation phenotype and p.G1961E mutation in STGD1. We also showed that STGD1 could phenocopy hydroxychloroquine (Plaquenil) maculopathy. The specific foveal sparing phenotype has quite unique conformation on SD-OCT, where foveal area is relatively well spared preserving a very good visual acuity. Detailed phenotypic descriptions and dynamic assessment via multimodal imaging as well as detecting new genotype-phenotype associations potentially improves the planning of pharmacological, gene- and stem cell-based treatment studies of STGD1

Confocal Microscopy and Nuclear Segmentation Algorithm for Quantitative Imaging of Epithelial Tissue

Carcinomas, cancers that originate in the epithelium, account for more than 80% of all cancers. When detected early, the 5-year survival rate is greatly increased. Biopsy and histopathology is the current gold standard for diagnosis of epithelial carcinomas which is an invasive, time-intensive, and stressful procedure. In vivo confocal microscopy has the potential to non-invasively image epithelial tissue in near-real time. This dissertation describes the development of a confocal microscope for imaging epithelial tissues and an image processing algorithm for segmentation of epithelial nuclei. A rapid beam and stage scanning combination was used to acquire fluorescence confocal images of cellular and tissue features along the length of excised mouse colon. A single 1 × 60 mm2 field of view is acquired in 10 seconds. Disruption of crypt structure such as size, shape, and distribution is visualized in images of inflamed colon tissue, while the normal mouse colon exhibited uniform crypt structure and distribution. An automated pulse coupled neural network segmentation algorithm was developed for epithelial nuclei segmentation. An increase in nuclear size and the nuclear-to-cytoplasmic ratio is a potential precursor to pre-cancer development. The spiking cortical model algorithm was evaluated using a developed confocal image model of epithelial tissues with varying contrast. It was further validated on reflectance confocal images of porcine and human oral tissue from two separate confocal imaging systems. Biopsies of human oral mucosa are used to determine the tissue and system effects on measurements of nuclear-to-cytoplasmic ratio

Laser doppler assessment of gastric mucosal blood flow in normals and its relationship to the systemic activity of growth peptides in healing and non healing gastric ulcers.

Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.The pattern of mucosal blood flow in normal human stomachs, and benign gastric ulcers was assesed with laser Doppler flowmetry and the relationship between a single determination of ulcer blood flow and the systemic level of growth factors was investigated. A significant ascending gradient in mucosal blood flow from the antrum to fundus was demonstrated. Different levels of cellular activity in the regions of the stomach may explain this gradient. In the gastric ulcers that healed on standard medical therapy mucosal blood flow was significantly increased in comparison to normal stomachs. In the ulcers that were refractory to standard medical therapy mucosal blood flow was significantly lower than in normal stomachs and healing ulcers. Higher systemic levels of the growth factor bFGF were demonstrated in healing ulcers compared to non-healing ulcers. Gastric mucosal blood flow can increase in response to the increased metabolic demands of healing, however impairment of this response may be an important factor preventing healing of benign gastric ulcers. It would appear that non-healing of gastric ulcers can be predicted at initial diagnosis by reduced peri-ulcer gastric mucosal blood flow and low blood levels of bFGF