High-resolution imaging for cancer detection with a fiber bundle microendoscope

Dysplasia and cancer of epithelial tissues, including the oral cavity and esophagus, typically have much higher survival rates if diagnosed at an early stage. Unfortunately, the clinical appearance of lesions in these tissues can be highly variable. To achieve a definitive diagnosis of a suspected lesion at these sites, an excisional biopsy must be examined at high-resolution. These procedures can be costly and timeconsuming, and in the case of Barrett's esophagus, surveillance biopsy strategies may not be entirely effective. Optical imaging modalities have the potential to yield qualitative and quantitative high-resolution data at low cost, enabling clinicians to improve early detection rate. This dissertation presents a low-cost high-resolution microendoscopy system based on a fiber optic bundle image guide. In combination with a topical fluorescent dye, the fiber bundle can be placed into contact with the tissue to be observed. A high-resolution image is then projected onto a CCD camera and stored on a PC. A pilot study was performed on both resected esophageal tissue containing intestinal metaplasia (a condition known as Barrett's esophagus, which can transform to esophageal adenocarcinoma) and resected oral tissue following surgical removal of cancer. Qualitative image analysis demonstrated similar features were visible in both microendoscope images and standard histology images, and quantitative image processing and analysis yielded an objective classification algorithm. The classification algorithm was developed to discriminate between neoplastic and non-neoplastic imaging sites. The performance of this algorithm was monitored by comparing the predicted results to the pathology diagnosis at each measurement site. In the oral cancer pilot study, the classifier achieved 85% sensitivity and 78% specificity with 141 independent measurement sites. In the Barrett's metaplasia pilot study, 87% sensitivity and 85% specificity were achieved with 128 independent measurement sites. The work presented in this dissertation outlines the design, testing, and initial validation of the high-resolution microendoscope system. This microendoscope system has demonstrated potential utility over a wide range of modalities, including small animal imaging, molecular-specific imaging, ex vivo and ultimately in vivo imaging

Optical Imaging Techniques for the Detection of Esophageal Neoplasia in Barrett’s Esophagus

The main objective of this research was to develop a two-stage optical imaging platform to improve detection of cancer in Barrett’s esophagus (BE). BE caused by chronic reflux and patients with BE are at a higher risk for developing esophageal adenocarcinoma (EAC). However, neoplasia in BE is often unidentifiable under standard endoscopy, and studies have shown nearly half of early cancers can go unidentified by this method. Widefield imaging (resolves ~100 microns) allows efficient surveillance of large BE segments. Two widefield imaging techniques were identified to improve contrast between benign and abnormal lesions during an ex vivo 15 patient feasibility study. Cross-polarized imaging (CPI) reduced specular reflection and improved vascular contrast. Vital-dye fluorescence imaging (VFI) using topically-applied proflavine improved visualization of glandular pattern. Moreover, relevant pathologic features visible during VFI were seen in corresponding histology slides as well as high resolution images of the same sites. Based on these results, a cap-based Multispectral Digital Endoscope (MDE) was designed and built. The MDE can image in three different imaging modes: white light imaging, CPI, and VFI. Modifications to a Pentax EPK-i video processor and a Pentax endoscope were made to incorporate these imaging modes into one system. A 21 patient in vivo pilot study with 65 pathologically correlated sites demonstrated the feasibility of using this system in vivo; image criteria were developed to classify neoplasia with a sensitivity and specificity of 100% and 76% respectively. High resolution imaging (resolves ~2-5 micron) may verify the disease presence in suspicious areas identified using widefield techniques. 2-NBDG, a fluorescent metabolic marker, was used as to identify neoplastic biopsies. In a study with 21 patients yielding 38 pathologically correlated biopsies and 158 image sites, 2-NBDG imaging allowed classification of cancerous biopsies with a sensitivity of 96% and specificity of 90%. The unique contributions of these results is the development of a multimodal cap-based endoscopic system to identify suspicious areas in BE, and using a metabolic marker to verify the presence of disease. This application extends beyond esophageal cancer detection and may be explored for cancer detection in other organ sites characterized by columnar epithelium

Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future

Intratumor heterogeneity, which fosters tumor evolution, is a key challenge in cancer medicine. Here, we review data and technologies that have revealed intra-tumor heterogeneity across cancer types and the dynamics, constraints, and contingencies inherent to tumor evolution. We emphasize the importance of macro-evolutionary leaps, often involving large-scale chromosomal alterations, in driving tumor evolution and metastasis and consider the role of the tumor microenvironment in engendering heterogeneity and drug resistance. We suggest that bold approaches to drug development, harnessing the adaptive properties of the immune-microenvironment while limiting those of the tumor, combined with advances in clinical trial-design, will improve patient outcome

The promoters of human cell cycle genes integrate signals from two tumor suppressive pathways during cellular transformation

Deciphering regulatory events that drive malignant transformation represents a major challenge for systems biology. Here we analyzed genome-wide transcription profiling of an in-vitro transformation process. We focused on a cluster of genes whose expression levels increased as a function of p53 and p16INK4A tumor suppressors inactivation. This cluster predominantly consists of cell cycle genes and constitutes a signature of a diversity of cancers. By linking expression profiles of the genes in the cluster with the dynamic behavior of p53 and p16INK4A, we identified a promoter architecture that integrates signals from the two tumor suppressive channels and that maps their activity onto distinct levels of expression of the cell cycle genes, which in turn, correspond to different cellular proliferation rates. Taking components of the mitotic spindle as an example, we experimentally verified our predictions that p53-mediated transcriptional repression of several of these novel targets is dependent on the activities of p21, NFY and E2F. Our study demonstrates how a well-controlled transformation process allows linking between gene expression, promoter architecture and activity of upstream signaling molecules.Comment: To appear in Molecular Systems Biolog

Spectroscopy systems for the detection of gastrointestinal dysplasia

Programa doutoral em BioengenhariaThe detection of gastrointestinal (GI) dysplasia is essential to improve the patient’s survival rate. The diagnosis of this condition can be performed using spectroscopy techniques, such as fluorescence and diffuse-reflectance, which have the potential to provide morphological and biochemical information regarding normal and dysplastic tissue. Research prototypes currently used for those clinical spectroscopy techniques have associated a few drawbacks: they are costly, bulky, too sophisticated and they use optical fibers, which are usually related with low-collection efficiency. Moreover, these catheterbased instruments are invasive and very uncomfortable for the patient. In this context, the present work had the purpose to develop a miniaturized spectroscopy system, based on those two techniques, that features low-complexity and costeffectiveness. Furthermore, the integration of optical components in a single chip allows a high level of reliability. Such a system can be integrated in less-invasive devices (e.g., the endoscopic capsules), for an effective and comfortable detection of GI dysplastic lesions. The developed spectroscopy system core is based on thin-film optical filters and lowcost silicon photodiodes, used for the selection and detection of a few light wavelengths significant for the diagnosis of dysplasia. Thin-film optical filters, centered at specific wavelengths, were designed, fabricated and characterized after the successful demonstration that the use of only 16 spectral bands (within the 350 to 750 nm spectral range) enabled an accurate extraction of tissue information. The feasibility of using the fabricated filters to establish a quantitative spectroscopy diagnosis was proved with measurements on tissue phantoms. Additionally, an even more compact device is proposed for qualitative diagnosis using only two different spectral bands, centered at 420 nm for fluorescence measurements and at 540 nm for diffuse-reflectance measurements. This approach enabled the construction of a diagnostic algorithm for the identification of dysplastic tissues, with a sensitivity and specificity of 77.8% and 97.6%, respectively. This thesis work was also directed towards the development of a fluorescence and diffuse-reflectance spectroscopy imaging system for excised tissue margins assessment, which results from mucosal resections in the GI tract. In the developed system, wide area imaging is achieved by mechanically scanning an optical probe along the tissue surface, with variable spatial resolution. This medical device can provide real-time feedback regarding the resected mucosal margins, which represents a huge impact in intra-operative diagnosis. The clinical utility of the spectroscopy imaging prototype was successfully demonstrated using biological samples, i.e., different images containing reliable quantitative tissue information were obtained using analytical models. These images can be subsequently used to establish a diagnosis. This system may enable the reduction of the patient anxiety, avoiding the follow-up surgery, once a fast and real-time data analysis can be performed inside the operating room.A deteção de displasia gastrointestinal (GI) é essencial para melhorar a taxa de sobrevivência dos pacientes. O diagnóstico desta condição pode ser efetuado utilizando técnicas de espectroscopia, como a fluorescência e a reflectância difusa, que têm a capacidade de proporcionar informação morfológica e bioquímica acerca dos tecidos normais e dos tecidos com displasia. Os protótipos, ainda em investigação, actualmente utilizados em espetroscopia clínica e que implementam as técnicas mencionadas, têm associadas várias desvantagens, como por exemplo: têm elevado custo; são demasiado sofisticados e volumosos; e utilizam fibras óticas, que normalmente estão associadas a uma baixa eficiência na recolha de sinal. Para além disso, estes instrumentos baseiam-se em catéteres, o que os torna invasivos e, por isso, desconfortáveis para o paciente. Neste contexto, o presente trabalho teve como objetivo o desenvolvimento de um sistema baseado nas duas técnicas de espectroscopia acima referidas, que seja simples, de baixo custo e miniaturizado. A integração de componentes óticos num único chip possibilita a obtenção dispositivos óticos mais fiáveis e estáveis. Um sistema com estas características pode ser integrado em dispositivos de diagnóstico menos invasivos (por exemplo, nas cápsulas endoscópicas) para uma deteção eficaz e confortável das lesões displásticas do trato GI. O elemento principal deste sistema miniaturizado de espetroscopia consiste em filtros óticos baseados em filmes finos e fotodíodos de silício de baixo custo, ambos utilizados para a seleção e deteção de alguns comprimentos de onda importantes para o diagnóstico de displasia. Estes filtros óticos, centrados em comprimentos de onda específicos, foram desenhados, fabricados e caracterizados após ter sido demonstrado que a utilização de apenas 16 bandas espetrais (na gama espetral compreendida entre os 350 e os 750 nm) permitia uma extração correta da informação dos tecidos. A viabilidade de utilizar os filtros fabricados para o estabelecimento de um diagnóstico espetroscópico quantitativo foi também demonstrada com êxito através de medições realizadas em fantomas. Adicionalmente, e tendo em vista o desenvolvimento de um dispositivo ainda mais compacto, foi proposto um diagnóstico qualitativo, ou empírico, utilizando apenas duas bandas espetrais, centradas em 420 nm no caso das medições de fluorescência e em 540 nm no caso das medições de reflectância difusa. Esta abordagem permitiu a construção de um algoritmo de diagnóstico para a identificação de displasia com uma sensibilidade e uma especificidade de 77,8% e de 97,6%, respetivamente. Desenvolveu-se, igualmente, um sistema espetroscópico de imagem (envolvendo fluorescência e reflectância difusa) para a análise de margens de tecido excisado que resultam de ressecções da mucosa do tracto GI. Este sistema, através do varrimento mecânico de uma sonda de fibra ótica ao longo da superfície do tecido, com uma resolução espacial variável, possibilita a aquisição de imagens numa vasta área de tecido. Este dispositivo médico pode fornecer uma resposta em tempo real acerca das margens de tecido excisado, o que representa um enorme impacto no diagnóstico intra-operatório. A sua utilidade clínica foi demonstrada, com bons resultados, utilizando amostras biológicas, isto é, foi possível obter imagens contendo informação quantitativa fidedigna acerca dos tecidos, que pode ser subsequentemente utilizada para estabelecer um diagnóstico. Um sistema como este poderá reduzir a ansiedade dos pacientes e evitar a realização de novas cirurgias, uma vez que logo após a cirurgia, e ainda na sala de operações, pode efectuar-se uma análise aos tecidos.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/38978/2007MIT Portugal Progra

A deep-learning approach to aid in diagnosing Barrett’s oesophagus related dysplasia

Barrett's oesophagus is the only known precursor to oesophagus carcinoma. Histologically, it is defined as a condition of columnar cells replacing the standard squamous lining. Those altered cells are prone to cytological and architectural abnormalities, known as dysplasia. The dysplastic degree varies from low to high grade and can evolve into invasive carcinoma or adenocarcinoma. Thus, detecting high-grade and intramucosal carcinoma during the surveillance of Barrett's oesophagus patients is vital so they can be treated by surgical resection. Unfortunately, the achieved interobserver agreement for grading dysplasia among pathologists is only fair to moderate. Nowadays, grading Barrett's dysplasia is limited to visual examination by pathologists for glass or virtual slides. This work aims to diagnose different grades of dysplasia in Barrett’s oesophagus, particularly high-grade dysplasia, from virtual histopathological slides of oesophagus tissue. In the first approach, virtual slides were analysed at a low magnification to detect regions of interest and predict the grade of dysplasia based on the analysis of the virtual slides at 10X magnification. Transfer learning was employed to partially fine-tune two deep-learning networks using healthy and Barrett’s oesophagus tissue. Then, the two networks were connected. The proposed model achieved 0.57 sensitivity, 0.79 specificity and moderate agreement with a pathologist. On the contrary, the second approach processed the slides at a higher magnification (40X magnification). It adapted novelty detection and local outlier factor alongside transfer learning to solve the multiple instances learning problem. It increased the performance of the diagnosis to 0.84 sensitivity and 0.92 specificity, and the interobserver agreement reached a substantial level. Finally, the last approach mimics the pathologists’ procedure to diagnose dysplasia, relying on both magnifications. Thus, their behaviours during the assessment were analysed. As a result, it was found that employing a multi-scale approach to detect dysplastic tissue using a low magnification level (10X magnification) and grade dysplasia at a higher level (40X magnification). The proposed computer-aided diagnosis system was built using networks from the first two approaches. It scored 0.90 sensitivity, 0.94 specificity and a substantial agreement with the pathologist and a moderate agreement with the other expert

Endoscopic multimodal imaging in Barrett's oesophagus

The incidence of oesophageal adenocarcinoma (OA) has increased exponentially in the western world over the past few decades. Barrett's oesophagus (BO) is a well known precursor of OA with a risk approximately 20 times more than that of background population. Regular endoscopic surveillance in patients with BO is recommended by most of the national gastroenterological societies. The advantage of Barrett's surveillance is to identify early subtle lesions which could then be managed early to avoid symptomatic and advanced cancers. The detection of such early lesions are challenging as they could be flat and inconspicuous on routine endoscopic examination. In the absence of any lesions, four quadrant biopsies every 1-2 cm of the whole length of Barrett's oesophagus is advised. This technique would map only 5-10% of the surface area of Barrett's segment and hence it is associated with significant sampling error. The improvement in electronics over the past decade has led to the production of endoscopes with better charged coupled devices and image enhancement techniques by altering the spectrum of light. This thesis examines the role of multi modal imaging in Barrett's oesophagus with a focus on detecting dysplasia and early cancer (EC). Firstly, the role of high definition (HD) imaging in routine clinical setting was studied using data from patients who have undergone Barrett's· surveillance. The yield of dysplasia by HD endoscopy was compared to standard definition (SD) endoscopy in this study. The role of narrow band imaging (NBI) with magnification in characterising abnormal lesions detected during BO surveillance was evaluated by performing a meta- analysis of clinical studies. The role of autofluorescence imaging (AFI) in Barrett's oesophagus was examined in detail with a view to understand the biological basis of autofluorescence and to improve the specificity of this technique as it is associated with significant false positive results in clinical studies. A meta-analysis was performed to identify whether AFI has a clinical advantage over white light endoscopy in detecting Barrett's dysplasia and the inter-observer reliability of this technology was studied using AFI expert and AFI non-expert endoscopists. An objective method of measuring the autofluorescence intensity was proposed as a ratio of the red to the green colour tone (AF ratio) of the area of interest. When the AF ratio of the lesion was divided by the AF ratio of the background mucosa, an AF index is obtained. A pilot study was performed to identify a cut-off value of AF index to differentiate high grade dysplasia (HGD) and EC from non-dysplastic BO. Finally, the biological basis of AF intensity was examined using APCmin mouse colonic models. This study looked into the AF ratio of the colonic mucosal lesions and correlated it with the amount of collagen and elastin in the submucosal tissue. Collagen and elastin are known to be the strongest fluorophores of the gastrointestinal tract and the question addressed is whether the low AF intensity associated with dysplastic lesions is due to the thickening of mucosa or to a reduction of collagen and elastin

Endoscopic multimodal imaging in Barrett's oesophagus

The incidence of oesophageal adenocarcinoma (OA) has increased exponentially in the western world over the past few decades. Barrett's oesophagus (BO) is a well known precursor of OA with a risk approximately 20 times more than that of background population. Regular endoscopic surveillance in patients with BO is recommended by most of the national gastroenterological societies. The advantage of Barrett's surveillance is to identify early subtle lesions which could then be managed early to avoid symptomatic and advanced cancers. The detection of such early lesions are challenging as they could be flat and inconspicuous on routine endoscopic examination. In the absence of any lesions, four quadrant biopsies every 1-2 cm of the whole length of Barrett's oesophagus is advised. This technique would map only 5-10% of the surface area of Barrett's segment and hence it is associated with significant sampling error. The improvement in electronics over the past decade has led to the production of endoscopes with better charged coupled devices and image enhancement techniques by altering the spectrum of light. This thesis examines the role of multi modal imaging in Barrett's oesophagus with a focus on detecting dysplasia and early cancer (EC). Firstly, the role of high definition (HD) imaging in routine clinical setting was studied using data from patients who have undergone Barrett's· surveillance. The yield of dysplasia by HD endoscopy was compared to standard definition (SD) endoscopy in this study. The role of narrow band imaging (NBI) with magnification in characterising abnormal lesions detected during BO surveillance was evaluated by performing a meta- analysis of clinical studies. The role of autofluorescence imaging (AFI) in Barrett's oesophagus was examined in detail with a view to understand the biological basis of autofluorescence and to improve the specificity of this technique as it is associated with significant false positive results in clinical studies. A meta-analysis was performed to identify whether AFI has a clinical advantage over white light endoscopy in detecting Barrett's dysplasia and the inter-observer reliability of this technology was studied using AFI expert and AFI non-expert endoscopists. An objective method of measuring the autofluorescence intensity was proposed as a ratio of the red to the green colour tone (AF ratio) of the area of interest. When the AF ratio of the lesion was divided by the AF ratio of the background mucosa, an AF index is obtained. A pilot study was performed to identify a cut-off value of AF index to differentiate high grade dysplasia (HGD) and EC from non-dysplastic BO. Finally, the biological basis of AF intensity was examined using APCmin mouse colonic models. This study looked into the AF ratio of the colonic mucosal lesions and correlated it with the amount of collagen and elastin in the submucosal tissue. Collagen and elastin are known to be the strongest fluorophores of the gastrointestinal tract and the question addressed is whether the low AF intensity associated with dysplastic lesions is due to the thickening of mucosa or to a reduction of collagen and elastin