Recent advances and current issues in single-cell sequencing of tumors

AbstractIntratumoral heterogeneity is a recently recognized but important feature of cancer that underlies the various biocharacteristics of cancer tissues. The advent of next-generation sequencing technologies has facilitated large scale capture of genomic data, while the recent development of single-cell sequencing has allowed for more in-depth studies into the complex molecular mechanisms of intratumoral heterogeneity. In this review, the recent advances and current challenges in single-cell sequencing methodologies are discussed, highlighting the potential power of these data to provide insights into oncological processes, from tumorigenesis through progression to metastasis and therapy resistance

Next-Generation Sequencing: Acquisition, Analysis, and Assembly

The process of sequencing a genome involves many steps, and accordingly, this project contains work from each of those steps. Genome sequencing begins with acquisition of sequence data, therefore, a novel biochemistry was utilized and optimized for the Sequencing By Ligation (SBL) process. A cyclic SBL protocol was created that could be utilized to extend sequencing reads in both the 5\u27 and 3\u27 directions, for an increase in read length and thru-put. After sequence acquisition, there is the process of data analysis, and the focus shifted to creating software that could take sequence information and match up the individual reads to a reference genome with greater speed and efficiency than other commonly-used software. The Sequence Analysis Workbench Tool, SAWTooth, was written and shown to outperform contemporaries NOVOAlign and BOWTIE. Finally, the last aspect of genome sequencing is de novo assembly, prompting a comparative analysis of three assemblers: CLC Genomics Workbench, Velvet Assembler, and MIRA. Results were generated using Mauve to assess the general effects of different sequencing platforms on the final assembly

Identifying Patterns of Cancer Disease Mechanisms by Mining Alternative Representations of Genomic Alterations

Cancer is a complex disease driven by somatic genomic alterations (SGAs) that perturb signaling pathways and consequently cellular function. Identifying combinatorial patterns of pathway perturbations would provide insights into common disease mechanisms shared among tumors, which is important for guiding treatment and predicting outcome. However, identifying perturbed pathways is challenging, because different tumors can have the same perturbed pathways that are perturbed by different SGAs. We started off by designing a novel semantic representation that captures the functional similarity of distinct SGAs perturbing a common pathway in different tumors. This representation was used alongside the nested hierarchical Dirichlet process topic model in order to identify combinatorial patterns in altered signaling pathways. We found that the topic model was able to capture the functional relationships between topics. It was also able to identify cancer subtypes composed of tumors from different tissues of origin that exhibit different survival rates. These results led us to investigate the performance of the methodology on pan-cancer data, as well as in conjunction with cancer driver data. The results revealed that the framework was still able to identify clinically relevant features in pan-cancer. However, the addition of driver data decreased the noise in the data and improved the separation of tumors in the clustering results. This provided support for including the use of driver data in our methodology. In order to have gene representations independent of literature, we developed a biological representation that could identify functionally related genes. Its performance when used alongside topic modeling was tested. We found that the topic association patterns separated tumors by their tissue of origin. But, analyzing some of the cancer types on an individual basis still led to significant differences in survival. Our studies show the potential for using alternative representations in conjunction with topic modeling to investigate complex genomic diseases. With further research and refinement of this methodology, it has the potential to capture the relationship between pathways involved in cancer. This would contribute to a better understanding of cancer disease mechanisms and treatment

Dos genes à radiorresistência no cancro da cabeça e pescoço

Mestrado em Biomedicina MolecularHead and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.Cancro da Cabeça e Pescoço refere-se a um grupo de tumores que aparecem no trato aerodigestivo superior, sendo que o carcinoma das células escamosas da cabeça e pescoço (CCECP) corresponde a mais de 90% de todos os casos de cancro nesta região. Foi considerado o sexto tumor mais maligno em todo o mundo e, apesar de todos os avanços tecnológicos e clínicos, a taxa de sobrevivência a cinco anos não melhorou significativamente nas últimas décadas. Atualmente sabe-se que o CCECP é uma doença bastante heterogénea que se desenvolve devido à acumulação de alterações genéticas e epigenéticas. Alguns dos grandes problemas associados a este tipo de cancro são o diagnóstico em fase tardia da doença e os poucos resultados terapêuticos. Uma das escolhas terapêuticas para o CCECP é a radioterapia, no entanto, esta tem diversos inconvenientes, como a radioresistência adquirida por algumas células tumorais, que se associam a piores prognósticos. Um aumento do conhecimento na área da biologia da radiação é necessário para melhorar esta opção terapêutica, evitando futuros efeitos tóxicos e fornecendo uma melhor qualidade de vida nos anos subsequentes ao tratamento. Desta forma, a identificação de marcadores moleculares associados quer a uma resposta à radioterapia, quer a possíveis alterações celulares após tratamento com radiação, é essencial para melhorar o diagnóstico, taxa de sobrevivência e qualidade de vida destes doentes. Adicionalmente, existe uma grande falha no conhecimento em relação aos efeitos da radiação nas células, como tal, o principal objetivo deste estudo foi o de contribuir para um conhecimento mais alargado do efeito da radiação em doentes com CCECP. Para isso foram utilizadas duas linhas comerciais celulares, HSC-3 (derivada de um tumor metastático da língua) e BICR-10 (derivada de um tumor da mucosa bucal), que foram caracterizadas com recurso a aCGH, MS-MLPA e citogenética convencional. Estas linhas foram submetidas a diferentes doses de radiação e as alterações genéticas e de metilação pós tratamento foram determinadas. Estes resultados demonstraram uma grande variação de resposta à radiação para estas duas linhas celulares, permitindo a conclusão que a linha HSC-3 é mais radiossensível que a linha BICR-10. Tendo isto em mente, procedeu-se a análise da morte celular, ciclo celular e danos no DNA de forma a tentar compreender esta diferença. A caracterização genética de ambas as linhas celulares permitiu corroborar que a linha HSC-3 era derivada de um tumor metastático e sugeriu que a linha celular BICR-10 estaria associada a um estado de displasia. Para além disto, foi possível analisar alterações genéticas e epigenéticas ocorridas após irradiação e associar determinados perfis genéticos a uma melhor ou pior resposta à radiação. Em suma, os nossos resultados contribuiram para um conhecimento mais aprofundado dos efeitos da radiação no CCECP possibilitando, no futuro, melhores opções de tratamento e uma melhor qualidade de vida para estes doentes