Multi-omics molecular profiling of lung tumours

Lung Cancer (LC) is one of the most common malignancies and is the leading cause of cancer death worldwide among both men and women. Current LC classifications are based on histopathological features which poorly reflect the molecular diversity of these tumours. Consequently, primary and secondary drug resistance are very frequent, and a high mortality is usual in LC patients. Despite the fact that LC has been intensively studied, there is a lack of effective biomarkers for early detection, stratification and prognosis. Integration of omics data is a powerful approach that can be used to identify molecular subgroups relevant in the clinical setting. This thesis addresses this challenge by characterising the molecular alterations accompanying LC at the genetic and DNA methylation level, using a combination of Whole-Exome Sequencing (WES), Targeted Capture Sequencing (TCS), Single Nucleotide Polymorphism (SNP) genotyping, Whole-Genome Bisulfite Sequencing and RNA-sequencing. The integration of different types of omics data first validated previous molecular alterations in frequently diagnosed LC tumours. This allowed comparison of the genomic and epigenomic landscapes between these common and rarer LC subtypes. Next, novel molecular subgroups of Non-Small Cell Lung Cancer (NSCLC) tumours with bad prognostic, as well as subgroups of Lung Carcinoids (L-CDs, an understudied LC subtype) have been identified and their molecular alterations and signatures characterised. Significant associations with histological features and gene expression programmes have been found by using several bioinformatic tools. These results show the value of multi-omics approaches to better understand the molecular mechanisms underlying LC and to identify new biomarkers. Importantly, some of these findings may be translatable and are likely to improve the detection, monitoring and stratification for targeted therapies in LC patients.Open Acces

Establishing methods for analysis of DNA methylation in breast cancer and cell-free circulating DNA

Breast cancer is the most common cancer in women worldwide. To date, diagnosis and metastasis monitoring are mainly carried out through tissue biopsy, a very invasive procedure limited only to certain locations and not always feasible in clinical practice. Tumour cells release DNA into the blood as circulating cell-­free DNA (cfDNA), which can be sampled from circulating blood, an approach known as liquid biopsy. This provides a resource for biomarkers that could allow the use of minimally invasive liquid biopsies for cancer-­related research, diagnostics, prognostics, and targeted therapy. The levels of cfDNA have already been shown to be higher in cancer individuals than healthy individuals, and correlate with tumour metastasis, response to therapy and recurrence. Recent technological advances have enabled the identification of both genetic and epigenetic aberrations in cfDNA that reflect changes also found in patients’ tumours. The host group performed methylation analysis using the Illumina EPIC Methylation Array, which interrogated CpG dinucleotide methylation at over 850,000 DNA sites. A total of 3172 CpGs showed median methylation differences of more than 40% between tumour and buffy coat of patients with breast cancer. This MRes project aimed to establish methods for detecting these methylation changes between matched tumour samples and leucocytes of breast cancer patients, and in cfDNA by using the Fluidigm 48.48 Access Array microfluidics system and the Illumina MiSeq sequencer. This approach provided quantitative, medium-­throughput targeted measurement of DNA methylation at single nucleotide resolution. Finally, bisulfite pyrosequencing was used as a sensitive validation technique for detecting differences in CpG methylation, providing a set of potential biomarkers that could be reliably detected by circulating tumour DNA-­based tests. Translating the alterations that are seen in the primary tumour into an assay that is applicable to cfDNA will have important diagnostic implications, such as monitoring tumour progression, drug response and disease recurrence, as well as the early detection of cancer, which could ultimately complement or even avoid the need for tumour tissue biopsies