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Project Description

PhD1 - Study of ctDNA biomarkers in the context of bowel cancer screening  programmes.

Background: Colorectal cancer is one of the most prevalent tumours and an important disease burden. Current prevention strategies largely rely on the implementation of population-wide screening programmes that rely on FIT as a proxy for bowel malignancy. Nevertheless, the sensitivity of FIT testing could be complemented with other molecular tests to optimise detection rates.

Hypothesis: we believe that liquid biopsy strategies could make a relevant contribution for risk biomarker development in the context of screening.

Objectives: The main aim of this project is to assess the usefulness of ctDNA mutation detection for patients undergoing CRC screening. We aim to describe the sensitivity and specificity of these biomarkers to be detected in the circulation as opposed to the primary bowel neoplasia, particularly in the context of pre-malignant lesions (polyps). We will also inspect the usefulness of ctDNA mutation detection for improved screening in combination with heritable genetic risk predictors.

Methodology: Patients will be categorised into risk groups depending on the endoscopic and histological findings, and following the guidelines of the Spanish Gastroenterology Association. PhD1 will evaluate the different ctDNA available technologies (NGS-based, ddPCR-based) and identify that with the highest sensitivity/specificity for early stages (pre-malignant polyps and localised tumours). Then, for each of the 150 patients, he/she will obtain the genomic profiles of: a) somatic mutation of the primary growth (including hotspot driver mutations; and b) the circulatory somatic mutation profile. The ctDNA predictivity will also be compared and/or used in conjunction with the a priori genetic polygenic risk score obtained from the genotyping of the 200+ SNPs that influence CRC risk. Models will be created to conjugate the fixed predisposition component together with epidemiological risk factors and dynamic ctDNA values to identify patients that should undergo colonoscopy. 

PhD2 - A multi-omics and pathway centric investigation of the relationship between lifestyle factors & CRC risk.

Background: Colorectal cancer (CRC) is one of the most prevalent tumours and an important disease burden. Current prevention strategies largely rely on the implementation of populationwide screening programmes (BSCP), that rely on FIT as a proxy for bowel malignancy. Nevertheless, BCSP has low adherence and the sensitivity of FIT testing could be complemented with other molecular tests to optimise detection rates.

Hypothesis: We believe that metabolomics data together with lifestyle and other omics data available in large prospective studies can help identify biomarkers predictive of CRC risk development, which could be relevant in the context of BCSP.

Objectives: To use available metabolomics and other multi-omics data, together with detailed anthropometric and lifestyle data from 1,120 matched case-control pairs nested within the EPIC cohort and other datasets available from ColoMARK  to understand CRC development. This project has received funding from the European Union´s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie Doctoral Network grant agreement No. 101072448  from the perspective of metabolic perturbations from its earliest stages.

Methodology: PhD2 will develop and apply supervised and unsupervised machine learning methods for the construction of omics and multi-omics signatures associated with CRC risk. Multi-omics signatures of CRC risk derived in different subgroups of individuals characterised by specific lifestyle patterns will lead to the identification of candidate metabolites and genes that might underpin the impact of lifestyle on colorectal cancer development. The corresponding biological pathways will be explored making use of state-of-the art bioinformatics network-based tools to combine our results with prior knowledge from the literature and biochemical databases.

PhD3 - Exploring the role of the microbiome, gut barrier dysfunction, host genetics, and metabolic interactions in the development of colorectal cancer.

Background: Accumulating research suggests that a disturbance in the gut microbiome can influence development of colorectal neoplasia from early lesions to tumours.

Hypothesis: Gut microbiome involvement in colorectal carcinogenesis may occur through inflammatory-induced weakening of the protective gut mucosal barrier by obesity, dietary/lifestyle, and microbiome metabolic factors that lead to exposure of the gut epithelium to pathogenic bacteria and their toxins.

Objective: To apply circulating (liquid biopsy) measures of gut barrier dysfunction and bacterial translocation into the circulation as biomarkers of colorectal carcinogenesis and disease progression. Blood-based detection of bacteria and gut-barrier health may allow novel screening strategies for CRC cancer prevention, diagnosis, and management.

Methodology: Measures of gut barrier dysfunction will be assessed by protein ELISA assays, while bacterial translocation and metabolic activity will be ascertained by bacterial antigen immunotyping, plasma metabolomics, and circulating bacterial DNA sequencing. The contribution of host genetics related to immune/microbiome and microbial metabolite interactions will be assessed using existing GWAS data. This will be conducted in patient casecontrol cohorts of colorectal adenomas and cancer, available in UCD and through ColoMARK partners. Additionally, the project will include data analysis integration with the metabolomics data, together with detailed anthropometric and lifestyle data, from 1,120 matched case-control pairs nested within the EPIC cohort in PhD2. Analytic approaches will include multivariable logistic regression and mediation analyses. Thus, this PhD will involve laboratory assays such as ELISA assays, qPCR, and metagenomic sequencing, but there will be a major focus on biostatistical and bioinformatic approaches. Together, this will help shed light on the involvement of microbial translocation and microbially derived metabolic perturbations on adenoma progression and CRC development.

PhD4 - Assessment of the correlation between primary tumour features and ctDNA release.

Background: CRC stage at diagnosis plays a key role in determining the outcome of disease treatment and overall survival. On average, the five-year survival for CRC is about 60%, but this varies greatly, from 90% for stage I patients to 10% for those diagnosed with metastatic disease, which make up to 20% of the diagnoses. Incidence is rising due to increasing population age and life. The Doctoral Network ColoMARK aims to find immediate solutions to better prevent, treat, and manage CRC. 
The analysis of ctDNA (cell-free circulating tumour DNA) is a very promising tool and is thought to revolutionize cancer care with respect to early detection, identification of minimal residual disease, assessment of treatment response, and monitoring tumour evolution (summarized in 1-5). However, a major challenge for ctDNA applications is the differentiation of circulating DNA derived from the tumour from non-tumour circulating DNA (cfDNA). In principle, attempts to use cfDNA/ctDNA as a cancer biomarker focus on two classes of alterations, i.e. quantitative and qualitative abnormalities. These approaches appeared powerful and achieved a good sensitivity, however prior knowledge about the tumour-associated alterations is necessary. Even though the detection of cancer-specific mutations offers a genotypic means to distinguish tumoral from non-tumoral plasma DNA, a major problem is that every cancer has a unique fingerprint and therefore there is no universal marker that can be used for cancer screening. Exact knowledge about the biology of cfDNA/ctDNA might reveal other useful parameters, which can be used in the early detection setting and might dramatically increase sensitivity/specificity for detecting early-stage disease. 

Hypothesis: We hypothesize that the rate of shedding of ctDNA into the circulation is dependent upon the location, size, and vascularity of the tumour and therefore leads to a high variability in levels across patients. A sharper picture of the biology and kinetics of ctDNA release and turnover should expand the utility of circulating nucleic acids as tumour markers.

Objective: Compared to the number of studies addressing the clinical applicability of ctDNA, data regarding the actual origin, the kinetics, and the mechanisms of release and clearance are limited and often contradictory. The aim of the proposed thesis is to close this gap. Although the ability to detect mutations or other cancer-specific alterations in plasma cell-free DNA (cfDNA) is thought to correlate with the tumour burden6-8, the exact size and the degree of vascularization for DNA release into the circulation have not yet been tested. The doctoral candidate will address this question using novel cutting-edge technologies for tumour profiling and ctDNA detection. 

Methodology: We will comprehensively analyse primary tumours from localized CRC patients at various omics-levels, whose tumours will be resected with curative intent. Genetic alterations at the genome-level will be tracked to in plasma. Tumours will be classified as ctDNA shedders and non-shedders and using bioinformatics and statistical methods such as PCA, correlation and regression analysis, we will search for genetic, histological, or phenotypic features that are associated with ctDNA release.

PhD5 - Retention of subtyping from tissues to liquid biopsies.

Background: Classical tissue biopsy approaches have proven to be pivotal for our fundamental as well as clinical understanding of colorectal cancer. However, such studies are hindered by several issues, including tumour heterogeneity, limiting representative biopsy sampling. The collection of study samples in this context is complex and generally requires invasive procedures (surgery, endoscopy), which preclude multiple sampling. As a result, progress is still to be made when it comes to effective prediction, diagnosis and treatment of colorectal cancer.

Hypothesis: The emergence of liquid biopsy approaches has reinvigorated tumour biomarkers research due to easier access, less invasiveness and allowing for serial sampling. This ultimately could contribute to a more accurate reflection of active disease (4-6), while posing much less of a burden for the patient.

Objective: To facilitate the transition from the classical, colorectal tissue biopsies approaches, to liquid biopsy approaches in the clinical setting, we will inspect consensus molecular subgroup (CMS) subtyping retention in liquid biopsies as a tool for tumour characterization.

Methodology: To this extent, the candidate will largely focus on the bioinformatic characterization of the primary/metastatic CMS, originating from tissue biopsy NGS data, and correlate this with matched liquid biopsy NGS data. In addition, we aim to generate a systems biology methodology that allows for liquid biopsy-based early diagnosis and/or subtyping of CRC patients. Lastly, the candidate will be heavily involved in collaborative projects that ultimately aim to push consortium-wide data integration.

PhD6 - Development of dynamic chemistry labelling assays to quantify circulating small non-coding RNAs directly from body fluids for the early detection of CRC.

Background: Small non-coding RNAs (sncRNAs) are important regulators of various biological processes. They are present in biological fluids with different expression levels under pathological conditions. sncRNAs are rapidly emerging as important biomarkers for clinical diagnosis and prognosis of various human diseases including Colorectal Cancer (CRC).

Hypothesis: Profiling of sncRNAs is one of the most active areas of research in the field of liquid biopsy. DESTINA offers a new way to interrogate sequences of sncRNAs through its unique Dynamic Chemistry Labelling (DCL) technology.

Objectives: The objectives of the present project is to develop novel DESTINA reagents and protocols to interrogate and quantify a panel of sncRNAs, both miRNAs and ctRNAs, in either singleplex or multiplex manner, for the early detection of CRC. The new set of reagents will be used to implement DCL on some of the major diagnostic platforms. Optimal diagnostic platform will be selected by depending on what limit of quantification is required for accurate and reliable direct testing of sncRNAs.

Methodology: The PhD candidate will perform the design and solid-phase synthesis of functionalized PNA oligomers (DESTINA probes) as well as novel tagged SMART-Bases. DESTINA probes will be designed in order to capture complementary RNA target molecules identified as biomarkers for early diagnosis of CRC. SMART-Bases will be biotinylated in order to perform the DCL on bead-based reading platforms such as Luminex MAGPIX, Quanterix SR-X and Merck SMCxPro. For the merging of DESTINA’s new reagents with the bead-based platforms. DESTINA probes will be coupled with magnetic beads so as to generate new assay kits capable of making an absolute quantification of target sncRNAs directly from body fluids. Protocols for the new kits will be implemented initially by interrogating  synthetic RNA mimic oligomers. The analytical performance of the assay kits will be compared with goal standard and SOPs will be produced.  Concluding, novel reagents, kits and assays will be employed to interrogate sncRNAs with early diagnostic value. The project includes assay optimization and validation with clinical samples provided by other ColoMARK members.

PhD7 - Circulating non-coding RNAs as a source of biomarkers for colorectal cancer prognosis and monitoring.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death. Although many improvements have been made in the last years, there is still a need to identify and develop novel, clinically useful biomarkers that can improve disease outcome. Small non-coding RNAs (sncRNAs) show altered expression patterns and play important regulatory roles in colorectal cancer development and progression. The fact that they can be detected in different body fluids in a stable manner makes them promising new non-invasive biomarkers.

Hypothesis: We believe that detection of sncRNAs in liquid biopsies could make a relevant contribution for biomarker development to improve CRC management.

Objectives & Methodology: The main aim of this project is to identify and validate novel sncRNA-based CRC biomarkers with prognostic value via liquid biopsy approaches by comparing different state-of-the-art technologies. To achieve that aim we will firstly select sncRNA candidates from next-generation sequencing followed by clinical performance assessment by RT-qPCR on liquid biopsies from CRC patients. The best set of sncRNA biomarkers will be also assessed with a different technology developed by another ColoMARK member and performance comparison will be carried out. Relevant results will be validated in independent cohorts from the ColoMARK consortium.

PhD8 - Diagnostic Leukapheresis and intraoperative samples to enhance CTC detection in early CRC.

Background: In localised CRC, the detection of CTCs could help to identify patients at risk for metastasis and could help to stratify them to adjuvant therapies.

Hypothesis: Beyond enumeration, the greater potential for CTC-based liquid biopsies lies in their subsequent molecular characterization to provide predictive information for molecular therapies. On the other hand, the analysis of CTCs can provide information on the mechanisms of tumour dissemination. However, the infrequent and unreliable detection of CTCs, poses a limitation for diagnostic applications in localised CRC. To increase CTC-detection, we have developed diagnostic leukapheresis (DLA) that enables the screening of litres of blood, and we have implemented a program for collection of blood samples during surgical intervention for tumour removal.

Objectives & methodology: With this project we aim specifically: 1) To test the value of Diagnostic Leukapheresis (DLA) and intraoperative samples (from the tumour-draining vein and central venous line) in localised CRC for CTC-detection and identification of patients at risk for metastatic disease. 2) To check the utility of DLA and intraoperatively collected CTCs for clinically relevant diagnostic tests (KRAS mutation testing, EGFR expression etc.). 3) To take advantage of the increased CTC yields in DLA products and intraoperative blood samples for comprehensive molecular analysis to dissect the biology of the potential precursor cells of metastatic relapse. 4) To test the value of DLA and intraoperative blood samples for detection of other circulating biomarkers (ctDNA, ctmiRNAs, microbiome and metabolomics). 5) Explore correlation between circulating tumour biomarkers (CTCs, ctDNA) and the integrity status of the gut-blood barrier.

PhD9 - Prognostic value of TP53 in exosomes from liquid biopsy studies.

Background: TP53 is the most frequently mutated gene in cancer and both germline and somatic mutations in the TP53 gene play an important role during tumorigenesis and TP53 mutations are a prognostic indicator in cancer. This has been demonstrated in prostate cancer, both in tissues  and in liquid biopsies.

Hypothesis: TP53 is a complex gene with different isoforms. Besides the canonical TP53, there are also the alternatively spliced p53 isoforms, p53β and p53γ. Together, all these variant codes for a multiprotein complex for which the functions are not fully understood. The composite P53 complex has functions in DNA repair, growth arrest and apoptosis. In several families with inherited cancer, mutations in the TP53β isoform have been shown to have an effect on TP53 functioning and possibly predispose to various cancer types including CRC. Interestingly, the TP53 isoforms p53β has been shown to be present in exosomes as well.

Objectives: We will study TP53 in ctDNA and exosomes in relation to prognosis in CRC, with a focus  on TP53β.

Methodology: A variety of techniques will be used, including RNA and protein expression analysis and NGS based ctDNA detection techniques. The most optimal exosome detection methods will be compared and investigated for our studies.

PhD10 - Single-cell RNA sequencing of circulating tumour cells (CTCs) in CRC patients to identify novel biomarkers of disease monitoring and progression.

Background: Colorectal cancer is one of the most prevalent tumours and an important disease burden. Mortality rates are especially relevant in CRC, with later metastatic stages responding poorly to conventional treatments.

Hypothesis: secondary tumours (metastases) are the main cause for CRC-related deaths. In order for these to develop, tumour cells must travel from the primary location to the distant organs through the circulation. The properties of these circulating tumour cells (CTCs) could therefore harbour important information on the CRC metastatic process and provide relevant clues on how to monitor and stop the disease.

Objectives: The main aim of this project is to utilise circulating tumour cells (CTCs and their transcriptomic landscape over the course of the disease and treatment to identify novel biomarkers that can help us guide therapeutic strategies.

Methodology: CRC patients will be recruited and single CTCs isolated to perform single-cell RNA sequencing. PhD10 will perform a comparison of CTC isolation procedures and techniques to obtain SOPs for the isolation of CTCs for downstream transcriptomic analyses. The dynamic CTC transcriptomic data will be mined to identify biomarkers that can help predict disease outcome and guide therapeutic strategies. The CTC results will be compared with those obtained from primary tumour analyses and other complementary biomarkers, to create optimal models. Then, results will be validated in additional ColoMARK datasets.

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