- Title
- Analysis of Fat1 cadherin and identification of novel biomarkers for acute leukemia
- Creator
- Ardjmand, Alireza
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Childhood leukaemia is one of the success stories of modern oncology with cure rates of around 80% although a significant number of cases still relapse and die. The therapeutic approaches for adult leukaemia’s are far less successful and collectively the unmet needs of these patients’ drives the need for improved clinical management approaches (diagnostics, prognostics and treatments). Here one of the main obstacles involves the specific identification of leukaemic progenitors but because they originate from their normal counterparts, the discovery of biomarkers that exclusively recognise leukaemic cells has proven to be a demanding task. Continuing the search for new and improved leukaemia biomarkers, this thesis addressed the general hypothesis that Fat1 represents a novel marker for both diagnostic and therapeutic applications in leukaemia. Fat1 is the largest known member of the cadherins, a large superfamily known for their ability to mediate cell-cell and cell substrate adhesion. Seminal studies have shown that the Drosophila homologue of Fat1 functions as a tumour suppressor and engages in fundamental signalling pathways controlling growth and differentiation during development. In Chapter 2, the expression of Fat1 was examined using quantitative PCR and Western blotting in a panel of leukaemic cell lines together with normal blood cells. Fat1 expression was identified in a number of acute lymphoid (ALL) and myeloid (AML) cell lines whereas no expression could be detected in PBMC’s or HSCs from peripheral blood and bone marrow of normal individuals. In silico mining of microarray expression data confirmed the expression of Fat1 in clinical ALL specimens, and further analyses using quantitative PCR in clinical samples revealed the presence of Fat1 mRNA transcripts in 11% of AML, 29% of B-ALL and 63% of T-ALL, respectively. An intensive search throughout normal haematopoiesis found mostly negligible levels of Fat1 mRNA in all of the major haematopoietic lineages, indicating that Fat1 represents a truly differentially expressed leukaemia-antigen. Further investigation across two independent matched diagnosis-relapse cohorts of pre-B ALL found that high Fat1 expression at the time of diagnosis was an independent prognostic marker of poor outcome (relapse-free and overall survival) therefore also suggesting some role for Fat1 in the biology of relapse. In the clinical setting, the measurement of MRD fulfils an important role in the clinical management of ALL, both as a prognostic feature and to direct the proper timing of therapy. To exploit the overexpression of Fat1 in pre-B- and T-ALL, Chapter 3 examined the potential of Fat1 to be used as a marker of MRD. Analysing microarray data from 125 matched diagnosis/relapse samples across three independent data sets, Fat1 mRNA was detectable in an average of 31.3% of diagnosed pre-B-ALL, of which 67.5% of cases remained positive at relapse. Furthermore, some 20% of cases with undetectable levels of Fat1 mRNA at diagnosis became positive upon relapse. Of note we also found that 15.6% of these cases lack any known chromosomal translocations and gene mutations. We also found 83.3% of T-ALL cases were positive for Fat1 expression at diagnosis which 77.7% of them remained positive at relapse. Towards proof of concept, a quantitative polymerase chain reaction assay was developed. Using standardised samples where Fat1-postive leukemic cells were mixed with normal peripheral blood cells, Fat1 mRNA could be detected at a sensitivity of 1 in 10,000 to 100,000 cells. Meeting the established clinical criteria, the results therefore suggest Fat1 could be employed as a marker of MRD for a major subset of patients with ALL including patients without known genetic aberrations. As illustrated by the example of Fat1, no one leukaemia marker is sufficient to identify all cases of the disease. Therefore in Chapter 4 it was hypothesised that a small number of overexpressed genes could provide complete MRD coverage for all patients. This idea was then tested using genome wide expression arrays in combination with a computational approach to identify compatible sets of genes. Using differentially expressed genes, i.e. those expressed in preB-ALL but not in normal B or hematopoietic stem cells, an integer-programming model was used to identify sets of genes that collectively identify the maximum number of preB-ALL cases. As a further refinement we validated the expression of candidate genes in distinct hematopoietic subpopulations for lack of expression in normal cells. Accordingly 13 overexpressed genes were identified in nine sets of three genes. Collectively each unique set provided potential MRD coverage for at least 99.3% of the entire disease cohort. None of the 13 genes have previously been reported in context of pediatric preB-ALL. However, the majority of the identified genes have been implicated directly or indirectly in tumorigenesis or have been recognised as tumor biomarkers in various cancer settings. We also characterised these genes for their continued overexpression in leukemic cells at relapse with a number of identified MRD sets of likely clinical utility.
- Subject
- Fat1; leukemia; biomarkers; cadherins; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1045339
- Identifier
- uon:14447
- Rights
- This thesis is currently under embargo and will be released at a date to be advised, Copyright 2014 Alireza Ardjmand
- Language
- eng
- Full Text
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