https://www.um.edu.mt/library/oar/handle/123456789/31663 2026-04-15T19:47:38Z https://www.um.edu.mt/library/oar/handle/123456789/143490 Title: Gene amplifications associated with primary colorectal cancer tumours and matched lymph nodes Abstract: Colorectal cancer is among the most commonly diagnosed malignancies worldwide, with metastasis being the leading cause of mortality. CRC is a genetically heterogenous disease driven by a multitude of genomic alterations. Gene amplifications contribute to tumour progression, metastasis and therapy resistance by increasing the copy number of specific DNA sequences, thus leading to overexpression of genes that can alter signalling pathways, cell proliferation and survival. This study aimed to investigate gene amplifications in primary CRC tumours and matched metastatic lymph nodes to better understand their role in metastatic progression. While many studies focus on gene mutations in CRC, there is a relative lack of research on gene amplifications and their correlations with gene expression in matched tissue samples. Gene expression analysis of both the primary tumours and the matched metastatic lymph nodes was performed using the QuantiGene™ Plex assay, revealing high expression of several genes in the primary tumours, namely MET, GNAS, IGF2, TOP1, MTIF3, CDX2, MYC, MCL1 and KLF5. Four genes – MET, GNAS, IGF2 and TOP1 – were selected for analysis on matched lymph nodes with ddPCR using primer/probe sets designed in-house. IHC staining with FN1 was also done on a select number of cases to assess protein expression levels. Of interest, comparing the presence of amplifications with the gene expression in matched primary tumours, high IGF2 expression in the primary tumour was observed in the amplified samples. ddPCR analysis of CNVs in lymph nodes, resulted in amplification frequency of MET (58.3%), GNAS (45.8%), IGF2 (6.25%) and TOP1 (95.8%) in lymph nodes (n=48). Overall, correlation of gene expression and CNV was low and hence, these findings suggest that measuring both will give a more comprehensive understanding of metastatic progression. Study limitations included degradation over the years in the FFPE material used and a limited sample size, restricting the statistical power and limiting data analysis to visual interpretation via box plots. Despite these consstraints, this study establishes the basis for further studies, primarily the comparison of matched primary tumours and lymph nodes, including comparisons with matched invasion fronts and tumour buds, an emerging histological characteristic that is currently being investigated. Description: M.Sc.(Melit.) 2025-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/140736 Title: Increasing platelet concentrate shelf life from 5 to 7 days : a pilot study Abstract: According to current guidelines, platelets are stored at 22°C±2°C with constant agitation in bags that permit proper gaseous exchange for up to 5 days. The need and demand for platelet transfusions is constantly on the rise because of their function in haemostasis, and being a vital part of cancer treatment, as well as prophylactic administration in certain cases. The short shelf life of platelets poses a problem in terms of availability and a costly waste of resources, equipment, and personnel time management. Increasing the shelf life from the current 5-day storage period to 7 days is one way of managing this problem. A 7-day storage shelf life is already implemented by various blood banks worldwide. It is well known that there are certain risks in extending the shelf life of this blood component. Being stored at a temperature of 22±°C makes platelets an optimal medium for bacteria to flourish and may also subject them to what is known as platelet storage lesions, two factors that need to be considered before addressing an extension in shelf life. This study evaluated the impact of extending platelet storage from 5 to 7 days, focusing on key safety and quality parameters. 20 pooled platelet concentrates were monitored at days 1, 5 and 7 for platelet count and indices, pH, glucose, and lactate dehydrogenase, which are established markers of cell viability and function. Cytokine levels of platelet factor 4 (PF4), interleukin-6 (IL-6), interleukin-8 (IL-8), and transforming growth factor-beta (TGF-β) were measured using ELISA to assess cytokine accumulation linked to febrile transfusion reactions. Sterility testing via BacT/ALERT bottles was performed on day 7 to check for microbial contamination. Statistical analysis of the results obtained, revealed no significant differences between days 5 and 7 across all parameters, with p-values all above the 0.05 level of significance. No bacterial growth was detected in any samples. These findings suggest that extending the shelf life of platelets from 5 to 7 days may be feasible without compromising safety or quality, supporting potential implementation by the local blood establishment. Description: M.Sc.(Hons.)(Melit.) 2025-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/132386 Title: Investigating the viability of cold-stored platelet concentrates and the effects of prolonged incubation Abstract: Thrombocytes, more commonly referred to as platelets, are cellular fragments produced within the bone marrow that play a pivotal role in hemostasis. Cancer patients, trauma patients and individuals who suffer from platelet disorders can benefit from platelet transfusions to ameliorate their condition and state of health. Upon donation, platelet concentrates (PC) are stored at a temperature of 22oC ± 2 oC in gas permeant bags under constant agitation for five days. Currently there are no guidelines available for cold storage and clinical trials are already underway. This study addressed issues related to logistics and blood product wastage by determining whether the quality and viability of platelets are affected if a lower temperature and a longer incubation period is put into practice. In this study, PC were divided into two cohorts, each of which were stored at different temperatures, one in refrigerated conditions (2oC to 6oC) and the other one at room temperature (22oC ± 2 oC). Each aliquot was analysed at different time intervals for parameters which literature has identified as markers of safety and quality. Results were statistically compared to determine whether there was any significant difference between the two cohorts. The investigation yielded predominantly positive results across three main aspects of platelet analysis. Parameters such as the Platelet count (PLT) remained stable, whereas the Mean Platelet Volume (MPV) and the Plateletcrit (PCT) showed significant variations. Platelet metabolism was evident in both room temperature and cold storage, with a notable decreased rate in the latter. pH levels remained within acceptable transfusion thresholds under both storage conditions. No statistically significant differences in cytokine levels were observed between room temperature and cold storage or across different incubation days, although mean cytokine levels were higher at room temperature. In conclusion, an overall positive result was collected for cold-stored platelets however, further studies are still required to fully establish their efficacy in real-life in-vivo scenarios. Description: M.Sc.(Melit.) 2024-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/131933 Title: Investigation of the stem cell compartment in patients with chronic cytopenias and low-risk MDS Abstract: Myelodysplastic syndromes (MDS) are defined by the WHO as a group of clonal haematopoietic stem cell (HSC) disorders which are characterised by cytopenias, ineffective haematopoiesis, dysplasia, the presence of blasts and an increased risk of developing Acute Myeloid Leukaemia (AML) (WHO,2022) Patients with persistent cytopenias are relatively frequent encounters in routine Haematology screening and the clinical management of these patients can be challenging due to unpredictable clinical course. This study aims to investigate the stem cell compartment of patients with persistent, unexplained cytopenias, and provide insights into the clinical behaviour of these conditions, aiding in better clinical management. In this study, the stem cell compartment of 53 patients was investigated in two separate cohorts: Cohort A (n=30) included patients with persistent cytopenias (potential pre-MDS conditions, or low-risk MDS) and Cohort B (n=23) patients with high-risk MDS and AML, as a control group. A one-tube flow cytometric assay was used for the detection of leukaemic stem cells (LSCs) using a combination of 13 different monoclonal antibodies, to identify immunophenotypic aberrancies. Molecular studies by next-generation sequencing (NGS) were carried out using a targeted myeloid NGS panel to detect any molecular aberrations. Immunophenotypic findings were then correlated with the molecular findings to confirm or otherwise the clonal nature of cytopenias. LSCs were found in 60% of patients from Cohort A and 91% of patients from Cohort B with the most common LSC markers being CD45RA and Combi markers. LSCs were detected at higher percentages in Cohort B. Various molecular aberrations which are commonly associated with MDS and AML were also detected in both Cohorts. There was high agreement between Immunophenotyping and Molecular results in Cohort A (56.6%) and Cohort B (91.3%). Cohort A was further sub-classified into low-risk MDS (50%), ICUS (33%), CCUS (7%) and ‘Other’ (10%) based on cytopenias, dysplasia and the presence of molecular aberrations. The presence of LSCs in 80% of LR-MDS patients, is an important adjunct finding that may prompt clinicians to monitor these patients more closely, with early therapeutic interventions in certain cases. In conclusion, patients with persistent cytopenias together with the presence of LSCs and molecular aberrations might have an increased risk of leukaemic progression and should be monitored more closely. The LSC assay provides valuable information on the stem cell compartment, better guiding clinicians on the course of action for patients with persistent cytopenias. Detection of LSCs is also important in view of the development of therapeutic targets such as immunotherapy targeted towards aberrant markers including CD33, CD123, TIM-3 and CLL-1 leading to more specific and personalised treatments (Hansen et al., 2022). Molecular analysis is also very important for patient stratification, prognosis and targeted therapy. The strong concordance between immunophenotyping and molecular results shows the importance of using a holistic approach when investigating patients with persistent cytopenia and suspected MDS. Description: M.Sc.(Melit.) 2024-01-01T00:00:00Z