Retrospective Analsyis of Clonal Transformation (RETROCLONE):
The RETROCLONE proposal involves the analysis of the BCR repertoire and genetic aberrations of at least 50 patients with a B-LPDs and their comparison with a matched paired sample of the same patient, collected in an average 10 years prior to diagnosis (when the patient was registered as a volunteer bone marrow donor at Karaiskakio Foundation, PA1). Patients follow up samples at several time points will be also analysed and results will be compared with diagnosis and pre-diagnosis time point. Results will be also paralleled to paired samples from age matched normal controls and patients with MBL. The unique opportunity of CSHM / Karaiskakio Foundation to access samples of patients collected many years prior to their diagnosis and post diagnosis with a haematological malignancy provides a distinctive opportunity to carry out a Retrospective Analsyis of Clonal Transformation (RETROCLONE) events involved in leukogenesis. We hypothesize that one or more populations of pre-leukemic clones with distinct BCR sequences many years prior to diagnosis, evolve distinctively based on a sequence of specific mutation events and emerged at presentation as the predominant malignant clone. Our aims are a) to apply a novel NGS method coupled with network construction and population analysis of the BCR rearrangement sequences, in B-LPDs diagnostic, pre-diagnostic and post-diagnosis samples, b) test the Somatic Hypermutation (SHM) status and B-cell immunoglobulin stereotypy, c) Investigate gene mutation evolution in B-LPDs applying NGS sequencing for genes correlated with LPDs and d) assess the expression of crucial markers for LPDs prognosis in order to identify their association with pre-diagnostic clone characteristics. The outcome of this top quality high-impact project is expected to shed light on the mechanisms of disease evolution. This information will be of critical importance for improving patients’ monitoring and clinical outcome.
Enhancing Adoptive T Cell Therapy via T Cell Costimulation: (ACT-Costimulation):
The ability to harness the immune system has revolutionized cancer therapeutics giving rise to a new group of treatments termed cancer immunotherapy (CI). CI includes adoptive T cell therapy (ACT) whereby autologous tumour specific T cells are isolated from the patient, expanded in vitro and re-infused back to the patient, the use of autologous but genetically engineered T cells for ACT, dendritic cell based cancer vaccines and immune checkpoint blockade therapy. ACT showed great promise and success in treating B-cell malignancies. ACT in solid cancers showed initial promise in metastatic melanoma but then faced a number of pitfalls in other types of solid tumours related to the properties of the tumours and the tumour microenvironment. A major parameter controlling the response of transferred T cells is their status (proliferative capacity and anti-tumour cytotoxicity) at the end of the in vitro manipulation that could involve only in vitro expansion and/or genetic manipulation to introduce a chimeric antigen receptor. We hypothesize that expanding mouse T cells in vitro using T cell receptor(TCR)/CD3 stimulation in the presence of additional stimulation via the CD2 costimulatory receptor generates T cells that are in a much better molecular and cellular state that translates to a more efficacious ACT modelled using in a triple negative breast mouse model compared to only TCR/CD3 stimulation (current common expansion protocol). We also hypothesize that the aforementioned T cell expansion protocol will benefit further, with increased in vivo efficacy, from the normalization of the tumour microenvironment mediated via in vivo pharmacological inhibition of transforming growth factor-β. Finally, we are investigating the role of in vivo CD2 costimulation in the efficacy of ACT with T cells expanded using the aforementioned in vitro protocol.
Suppressing tumour Invasion and vessel Emergence through Genetic Engineering (SIEGE):
The main feature of cancer is the uncontrolled cell proliferation that leads to the formation and accumulation of agglomerates, known as “malignant tumors”. These tumors can grow without restriction and migrate from one tissue to another affecting the various organs of the body, a condition known as “metastasis”. However, there is limited knowledge about what induces metastasis. This is the question of the SIEGE research project. SIEGE aims to investigate breast cancer metastatic drivers such as cell migration and escape from dormancy as well as other biological attributes like regulation of expression of certain surface markers and genetic vulnerabilities that could serve as potential therapeutic targets. The cancer type used in the study is Basal-Like Breast Cancer (BLBC), which is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. To perform these dropout, positive and functional screens we use breast cancer cell lines and a CRISPR functional screening toolkit composed of lentiviral gRNA expression vectors, Cas9 activity reporters and a human genome-wide CRISPR library consisting of 90,709 gRNAs targeting a total of 18,010 genes. For selection of cells harboring specific phenotypes, cells are sorted on different time points for specific surface markers. Following sorting, cells are pelleted, lysed, and undergo DNA extraction. gRNA cassettes are then PCR amplified and sequenced on an Illumina Next-Seq machine. Similarly, for drop-out screens, the entire cell populations are harvested for DNA extraction and sequenced. The in vivo part of the study involves the harvesting and injection of transduced cells in a previously established mouse model via a tail vein injection, to observe the metastatic potential of these breast cancer cells to the lungs. Post injection, mice are imaged once a week. At different time points, tumors are isolated, DNA extracted, gRNA cassettes PCR amplified and sequenced on an Illumina Next-Seq machine. In vitro, clonal expansion experiments are performed to identify fitness promoting knockouts, and the expanded cells are injected into mice to determine metastatic potential of the expanded clones. The Sequencing data from these experiments are bioinformatically analyzed for the expression of gRNA signatures present in the positive selection screens or absent in the dropout screens. We are also performing ranking of candidate genes putatively involved in BLBC metastasis as well as gene ontology and pathway analysis on all resulting hits. SIEGE is innovative because it aims to establish CRISPR-Cas9, a powerful gene-editing platform, as an approach to study escape of dormancy and cancer metastasis both in vitro and in vivo. We anticipate that our results will lead to the discovery of genetic pathways linked with escape of dormancy-metastasis in breast cancer. The elucidation of such pathways may lead to the finding of common metastatic pathways amongst different cancers and potentially contribute to the discovery of new therapeutic targets.
The Cyprus Genome Project:
Human genetic variation is vital for our understanding of the biological differences between populations and the susceptability and response to different diseases. Databases of genetic variants among different populations are maintained around the world to enable medical research. This is a population-level genetic variation study in the Cypriot population based on the DNA sequencing of 10,000 people in Cyprus.The Cyprus Genome variants are uploaded in Clinical Varsome and Franklin as a population variant filter. It is made available to the entire research community through an intuitive web-based browser www.cyprusgenome.org.