ALBERT

Description: T cell cancers are common in pediatric oncology, and include the clinical entities T cell acute lymphoblastic leukemia (T-ALL) and the Non-Hodgkin Lymphoma, T cell lymphoblastic lymphoma (T-LBL). These diseases carry worse prognoses than their B cell counterparts, and the regimens used in their treatment confer significant short- and long-term morbidities. Unlike many pediatric malignancies, which have characteristic chromosomal translocations, most T cell-derived cancers carry no such cytogenetic hallmark. Because such straightforward oncogenic lesions are typically absent, insights into the genetic underpinnings and molecular pathogenesis of these T cell diseases are also less comprehensive. To address these deficiencies, we have performed a forward genetic screen for T cell malignancies using the vertebrate model organism, zebrafish (Danio rerio). Although others have used transgenic zebrafish to show that known mammalian oncogenes can induce zebrafish leukemias, to date, this powerful animal model has not been used as a discovery tool to elucidate novel genetic mutations underlying de novo T cell malignancies. Zebrafish share ontogeny with other vertebrates, and possess an adaptive immune system strikingly similar to mammals. However, unlike other vertebrate models, zebrafish are amenable to forward genetic approaches, where animals are randomly mutagenized, and then screened using high-throughput strategies to detect rarely-occurring phenotypes. Using a zebrafish line with T lymphocyte-specific expression of green fluorescent protein (GFP), we have conducted a germline mutagenesis screen to identify animals that develop T cell malignancy. Thus far, we have isolated three independent lines with a heritable predisposition to developing T cell cancers. In all three lines, age of onset, patterns of spread, and morphologic appearance resemble human T-ALL/-LBL. In addition, we have compiled histologic and immunohistochemical evidence further characterizing these diseases. Analysis of T cell receptor (TCR) β gene sequences from malignant tissue has verified the monoclonality of malignant cells, based on identical VDJ rearrangements. Malignant cells have also been successfully transplanted serially into sub-lethally irradiated allogeneic recipients, using as few as 2500 donor cells to transfer disease. Furthermore, we have shown the malignancies themselves to be radiation sensitive, like their human correlates. Soon after radiation-induced remissions are achieved, however, high rates of aggressive disease relapse are seen, modeling recurrent human T-ALL/-LBL. In summary, we have identified three independent zebrafish mutant lines, which each recapitulate human T lymphocyte malignancy, and that are all heritably transmissible from one generation to the next. The positional cloning of the underlying genetic mutation of each line will provide exciting candidates for study in these human diseases. Moreover, using these lines as subjects in ongoing investigations, we are examining the genomic, epigenetic, and transcriptional profiles of these malignant cell populations, comparing pre- and post-transplant samples, pre- and post-irradiation isolates, and following other accepted and investigational therapeutic interventions. Finally, these lines are now poised for utilization in additional high-throughput screening approaches, including forward genetic suppressor and enhancer mutagenesis projects, as well as small molecule drug screens. Collectively, these new vertebrate models of T-ALL/-LBL should provide vital experimental platforms for studies of this important class of human cancers.

Description: Abstract 1192 T cell acute lymphoblastic leukemia (T-ALL) is a challenging clinical entity. Over half of adult, and about 20% of pediatric, T-ALL patients either relapse or fail to achieve remission, and despite salvage attempts outcomes are poor. To discover new acquired genetic changes that occur in T-ALL, as well as those that contribute to disease relapse, we studied zebrafish (Danio rerio) T-ALL samples using array comparative genomic hybridization (aCGH). Five different D. rerio T-ALL models have been described, and all 5 develop neoplasias that clinically and molecularly resemble human T-ALL. We performed aCGH on 14 de novo T-ALLs representing 4 of these models. To evaluate possible similarities between human and zebrafish copy number aberrations (CNAs) in T-ALL, we compared all D. rerio genes found in any CNA in our 14 zebrafish cancers to a cohort comprised of 61 published primary human T-ALLs analyzed by aCGH. In these D. rerio CNAs, we identified 764 genes with human homologues. Of these, we found significant overlap (62%) with genes in CNAs from the human T-ALL dataset. In addition, 10 genes recurrently altered (〉3 samples) in zebrafish T-ALL were also seen in CNAs from 5 or more human T-ALL cases, suggesting a conserved role for these loci in T-ALL transformation across vertebrate species. In addition to studying primary T-ALL, we also conducted iterative allo-transplantations with 3 zebrafish malignancies. This technique selects for particularly aggressive disease, increasing engraftment rates and also resulting in shorter time to death of recipient animals in successive transplant rounds. Because these passaged cancers show more malignant behavior in vivo, this procedure models refractory and relapsed T-ALL. In these 3 serially-passaged cancers, 55% of the original CNAs were preserved after iterative transplantation, demonstrating the clonal relationship between the primary and passaged leukemia. In addition, 101 CNAs were acquired during passaging of the 3 T-ALLs. Genes in these loci may underlie the enhanced malignant behavior of these neoplasias. We also compared genes found in CNAs from passaged zebrafish malignancies to human aCGH results from 50 T-ALL patients who failed induction, went on to relapse, or had already relapsed. Again, many genes (n=76) were present in both datasets. Four genes altered in 2 of 3 D. rerio samples were also found in 〉4 cases from the human dataset. Collectively, these results suggest that zebrafish and human T-ALL are similar at the genomic level, with several homologous genes commonly gained or lost by cancers from both organisms. Genes from recurring CNAs in disease samples from both species may have particular relevance, as these candidates likely have conserved roles in T cell oncogenesis or T-ALL disease progression. As the comprehensive list of genes in CNAs from human T-ALL cases is vast and heterogeneous (comprising 15,724 unique genes from just 75 clinical samples), zebrafish T-ALL models may permit a more expedient prioritization of these candidates for further investigation. Disclosures: No relevant conflicts of interest to declare.