Clonal origins of relapse in ETV6-RUNX1 acute lymphoblastic leukemia.

B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1(+) acute lymphoblastic leukemia.

The genomic landscape of teenage and young adult T-cell acute lymphoblastic leukemia.

BACKGROUND: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T-cell acute lymphoblastic leukemia (T-ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T-ALL has a specific biological-molecular profile distinct from pediatric or adult T-ALL. METHODS: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15-26 years with primary or relapsed T-ALL, using a combination of Genome-Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real-time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1-14 years), 439 TYA (15-24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search. RESULTS: Genomic characterization of this large cohort of TYA T-ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation - relapsed T-ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways. CONCLUSIONS: All genetic aberrations in TYA T-ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T-ALL exhibits a transitional genomic profile. Analysis of matched presentation - relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub-clones associated with drug resistance (NT5C2 and TP53 mutations) and re-iterative mutation of known key T-ALL drivers, including NOTCH1.

Specialized fluorescence in situ hybridization (FISH) techniques for leukaemia research.

Fluorescence in situ hybridization (FISH) provides one of the few ways of analysing the genotype of individual cells, an important consideration for mixed cell populations such as those found in leukaemia. A more sophisticated variation combines fluorescence immunophenotyping and FISH for specific leukaemia-associated chromosome rearrangements. Combined immunophenotyping and FISH is a powerful tool to identify the cell lineage in which the leukaemia-specific chromosome rearrangement occurs and has been used to identify putative pre-leukaemic cells in normal cord blood. Another valuable FISH-based research technique is multi-fluor FISH (M-FISH). This multicolour approach is effectively a molecular karyotype of individual cells and has a range of applications, from chromosome breakage studies and characterising mouse models of leukaemia, to providing a perfect complementary approach to the emerging genomic microarray technologies.

Genetic and functional diversity of propagating cells in glioblastoma.

Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure. This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.

Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia.

Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.