Ian Titley

Genetic variegation of clonal architecture and propagating cells in leukaemia

Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6–RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or ‘driver’ copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγnull mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1–positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma

Although intratumor heterogeneity has been inferred in multiple myeloma (MM), little is known about its subclonal phylogeny. To describe such phylogenetic trees in a series of patients with MM, we perform whole-exome sequencing and single-cell genetic analysis. Our results demonstrate that at presentation myeloma is composed of two to six different major clones, which are related by linear and branching phylogenies. Remarkably, the earliest myeloma-initiating clones, some of which only had the initiating t(11;14), were still present at low frequencies at the time of diagnosis. For the first time in myeloma, we demonstrate parallel evolution whereby two independent clones activate the RAS/MAPK pathway through RAS mutations and give rise subsequently to distinct subclonal lineages. We also report the co-occurrence of RAS and interferon regulatory factor 4 (IRF4) p.K123R mutations in 4% of myeloma patients. Lastly, we describe the fluctuations of myeloma subclonal architecture in a patient analyzed at presentation and relapse and in NOD/SCID-IL2Rγnull xenografts, revealing clonal extinction and the emergence of new clones that acquire additional mutations. This study confirms that myeloma subclones exhibit different survival properties during treatment or mouse engraftment. We conclude that clonal diversity combined with varying selective pressures is the essential foundation for tumor progression and treatment resistance in myeloma.

Single cell mutational profiling and clonal phylogeny in cancer

The development of cancer is a dynamic evolutionary process in which intraclonal, genetic diversity provides a substrate for clonal selection and a source of therapeutic escape. The complexity and topography of intraclonal genetic architectures have major implications for biopsy-based prognosis and for targeted therapy. High-depth, next-generation sequencing (NGS) efficiently captures the mutational load of individual tumors or biopsies. But, being a snapshot portrait of total DNA, it disguises the fundamental features of subclonal variegation of genetic lesions and of clonal phylogeny. Single-cell genetic profiling provides a potential resolution to this problem, but methods developed to date all have limitations. We present a novel solution to this challenge using leukemic cells with known mutational spectra as a tractable model. DNA from flow-sorted single cells is screened using multiplex targeted Q-PCR within a microfluidic platform allowing unbiased single-cell selection, high-throughput, and comprehensive analysis for all main varieties of genetic abnormalities: chimeric gene fusions, copy number alterations, and single-nucleotide variants. We show, in this proof-of-principle study, that the method has a low error rate and can provide detailed subclonal genetic architectures and phylogenies.

Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies

The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders. This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker. We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies. The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method. Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry. Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15). There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%). We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.

Clonal origins of ETV6-RUNX1⁺ acute lymphoblastic leukemia: studies in monozygotic twins.

Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage.

Regulation of the apoptotic response to radiation damage in B cell development

B lymphocyte precursor cells are ultrasensitive to DNA damage induced by irradiation and drugs and die by apoptosis at very low levels of exposure. Previous studies have shown that this high level sensitivity is p53-dependent, associated with very low level expression of Bcl-2 protein and can be reversed by expression of a bcl-2 transgene. We show here that transition from the pro-B to pre-B and then mature B cell stages of murine lymphopoiesis is accompanied by changes in proliferating cells in sensitivity to X-irradiation induced apoptosis and that this is paralleled by variation in the ratio of anti-(Bcl-2/Bcl-χL) to pro-(Bax) apoptotic proteins. These are however not fixed or invariant features of developmental stage as they can be modulated by interactions via adhesive interactions with stromal cells, stromal proteins and growth factors. We interpret these data in the context of the stringent developmental regulation of clonal lymphopoiesis and the contingency programming of cells that have extensive proliferative potential with a very low threshold for apoptosis following DNA damage.

Tyrosine kinase inhibitor insensitivity of non‐cycling CD34+ human acute myeloid leukaemia cells with FMS‐like tyrosine kinase 3 mutations

The efficacy of tyrosine kinase (TK) inhibitors on non‐cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non‐cycling cells from a small series of human FMS‐like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non‐cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non‐cycling cells, indicating their leukaemic origin. Significantly, the FLT3‐internal tandem duplication (ITD) mutation was found in the non‐cycling fraction of four out of five cases. Exposure to the FLT3‐directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short‐term cultures and colony‐forming unit assays. Crucially, non‐cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer‐term cultures, TKI258‐treated non‐cycling cells showed no growth impairment compared to treatment‐naive non‐cycling cells. These findings suggest that non‐cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Successful treatment of aplastic anemia with G-CSF and high dose erythropoietin.

We report the successful treatment of pancytopenia with G-CSF and high dose erythropoietin (Epo) in an elderly patient diagnosed with aplastic anemia (AA). Furthermore this effect is dose dependent for Epo in vivo. Detection of apoptosis by gel electrophoresis shows that high dose Epo protects bone marrow mononuclear cells from spontaneous apoptosis in vitro. These findings may explain some of the mechanisms of aplastic anemia.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.