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Monitoring of NPM1 mutant (NPM1mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients who are treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as an MRD transcript level <1% to 2% with a <1-log change between any 2 positive samples collected after the end of treatment (EOT). Because the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received ≥2 cycles of intensive chemotherapy were included if bone marrow was NPM1mut MRD positive at the EOT, and they were not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year, either spontaneously achieving complete molecular remission (CRMRD-; 30%) or retaining a low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow-up). Forty percent met the criteria for MP-LCN. Preemptive salvage therapy significantly prolonged relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-internal tandem duplication at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy., Competing Interests: Competing Interest M.L was an employee of Fluidigm Corporation at the time of the study.

Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1 mut measurable residual disease (MRD) using off-label venetoclax in combination with low-dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CR MRD- ) without transplantation. Six of seven patients with molecular relapse/progression achieved CR MRD- after 1-2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax-based therapy to reduce the relapse risk in patients with persistent or rising NPM1 mut MRD., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice., Methods: We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes., Results: We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials., Conclusions: The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

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.

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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-IL2Rγ(null) 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.

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.

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.

We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.

Few studies on the biologic and molecular properties of pediatric glioblastoma have been performed. Until now, differential genomic analysis of CD133(+)ve and CD133(-)ve fractions has not been described in pediatric glioma. We hypothesize not only that the presence of CD133 could be the source of tumor resistance but also that maintenance of this molecule by hypoxia dictates cellular and molecular behavior. From a series of human glioblastoma biopsies investigated, only one, IN699 (from a pediatric glioblastoma), generated greater than 4% of the total cell volume as CD133(+)ve cells. Using this pediatric glioblastoma, containing unprecedented high levels of the putative brain tumor stem cell marker CD133, as a study model, we report biologic and molecular characteristics of the parent culture and of CD133(+)ve and CD133(-)ve populations derived therefrom under atmospheric and hypoxic culture conditions. Immunocytochemistry and flow cytometry were performed with antigenic markers known to characterize neural stem cells and associated glioma behavior. Behavioral analysis was carried out using proliferation, adhesion, migration, and invasion assays. Cell cycle analysis and array comparative genomic hybridization were used to assess copy number profiles for parental cells and CD133(+)ve and CD133(-)ve fractions, respectively. With regard to invasion and proliferation, CD133(+)ve and CD133(-)ve fractions were inversely proportional, with a significant increase in invasive propensity within the CD133(-)ve cells (P < .005) and a significant increase in proliferation within CD133(+)ve cells (P < .005). Our observations indicate identical genomic imbalances between CD133(+)ve and CD133(-)ve fractions. Furthermore, our research documents a direct link between decreasing oxygen tension and CD133 expression.

The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.