Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Activating STAT6 mutations in follicular lymphoma.

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell-based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) -induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4-induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis.

Mutations in linker histone genes HIST1H1 B, C, D, and E; OCT2 (POU2F2); IRF8; and ARID1A underlying the pathogenesis of follicular lymphoma.

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and single nucleotide polymorphism 6.0 array genomic profiling of 11 highly purified FL cases, and 1 transformed FL case and the validation of selected mutations in 102 FL cases. We report the identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-E affected binding to DNMT3B, and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stably transduced cell lines, and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.

Clonal evolution and devolution after chemotherapy in adult acute myelogenous leukemia.

The frequent occurrence of persistent or relapsed disease after induction chemotherapy in AML necessitates a better understanding of the clonal relationship of AML in various disease phases. In this study, we used SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients who achieved complete remission with chemotherapy and subsequently relapsed and 11 sample pairs from patients with persistent disease after induction chemotherapy. Through review of aCNA/cnLOH and gene mutation profiles in informative cases, we demonstrate that relapsed AML invariably represents re-emergence or evolution of a founder clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered cases with 2 coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant, respectively. These data support the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research.

MDI 301 suppresses myeloid leukemia cell growth in vitro and in vivo without the toxicity associated with all-trans retinoic acid therapy.

MDI 301 is a novel 9-cis retinoic acid derivative in which the terminal carboxylic acid group has been replaced by a picolinate ester. MDI 301, a retinoic acid receptor-α - agonist, suppressed the growth of several human myeloid leukemia cell lines (HL60, NB4, OCI-M2, and K562) in vitro and induced cell-substrate adhesion in conjunction with upregulation of CD11b. Tumor growth in HL60-injected athymic nude mice was reduced. In vitro, MDI 301 was comparable to all-trans retinoic acid (ATRA) whereas in vivo, MDI 301 was slightly more efficacious than ATRA. Most importantly, unlike what was found with ATRA treatment, MDI 301 did not induce a cytokine response in the treated animals and the severe inflammatory changes and systemic toxicity seen with ATRA did not occur. A retinoid with these characteristics might be valuable in the treatment of promyelocytic leukemia, or, perhaps, other forms of myeloid leukemia.

Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia.

Genomic aberrations are of predominant importance to the biology and clinical outcome of patients with chronic lymphocytic leukemia (CLL), and FISH-based genomic risk classifications are routinely used in clinical decision making in CLL. One of the known limitations of CLL FISH is the inability to comprehensively interrogate the CLL genome for genomic changes. In an effort at overcoming the existing limitations in CLL genome analysis, we have analyzed high-purity DNA isolated from FACS-sorted CD19(+) cells and paired CD3(+) or buccal cells from 255 patients with CLL for acquired genomic copy number aberrations (aCNAs) with the use of ultra-high-density Affymetrix SNP 6.0 arrays. Overall, ≥ 2 subchromosomal aCNAs were found in 39% (100 of 255) of all cases analyzed, whereas ≥ 3 subchromosomal aCNAs were detected in 20% (50 of 255) of cases. Subsequently, we have correlated genomic lesion loads (genomic complexity) with the clinical outcome measures time to first therapy and overall survival. With the use of multivariate analyses incorporating the most important prognostic factors in CLL together with SNP 6.0 array-based genomic lesion loads at various thresholds, we identify elevated CLL genomic complexity as an independent and powerful marker for the identification of patients with aggressive CLL and short survival.

Somatic mutations in the transcriptional corepressor gene BCORL1 in adult acute myelogenous leukemia.

To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML.

Acquired genomic copy number aberrations in CLL.

Approximately 80 % of chronic lymphocytic leukemia (CLL) carries somatically acquired genomic copy number aberrations (aCNAs). These include gains of entire chromosomes (trisomy 12) and recurrent genomic losses, including interstitial deletions of various lengths at 13q14, 11q, and of more uniform length at 17p. In addition, approximately 10-15 second-tier aCNAs, with frequencies of 1-5 %, have been identified. In this chapter, we will discuss the biology and clinical significance of these CLL-associated aCNAs in detail and also discuss generic aspects of aCNAs relevant to all cancer cells. The hypothesis is advanced that most if not all aCNAs in CLL deregulate multiple target genes as a consequence of aCNA-associated gene mutations and through stable deregulation of gene expression. The concept of elevated genomic complexity (multiple aCNAs per CLL case) is reinforced as one of the strongest biological traits associated with aggressive CLL with short survival. Further, all inherited polymorphic copy number variations as detected through SNP 6.0 array profiling of T-cell-derived DNA of 255 CLL patients are listed to allow the reader a more critical appraisal of the somatic status of CLL-associated aCNAs as reported in the literature. Finally, given that aCNAs and gene mutations coexist in many CLL cells, we stress the importance of understanding in detail the relative biological and clinical roles each mutation type serves in individual CLL patients; this is a research area in need of more in-depth investigation.

Incidence and clinical implications of ATM aberrations in chronic lymphocytic leukemia.

A subset of chronic lymphocytic leukemia (CLL) carries mutations in ataxia telangiectasia mutated (ATM). Such ATM mutations may be particularly relevant in the setting of del11q, which invariably results in the deletion of one ATM allele. To improve our understanding of the frequency and type of ATM mutations that exist in CLL, we resequenced all ATM coding exons in 24 CLL with del11q using direct sequencing. We detected two missense mutations, resulting in an ATM mutation frequency of 8%; nonsense and frameshift mutations were not identified. Given the low ATM mutation frequency detected in this cohort, we proceeded with measurements of nonmutational ATM aberrations in CLL through analysis of the activation state of ATM in response to external irradiation. The phosphorylation state of ATM at Ser-1981 was measured using quantitative immunoblotting in purified CLL cells isolated from 251 CLL patients; data were normalized to simultaneous measurements of total ATM protein and actin. Resulting p-ATM/ATM and p-ATM/actin ratios were subsequently analyzed for prognostic significance inclusive and exclusive of TP53 exons 2-10 mutations. From these analyses, conducted in a large prospectively enrolled CLL patient cohort, neither the p-ATM/ATM nor the p-ATM/actin ratios were found to be prognostic for short survival. These data in aggregate demonstrate a low frequency of ATM aberrations in an unselected CLL cohort and do not support a major prognostic role for ATM aberrations in CLL, thus motivating renewed research efforts aimed at understanding the pathobiology of 11q deletions in CLL. © 2012 Wiley Periodicals, Inc.

Multiple distinct molecular mechanisms influence sensitivity and resistance to MDM2 inhibitors in adult acute myelogenous leukemia.

The survival of most patients with acute myelogenous leukemia (AML) remains poor, and novel therapeutic approaches are needed to improve outcomes. Given that the fraction of AML with mutated p53 is small ( approximately 10%), it appears rational to study MDM2 inhibitors as therapy for AML. Here, we report results of a detailed characterization of sensitivity and resistance to treatment ex vivo with the MDM2 inhibitor MI219 in AML blasts from 109 patients. In line with previous observations, all AML cases with mutated p53 were resistant to MI219. Importantly, approximately 30% of AML cases with unmutated p53 also demonstrated primary resistance to MI219. Analysis of potential mechanisms associated with MI219 resistance in AML blasts with wild-type p53 uncovered distinct molecular defects, including low or absent p53 protein induction after MDM2 inhibitor treatment or external irradiation. Furthermore, a separate subset of resistant blasts displayed robust p53 protein induction after MI219 treatment, indicative of defective p53 protein function or defects in the apoptotic p53 network. Finally, analysis of very sensitive AML cases uncovered a strong and significant association with mutated Flt3 status (Flt3-ITD), which for the first time identified a clinically high-risk group of AML that may particularly benefit from MDM2 inhibitor treatment.

Acquired genomic copy number aberrations and survival in adult acute myelogenous leukemia.

Genomic aberrations are of predominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML), and conventional karyotype-based risk classifications are routinely used in clinical decision making in AML. One of the known limitations of cytogenetic analysis is the inability to detect genomic abnormalities less than 5 Mb in size, and it is currently unclear whether overcoming this limitation with high-resolution genomic single-nucleotide polymorphism (SNP) array analysis would be clinically relevant. Furthermore, given the heterogeneity of molecular mechanisms/aberrations that underlie the conventional karyotype-based risk classifications, it is likely that further refinements in genomic risk prognostication can be achieved. In this study, we analyzed flow cytometer-sorted, AML blast-derived, and paired, buccal DNA from 114 previously untreated prospectively enrolled AML patients for acquired genomic copy number changes and loss of heterozygosity using Affymetrix SNP 6.0 arrays, and we correlated genomic lesion load and specific chromosomal abnormalities with patient survival. Using multivariate analyses, we found that having ≥ 2 genomic lesions detected through SNP 6.0 array profiling approximately doubles the risk of death when controlling for age- and karyotype-based risk. Finally, we identified an independent negative prognostic impact of p53 mutations, or p53 mutations and 17p-loss of heterozygosity combined on survival in AML.

Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients enrolled in a prospective study at the University of Michigan, of whom 139 and 39 were previously untreated and previously treated, respectively. We obtained unbiased, high-density, genome-wide measurements of subchromosomal copy number changes in highly purified DNA from sorted CD19(+) cells and buccal cells using the Affymetrix 50kXbaI SNP array platform (Santa Clara, CA). Genomic complexity scores were derived and correlated with the surrogate clinical end points time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pretreated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic subchromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis.

Display Characteristics and Their Impact on Digital Pathology: A Current Review of Pathologists' Future "Microscope".

Digital displays (monitors) are an indispensable component of a pathologists' daily workflow, from writing reports, viewing whole-slide images, or browsing the Internet. Due to a paucity of literature and experience surrounding display use and standardization in pathology, the Food and Drug Administration's (FDA) has currently restricted FDA-cleared whole-slide imaging systems to a specific model of display for each system, which at this time consists of only medical-grade (MG) displays. Further, given that a pathologists' display will essentially become their new surrogate "microscope," it becomes exceedingly important that all pathologists have a basic understanding of fundamental display properties and their functional consequences. This review seeks to: (a) define and summarize the current and emerging display technology, terminology, features, and regulation as they pertain to pathologists and review the current literature on the impact of different display types (e.g. MG vs. consumer off the shelf vs. professional grade) on pathologists' diagnostic performance and (b) discuss the impact of the recent digital pathology device componentization and the coronavirus disease 2019 public emergency on the pixel pathway and display use for remote digital pathology. Display technology has changed dramatically over the past 20 years and continues to change at a rapid rate. There is a paucity of published studies to date that investigate how display type affects pathologist performance, with more research necessary in order to develop standards and minimum specifications for displays in digital pathology. Given the complexity of modern displays, pathologists must become better informed regarding display technology if they wish to have more choice over their future "microscopes."

Automated histologic diagnosis of CNS tumors with machine learning.

The discovery of a new mass involving the brain or spine typically prompts referral to a neurosurgeon to consider biopsy or surgical resection. Intraoperative decision-making depends significantly on the histologic diagnosis, which is often established when a small specimen is sent for immediate interpretation by a neuropathologist. Access to neuropathologists may be limited in resource-poor settings, which has prompted several groups to develop machine learning algorithms for automated interpretation. Most attempts have focused on fixed histopathology specimens, which do not apply in the intraoperative setting. The greatest potential for clinical impact probably lies in the automated diagnosis of intraoperative specimens. Successful future studies may use machine learning to automatically classify whole-slide intraoperative specimens among a wide array of potential diagnoses.

Comprehensive analysis of copy number and allele status identifies multiple chromosome defects underlying follicular lymphoma pathogenesis.

PURPOSE: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. EXPERIMENTAL DESIGN: We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism-chip platform to interrogate the genomes of 58 fluorescence-activated cell-sorted (FACS) FL specimens for chromosomal copy number changes and 46 specimens for loss of heterozygosity (LOH). RESULTS: We report (a) previously unknown high-frequency copy-neutral LOH (uniparental disomy) in FL on chromosomes 1p (approximately 50%) and 6p (approximately 30%); (b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, approximately 8% of FL specimens contain a homozygous deletion at 6q23.3-24.1 that spans the negative NFkappaB regulator A20 and the p53 apoptosis effector PERP; (c) that combined analysis of chromosome 17p for LOH, copy number, and p53 mutations shows that most p53 mutations in FL do not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromosomes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. CONCLUSIONS: This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of genes relevant to FL biology and clinical behavior.

Integrated genomic profiling, therapy response, and survival in adult acute myelogenous leukemia.

PURPOSE: Recurrent gene mutations, chromosomal translocations, and acquired genomic copy number aberrations (aCNA) have been variously associated with acute myelogenous leukemia (AML) patient outcome. However, knowledge of the co-occurrence of such lesions and the relative influence of different types of genomic alterations on clinical outcomes in AML is still evolving. EXPERIMENTAL DESIGN: We performed SNP 6.0 array-based genomic profiling of aCNA/copy neutral loss-of-heterozygosity (cnLOH) along with sequence analysis of 13 commonly mutated genes on purified leukemic blast DNA from 156 prospectively enrolled non-FAB-M3 AML patients across the clinical spectrum of de novo, secondary, and therapy-related AML. RESULTS: TP53 and RUNX1 mutations are strongly associated with the presence of SNP-A-based aCNA/cnLOH, while FLT3 and NPM1 mutations are strongly associated with the absence of aCNA/cnLOH. The presence of mutations in RUNX1, ASXL1, and TP53, elevated SNP-A-based genomic complexity, and specific recurrent aCNAs predicted failure to achieve a complete response to induction chemotherapy. The presence of ≥1 aCNA/cnLOH and higher thresholds predicted for poor long-term survival irrespective of TP53 status, and the presence of ≥1 aCNA/cnLOH added negative prognostic information to knowledge of mutations in TET2, IDH1, NPM1, DNMT3A, and RUNX1. Results of multivariate analyses support a dominant role for TP53 mutations and a role for elevated genomic complexity as predictors of short survival in AML. CONCLUSIONS: Integrated genomic profiling of a clinically relevant adult AML cohort identified genomic aberrations most associated with SNP-A-based genomic complexity, resistance to intensive induction therapies, and shortened overall survival. Identifying SNP-A-based lesions adds prognostic value to the status of several recurrently mutated genes.

Genetics of follicular lymphoma transformation.

Follicular lymphoma (FL) is an indolent disease, but 30%-40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.

Analysis of acquired genomic copy number aberrations and regions of loss of heterozygosity in acute myelogenous leukemia genomes using Affymetrix SNP 6.0 arrays and supporting software tools.

The application of SNP array technology to the analysis of cancer genomes has greatly advanced our knowledge of the incidence and functional consequences of acquired genomic copy number aberrations (aCNA) and LOH in various malignancies. The major challenges of using SNP arrays are accurately identifying acquired genomic DNA aberrations in the raw array data with very high sensitivity and specificity and meaningfully assessing the associations between these aberrations and biological characteristics or patient outcomes. Critical to the success and valid interpretation of data derived from SNP array profiling are (1) the purity of cells used as a source of template DNA; (2) the analysis of paired DNA samples (tumor and normal); (3) use of validated software tools for data analysis; (4) access to an acceptable gold standard for aCNA and LOH, including FISH data, cytogenetic results, and Q-PCR data; and (5) statistical support to employ or develop algorithmic approaches to SNP array data analysis. Overcalling of lesions including lack of validation and undercalling of lesions that display low fractional allelic representations are common problems. This guide should help the reader establish this powerful technology in the laboratory and aims to stimulate transition of SNP array profiling into clinical applications.

Clonal evolution, genomic drivers, and effects of therapy in chronic lymphocytic leukemia.

PURPOSE: The identification of gene mutations and structural genomic aberrations that are critically involved in chronic lymphocytic leukemia (CLL) pathogenesis is still evolving. One may postulate that genomic driver lesions with effects on CLL cell proliferation, apoptosis thresholds, or chemotherapy resistance should increase in frequency over time when measured sequentially in a large CLL cohort. EXPERIMENTAL DESIGN: We sequentially sampled a large well-characterized CLL cohort at a mean of 4 years between samplings and measured acquired copy number aberrations (aCNA) and LOH using single-nucleotide polymorphism (SNP) 6.0 array profiling and the mutational state of TP53, NOTCH1, and SF3B1 using Sanger sequencing. The paired analysis included 156 patients, of whom 114 remained untreated and 42 received intercurrent therapies, predominantly potent chemoimmunotherapy, during the sampling interval. RESULTS: We identify a strong effect of intercurrent therapies on the frequency of acquisition of aCNAs in CLL. Importantly, the spectrum of acquired genomic changes was largely similar in patients who did or did not receive intercurrent therapies; therefore, various genomic changes that become part of the dominant clones are often already present in CLL cell populations before therapy. Furthermore, we provide evidence that therapy of CLL with preexisting TP53 mutations results in outgrowth of genomically very complex clones, which dominate at relapse. CONCLUSIONS: Using complementary technologies directed at the detection of genomic events that are present in substantial proportions of the clinically relevant CLL disease bulk, we capture aspects of genomic evolution in CLL over time, including increases in the frequency of genomic complexity, specific recurrent aCNAs, and TP53 mutations.