Dan A. Landau

Mutational heterogeneity in cancer and the search for new cancer-associated genes.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour–normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Evolution and Impact of Subclonal Mutations in Chronic Lymphocytic Leukemia

Clonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12, and del(13q)) or subclonal (e.g., SF3B1 and TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two time points. Ten of twelve CLL cases treated with chemotherapy (but only one of six without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1 and TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcomes.

Mutations driving CLL and their evolution in progression and relapse

Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized putative cancer drivers (RPS15, IKZF3), and collectively identify RNA processing and export, MYC activity, and MAPK signalling as central pathways involved in CLL. Clonality analysis of this large data set further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing data sets of clinically informative samples enable the discovery of novel genes associated with cancer, the network of relationships between the driver events, and their impact on disease relapse and clinical outcome.

Clonal evolution in hematological malignancies and therapeutic implications

The ability of cancer to evolve and adapt is a principal challenge to therapy in general and to the paradigm of targeted therapy in particular. This ability is fueled by the co-existence of multiple, genetically heterogeneous subpopulations within the cancer cell population. Increasing evidence has supported the idea that these subpopulations are selected in a Darwinian fashion, by which the genetic landscape of the tumor is continuously reshaped. Massively parallel sequencing has enabled a recent surge in our ability to study this process, adding to previous efforts using cytogenetic methods and targeted sequencing. Altogether, these studies reveal the complex evolutionary trajectories occurring across individual hematological malignancies. They also suggest that while clonal evolution may contribute to resistance to therapy, treatment may also hasten the evolutionary process. New insights into this process challenge us to understand the impact of treatment on clonal evolution and inspire the development of novel prognostic and therapeutic strategies.

Restoration of peripheral immune homeostasis after rituximab in mixed cryoglobulinemia vasculitis

Rituximab, an anti-CD20 monoclonal antibody, has been used to treat autoimmune disorders such as mixed cryoglobulinemia (MC). However, its mechanisms of action as well as the effects on cellular immunity remain poorly defined. We investigated the changes of peripheral blood B- and T-cell subsets, the clonal VH1-69 cells, as well as the cytokine profile following rituximab therapy. The study involved 21 patients with hepatitis C-related MC who received rituximab, of whom 14 achieved a complete response. Compared with healthy and hepatitis C virus (HCV) controls, pretreatment abnormalities in MC patients included a decreased percentage of naive B cells (P < .05) and CD4(+)CD25(+)FoxP3(+) regulatory T cells (P = .02) with an increase in memory B cells (P = .03) and plasmablasts (P < .05). These abnormalities were reverted at 12 months after rituximab. Clonal VH1-69(+) B cells dramatically decreased following treatment (32% +/- 6% versus 8% +/- 2%, P = .01). Complete responders of rituximab exhibited an expansion of regulatory T cells (P < .01) accompanied with a decrease in CD8(+) T-cell activation (P < .01) and decreased production of interleukin 12 (IL-12; P = .02) and interferon-gamma (IFN-gamma; P = .01). Our findings indicate that in patients with MC, response to B-cell depletion induced by rituximab effectively normalizes many of the disturbances in peripheral B- and T-lymphocyte homeostasis.

Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition

Resistance to the Brutons tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.

Resistance to checkpoint blockade therapy through inactivation of antigen presentation

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.Resistance to immune-checkpoint blockade often occurs in treated patients. Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

Chronic lymphocytic leukemia: molecular heterogeneity revealed by high-throughput genomics

Chronic lymphocytic leukemia (CLL) has been consistently at the forefront of genetic research owing to its prevalence and the accessibility of sample material. Recently, genome-wide technologies have been intensively applied to CLL genetics, with remarkable progress. Single nucleotide polymorphism arrays have identified recurring chromosomal aberrations, thereby focusing functional studies on discrete genomic lesions and leading to the first implication of somatic microRNA disruption in cancer. Next-generation sequencing (NGS) has further transformed our understanding of CLL by identifying novel recurrently mutated putative drivers, including the unexpected discovery of somatic mutations affecting spliceosome function. NGS has further enabled in-depth examination of the transcriptional and epigenetic changes in CLL that accompany genetic lesions, and has shed light on how different driver events appear at different stages of disease progression and clonally evolve with relapsed disease. In addition to providing important insights into disease biology, these discoveries have significant translational potential. They enhance prognosis by highlighting specific lesions associated with poor clinical outcomes (for example, driver events such as mutations in the splicing factor subunit gene SF3B1) or with increased clonal heterogeneity (for example, the presence of subclonal driver mutations). Here, we review new genomic discoveries in CLL and discuss their possible implications in the era of precision medicine.

Proceedings from the National Cancer Institute's Second International Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation: Part I. Biology of Relapse after Transplantation

In the National Cancer Institutes Second Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on the Biology of Relapse discussed recent advances in understanding some of the host-, disease-, and transplantation-related contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations. Factors influencing the biology of the therapeutic graft-versus-malignancy (GVM) effect-and relapse-include conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches, and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional immune competence; and genetic heterogeneity within the malignancy defining potential for clonal escape. Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with implications for prevention and treatment of relapse, as is the application of modern genome sequencing to defining the biologic basis of GVM, clonal escape, and relapse after HSCT.