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Hypermutation takes the driver’s seat
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Abstract
Most pediatric tumors have only very few somatic mutations. However, a recent study revealed that a subset of tumors from children with congenital biallelic deficiency of DNA mismatch repair exhibits a mutational load surpassing almost all other cancers. In these ultra-hypermutated tumors, somatic mutations in the proofreading DNA polymerases complement the congenital mismatch repair deficiency to completely abolish replication repair, thereby driving tumor development. These findings open several possibilities for exploiting ultra-hypermutation for cancer therapy.
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Classification of current anticancer immunotherapies
During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.
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Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers.
Adam Shlien, Brittany B Campbell, Richard de Borja … (2015)
DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.
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Hypermutation of the Inactive X Chromosome Is a Frequent Event in Cancer
Natalie Jäger, Matthias Schlesner, David Jones … (2013)
Introduction The process of somatic mutation is fundamental to cancer development. A number of causes for these mutations have been described, including intrinsic mutation processes such as damage from endogenous reactive oxygen species or incomplete fidelity of the DNA replication machinery and extrinsic factors such as environmental and lifestyle exposures. For example, UV light and tobacco exposure are both well-known factors adding to the mutational burden of somatic cells (Stratton et al., 2009). Human germline mutation rates are not constant across the genome, varying with factors such as base composition and transcription levels (Hodgkinson and Eyre-Walker, 2011; Ellegren et al., 2003). It is also known that the X chromosome typically shows reduced variation compared with the autosomes (Malcom et al., 2003). Only recently, however, have some studies elucidated the existence of variation in genome-wide somatic mutation rates and potential causes thereof. The mutation rate varies within a cancer genome according to underlying genomic features such as GC content, CpG islands, and recombination rate (Greenman et al., 2007). Regions that are actively transcribed have mutation rates at least 25% lower than nontranscribed regions (Chapman et al., 2011) due to mechanisms of transcription-coupled repair. Chromatin organization, specifically the level of heterochromatin-associated histone modification H3K9me3, has been reported to account for more than 40% of mutation-rate variation (Schuster-Böckler and Lehner, 2012). Late-replicating regions also have a higher mutation rate than early-replicating regions in cancer as well as in the germline (Liu et al., 2013; Stamatoyannopoulos et al., 2009). The inactive X chromosome (Xi) is one of the latest replicating regions of the human genome, being replicated distinctly later in S phase than the autosomes and its active X counterpart (Xa; Hansen et al., 1996; Morishima et al., 1962). In contrast to the autosomes, for which two active copies are present, both male and female cells carry only one active X chromosome. In mammals, dosage compensation between male and female cells is achieved by inactivating one of the two female X chromosomes (Chow and Heard, 2009; Lyon, 1961). This results in transcriptional silencing of most of the ∼1,500 genes located on the human X chromosome, although about 3%–15% of genes are known to escape X chromosome inactivation (XCI), depending on cell type (Carrel and Willard, 2005). XCI is initiated very early in embryonic stem cell differentiation and is characterized by a stochastic choice of the X chromosome subjected to inactivation (Barakat and Gribnau, 2012). The chosen inactivated copy (Xi) is then stably maintained through all subsequent cell divisions. The transcription of X-inactive-specific transcript (XIST) RNA, a 17 kb spliced and polyadenylated RNA with no coding capacity, is monoallelically upregulated at the onset of XCI and associates with the future Xi in cis (Brown et al., 1992). This XIST coating of the Xi provides the template for a series of histone modifications, including histone-H3 lysine 9 and 27 methylation and histone-H4 deacetylation and macroH2A accumulation, ultimately leading to heterochromatin formation (Plath et al., 2002). After XCI, XIST is expressed continuously and exclusively from the inactive copy of the X chromosome. In this study, we performed a cross-cancer analysis based on 402 whole-cancer genomes, including our own published and new cancer genome data sets from six different entities (medulloblastoma [Jones et al., 2012; M.K., D.T.W.J., N.J., P.A.N., M.D.T., R.E., S.M.P., and P.L., unpublished data], pilocytic astrocytoma [Jones et al., 2013], glioblastoma [S.M.P., M.K., D.T.W.J, P.A.N., M.D.T., R.E., P.L., and A.K., unpublished data], ependymoma [S.C.M., H.W., P.A.N., D.T.W.J., N.J., S.M.P., and M.D.T., unpublished data], B cell lymphoma [Richter et al., 2012; M.S., J.R., M.H., P.L., R.E., and R.S., unpublished data], and prostate carcinoma [Weischenfeldt et al., 2013]), in addition to published mutation call sets of six different cancer types: breast cancer (Nik-Zainal et al., 2012), neuroblastoma (Molenaar et al., 2012), chronic lymphocytic leukemia (CLL, Puente et al., 2011), acute myeloid leukemia (AML, Welch et al., 2012), colorectal carcinoma (Bass et al., 2011), and retinoblastoma (Zhang et al., 2012). In many female cancer genomes, we unexpectedly found hypermutation of the X chromosome—i.e., a clearly elevated density of mutations compared with the individual autosomes. We show that this hypermutation of the X chromosome is confined to the inactive X chromosome and involves single-nucleotide variants (SNVs) as well as small insertions and deletions (indels), which both show a marked increase in mutations along the X chromosome. Whole-genome sequencing of three independent clonal expansions of healthy hematopoietic stem/progenitor cells and one sample from myelodysplastic syndrome (MDS), which, although clonal, is considered a premalignant condition, revealed no X chromosome hypermutation. Thus, X chromosome hypermutation is a common feature of female cancer genomes occurring across a wide range of tumor types. Results The X Chromosome Accumulates Significantly More Mutations Than Autosomes in Medulloblastoma Genomes from Female Samples We analyzed the genome-wide distribution of somatic SNVs in 113 primary medulloblastoma samples collected within the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. The tumors, together with matched normal DNAs, were sequenced to average 30- to 40-fold coverage (Jones et al., 2012; M.K., D.T.W.J., N.J., P.A.N., M.D.T., R.E., S.M.P., and P.L., unpublished data; Table S1 available online). To analyze the distribution of mutations in the genome, the intermutation distance (the distance between a given somatic SNV and the SNV immediately upstream) was plotted for each sample. The mutational patterns revealed by this analysis are outlined below using an exemplary female and male genome (Figures 1A and 1B), which both belong to the same tumor subgroup (Sonic Hedgehog pathway-activated medulloblastoma). A lower mean intermutation distance on the X chromosome (0.33 Mb) compared with the autosomes (1.2 Mb) was observed in the female sample (Figure 1A), corresponding to a much higher number of mutations on the X chromosome than on any of the individual autosomes. In most cases, the X chromosome harbored a higher number of SNVs than both chromosomes 1 and 2 combined (e.g., MB56 in Figure 1A), even though each of them is much larger in size than the X chromosome. Further, MB56 has a total of 2,887 somatic SNVs in its genome, 469 (16%) of which are located on the X chromosome (Figure 1A). Given the size of the X chromosome, which at ∼155 megabases is very similar in size to chromosome 7, only about 5% of SNVs would be expected to occur on the X chromosome by chance, as is the case for chromosome 7 (n = 145 SNVs, 5.0%). In the male genome, no such difference in mutation rate between the X chromosome and the autosomes was observed (Figure 1B). Both exemplary tumor genomes are diploid, with no copy number or structural variations except for partial 10q loss in the female sample (Figure S1). Therefore, copy number changes of the chromosomes do not explain the large difference of SNVs on the X chromosome. To present mutational load per chromosome corrected for the size of the respective chromosome, the mutation rate per megabase was plotted (Figures 1C and 1D). Coloring of bars represents the proportions of the six possible nucleotide changes (C > A, C > G, C > T, T > A, T > C, and T > G) for each chromosome. This presentation of the chromosomal distribution of SNVs shows that the X chromosome accumulated 3.6-fold more somatic mutations per megabase compared to the mean of all autosomes in sample MB56 (Figure 1C). If the number of mutations per megabase on X is at least twice that of the mean mutation rate of the autosomes, we use the term “X chromosome hypermutation.” The majority of medulloblastoma cancer genomes of female patients (20/25, 80%) showed X chromosome hypermutation (Figure 2A). The expected number of SNVs depends on the copy number state of each chromosome. Thus, copy number changes of each chromosome have to be considered as confounding variables when comparing number of somatic SNVs per chromosome. To correct for copy number state of the autosomes, we considered only diploid medulloblastoma genomes with at most six copy number aberrant chromosomes and excluded the respective chromosomes per case; 49/113 cases fulfilled these criteria (Figure 2A). For all remaining cases, we can only infer X hypermutation to be present or not—we cannot accurately estimate the strength of X hypermutation. In general, male medulloblastoma genomes do not show X chromosome hypermutation (Figures 1D and 2A). Male genomes have only one copy of X in the germline; therefore, the amount of mutations on the X chromosome needs to be doubled in order to correct for copy number status. Even after correcting the mutation rate of the X chromosome in males, the difference between female versus male X chromosome mutation rate is highly significant in medulloblastoma (p = 8 × 10−11, t test) and in three other tumor types (Figures 2D–2G). We note that, after this correction, a few male samples enter the range of our definition of X chromosome hypermutation (Figure 2A). This might be explained by the fact that hemizygous mutations on the single X chromosome in males can be more readily called by mutation calling algorithms than heterozygous mutations in female samples, which will typically be supported by fewer reads. However, the ratio of X chromosome versus autosome mutation rate in males and in females with X chromosome loss ranges from about 0.5 to 1, or 1- to 2-fold after correcting for the single copy of X, whereas we observe a range of ∼2- to 4-fold higher mutation rates when both Xa and Xi are present. This suggests that Xi accumulates at least twice as many mutations as Xa. Note that the variation in strength of X chromosome hypermutation observed in the medulloblastoma female samples (Figure 2A) does not correlate with age at diagnosis (Figure 2B), which is in contrast to the finding that the overall number of somatic mutations in medulloblastoma strongly correlates with age (Jones et al., 2012). X Hypermutation Is Confined to the Inactive X Chromosome Most, but not all, medulloblastoma genomes from female patients display X chromosome hypermutation (Tables 1 and S1 and Figure 2A). We therefore further examined those cases not displaying this phenomenon. First, medulloblastoma genomes from females with loss of an X chromosome in the tumor (resulting in only one copy of the X chromosome) do not show X chromosome hypermutation (Figure 2A; for example, MB18). However, it is not the X copy number state in itself that determines X hypermutation. Tetraploid female sample MB6 has two copies of the X chromosome but has loss of heterozygosity and no XIST expression (Figure S2). Indeed, all medulloblastoma genomes from female samples that lost one copy of the X chromosome also show no XIST expression, indicating that Xa is kept and Xi is lost (Figure 2C). We did not observe X chromosome hypermutation in MB6 (with two copies of Xa; Figure S2B) and consistently found no X hypermutation in cases that lost Xi, regardless of the absolute copy number state of X. Further, X hypermutation is not present in male tetraploid genomes with more than one copy of the X chromosome. These analyses indicate that the copy number state of the X chromosome does not impact X hypermutation but rather indicates that it depends on the presence of Xi. Remarkably, a medulloblastoma from a male patient (MB139) with Klinefelter syndrome (47, XXY genome) also shows a trend toward X chromosome hypermutation (Figure 2A). The matching RNA data confirm XIST expression and therefore presence of Xi (Figure 2C). To further study the confinement of hypermutation to Xi, we performed two different approaches. First, for two samples (MB101 and lymphoma 4120193) with high mutational load and imbalanced copy number states of Xi versus Xa, we assigned individual SNVs to the active/inactive X chromosome by in silico haplotype phasing of mutations. Second, we performed chromatin immunoprecipitation sequencing (ChIP-seq) for histone marks H3K36me3 and histone variant macroH2A1 in order to haplotype the X chromosome of two additional samples (MB59 and GBM103). For haplotype phasing, we attempted to phase mutations that are sufficiently close together to be spanned by single read pairs. The first 52 megabases of chromosome arm Xp in female sample MB101 are present at three copies in the tumor. RNA sequencing (RNA-seq)-based allele frequencies of germline variants clearly identify Xi being present with two copies and Xa with a single copy. In total, 222 somatic SNVs were called in this region. Many somatic mutations were sufficiently close to heterozygous germline SNPs that individual sequence read pairs spanned both, thus allowing the mutation to be phased with the SNP. Here, a germline SNP with an allele frequency of about 1/3 indicates that this mutation is on Xa, whereas an allele frequency of 2/3 indicates that this mutation is located on Xi. In total, 58 somatic SNVs were haplotyped by this approach, of which 46 unambiguously mapped to Xi and 12 to Xa (p T mutations was found (Figures 1C and 4). Similar Mutation Spectrum on Autosomes and the Hypermutated X Within an individual cancer genome, there is no substantial variation between the autosomes and X chromosome in the relative contributions of each of the six classes of base substitution (C > A, C > G, C > T, T > A, T > C, and T > G) (Figure 4). Thus, the hypermutated X chromosome has the same mutation spectrum as the autosomes. This holds true even in those cases of lymphoma or neuroblastoma that have a very unique mutation spectrum (Figures 4C and 4D). To provide further insight into the underlying mutational processes, we incorporated the sequence context in which mutations occurred by considering the bases immediately 5′ and 3′ to each mutated base, giving 96 possible trinucleotide contexts for a mutation. The resulting heatmap reveals that the mutational signatures on the autosomes and the X chromosome are very similar (Figure S3). Further, principal component analysis (PCA) shows that the mutation spectrum of the autosomes and the hypermutated X chromosome of individual samples within one tumor type cluster closer together than the hypermutated X chromosomes of different tumors (Figure S4A). In addition, the distribution of mutations along the hypermutated X chromosome is very similar to the distribution observed on X in males of the same tumor type (e.g., for B cell lymphoma r2 = 0.64, p 5 of the respective gene to define an escape region (Figure 3, escape regions marked in gray). H3K36me3 and macroH2A1 ChIP-Seq of Frozen Tissue for Samples GBM103 and MB59 Frozen tissue was crushed with a pre-cooled douncer on dry ice. After crushing the tissue was fixed with freshly prepared 1% formaldehyde in PBS for 12 min. The reaction was stopped with 125 mM glycine for 5 min. To gain nuclei the fixed tissue was dounced again and washed 3 times with PBS. The tissue was then resuspended in MNase buffer (25 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris/HCl pH 7.4) and 10 U MNase per 15 mg tissue was added. After 15 min, incubation at 37°C MNase was stopped by adding 10x covaris buffer (100 mM Tris pH 8.0, 2 M NaCl, 10 mM EDTA, 5% N-lauroylsarcosine, 1% Na-deoxycholate, supplemented with protease inhibitors). The samples were sonicated for 25 min with the following parameters with a Covaris S2 system: burst 200, cycle 20%, and intensity 8. After centrifugation the supernatant was collected and directly used for IP. After IgG preclearance the sheared chromatin was incubated with protein G magnetic beads (Cell signaling, 9006) and 4 μg of either H3K36me3 (Abcam, ab9050) or macroH2A (Abcam, ab37264) antibody overnight. After washes with 1x covaris buffer (10 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na–deoxycholate), high-salt-buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na–deoxycholate, 0.1% SDS), lithium buffer (20 mM Tris–HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na–deoxycholate) and 10 mM Tris–HCl, chromatin was eluted from the magnetic beads (elution buffer: 50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and the crosslink was reversed overnight. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina sequencing platform. In brief, after DNA repair and A-addition NEBNext adapters (NEB, E7335) were ligated and digested with the USER enzyme. Barcodes (NEB, E7335) were introduced via PCR with a maximum of 14 cycles by the NEBNext polymerase (NEB, M0541). Size selection for mononucleosomal insert fragments was done with Ampure XP beads (Agencourt, A63880). Each ChIP-seq library was sequenced with two complete lanes on the Illumina HiSeq 2500 in the 101-bases paired-end rapid mode and aligned to hg19 using bwa. This resulted in the following coverage values (genome-wide, after deduplication, including all uniquely mapping reads): GBM103 macroH2A1: 17x H3K36me3: 20x MB59 macroH2A1: 11x H3K36me3: 11x Analysis of ChIP-Seq Data For peak calling, MACS version 1.4 (Zhang et al., 2008) was used without control and without local lambda calculation. On the X chromosome, a total of 8.8 Mbs of H3K36me3 peak regions were called in GBM103, and 4.5 Mbs in MB59. Peak calling for macroH2A1 was not possible, due to the uniform enrichment along the X chromosome (Mietton et al., 2009). Therefore, we restricted haplotype assignment (to Xi or Xa) of mutations to the H3K36me3 peak regions. MacroH2A1 is enriched on Xi (Mietton et al., 2009), while H3K36me3 is enriched in actively transcribed regions and therefore on Xa. Thus, a mutation located on Xi is expected to have a high allele frequency in the macroH2A1 data and a low allele frequency in H3K36me3, which we clearly observed for the heterozygous germline mutations on the X chromosome (data not shown). In addition, patient-matched RNA-seq data further supported this in silico haplotying approach of the X chromosome: heterozygous DNA mutations that showed ∼100% allele frequency in the RNA-seq data had high allele frequencies in H3K36me3 data (or ∼0% allele frequency in the RNA-seq correlating with low allele frequency in H3K36me3 data). PCA In order to compare the mutation spectrum of the combined autosomes and the hypermutated X chromosome of individual samples (as shown in Figure S4A), PCA was performed on a matrix of 68 rows representing 34 samples (separated each for the autosomes and the X chromosome) and six columns, corresponding to the ratio of the six possible nucleotide changes (C > A, C > G, C > T, T > A, T > C, and T > G). Calculations were performed in R using the prcomp function. All feature vectors were scaled to be zero-centered and to have unit variance. The first three principal components accounted for 84% variance of the data. RepliSeq-Based Replication Timing Analysis for the Autosomes We correlated the number of somatic mutations binned into windows of sizes 100Kb, 1Mb, and 5Mb with genome-wide replication timing data (Repli-Seq; Hansen et al., 2010). The Repli-Seq data used in this analysis are a wavelet-smoothed, weighted average signal where high (and low) values indicate early (and late) replication during S-phase. Values < 38 indicate late replication timing. RepliSeq replication timing data were downloaded from http://genome.ucsc.edu/ENCODE for 10 different cell lines: Gm06990, Gm12801, Gm12812, Gm12813, Gm12878, Hepg2, Huvec, K562, Mcf7, Nhek. We used the mean value of genomic regions that maintain similar replication timing between these different cell types, determined by low standard deviation per window. For B cell lymphoma (in Figures 6B and S5), we only used the Repli-Seq data for lymphoblastoid cell lines (n = 5) to match the tumor cell of origin: Gm06990, Gm12801, Gm12812, Gm12813, Gm12878.
Author and article information
Contributors
Matthias Schlesner: m.schlesner@dkfz-heidelberg.de
Roland Eils: r.eils@dkfz-heidelberg.de
Journal
Journal ID (nlm-ta): Genome Med
Journal ID (iso-abbrev): Genome Med
Title:
Genome Medicine
Publisher:
BioMed Central
(London
)
ISSN
(Electronic):
1756-994X
Publication date
(Electronic):
28
March
2015
Publication date PMC-release: 28
March
2015
Publication date Collection: 2015
Volume: 7
Issue: 1
Electronic Location Identifier: 31
Affiliations
[
]Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ),
Heidelberg, 69120 Germany
Article
Publisher ID: 159DOI: 10.1186/s13073-015-0159-x
PMC ID: 4376156
PubMed ID: 25821521
SO-VID: ee94a847-e93a-4e97-8e35-1c550fce27be
Copyright © © Schlesner and Eils; licensee BioMed Central. 2015
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
History
Date
received
: 9
March
2015
Date
accepted
: 13
March
2015
Categories
Subject:
Research Highlight
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ScienceOpen disciplines: Molecular medicine
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ScienceOpen disciplines: Molecular medicine