Author response: Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks (2014)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR)-deficient tumors and convey them with resistance to standard therapy. Using whole-genome and whole-exome sequencing, we here confirm an MMR-deficient mutation signature that is distinct from other tumor genomes, but surprisingly similar to germ-line DNA, indicating that a substantial fraction of human genetic variation arises through mutations escaping MMR. Moreover, we identify a large set of recurrent indels that may serve to detect microsatellite instability (MSI). Indeed, using endometrial tumors with immunohistochemically proven MMR deficiency, we optimize a novel marker set capable of detecting MSI and show it to have greater specificity and selectivity than standard MSI tests. Additionally, we show that recurrent indels are enriched for the ‘DNA double-strand break repair by homologous recombination’ pathway. Consequently, DSB repair is reduced in MMR-deficient tumors, triggering a dose-dependent sensitivity of MMR-deficient tumor cultures to DSB inducers. https://doi.org/10.7554/eLife.02725.001 eLife digest Before a cell divides, it must first copy all of its genetic material. Any mistakes that are made during this process are called mutations. Mutations can give rise to new traits but are mostly harmful to the cells, or cause cancer; therefore, cells have evolved tools that can efficiently spot these mistakes and repair them. One of the main tools is called mismatch repair (MMR). Defects in the cell's mismatch repair tools can wreak havoc as this allows many mutations to accumulate. Zhao et al. looked at the genomes of tumors where mismatch repair was not working properly to see what makes these ‘MMR-deficient tumors’ different from other tumors. This revealed that MMR-deficient tumors have similar patterns of mutations to those seen in egg and sperm cells. This was unexpected and suggests that mutations that are not corrected by mismatch repair are an important source of the genetic differences found between different humans, and between humans and their ancestors. Identifying cancerous tumors that are MMR-deficient is vital, as these tumors tend not to respond to commonly used cancer treatments. However, current clinical methods to identify MMR-deficient tumors often fail or produce results that are difficult to interpret. MMR-deficient tumors commonly contain mutations called indels, where short fragments of DNA are inserted or deleted into longer DNA sequences. Zhao et al. have found 59 indels that can be used to detect MMR-deficient tumors, where each indel had been identified in several tumors taken from different tissues. This new approach allowed MMR-deficiency to be identified in several types of tumor, including colon and ovarian cancers, with greater sensitivity and accuracy than the existing methods. Zhao et al. also found that the indels in MMR-deficient tumors reduce the ability of the tumors to repair a type of DNA damage called double-strand breaks. In these, both strands of DNA that make up the double helix are broken and the DNA chain is severed. As this kind of damage is very harmful to a cell, making more double-strand breaks could therefore form part of a more effective treatment against MMR-deficient tumors; further research is needed to investigate this possibility. https://doi.org/10.7554/eLife.02725.002 Introduction MMR-deficiency represents a well-established cause of Lynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility triggered by loss-of-function mutations in MMR genes (MLH1, MSH2, or MSH6) (Jiricny, 2006). Lynch syndrome is responsible for 2–5% of endometrial (EM) or colorectal (CRC) tumors. Additionally, epigenetic silencing of MLH1 contributes to another 15–28% of these tumors (Parsons et al., 2012; Peltomaki, 2014). Deficiency of the MMR machinery leads to DNA replication errors in the tumor tissue, but not the normal surrounding tissue. In particular, errors often accumulate as indel mutations in mono- and di-nucleotide repeats—a phenomenon referred to as microsatellite instability (MSI) (Pinol et al., 2005). MMR-deficient tumors exhibit a different prognosis and therapeutic outcome after standard chemotherapy (Ng and Schrag, 2010). Untreated CRC patients with MMR-deficient tumors have a modestly better prognosis, but do not seem to benefit from 5-fluorouracil-based adjuvant chemotherapy, which is the first-choice chemotherapy for CRC. In particular, in MMR-deficient tumors, mismatches induced by 5-fluorouracil are tolerated, leading to failure to induce cell death (Fischer et al., 2007). MMR-deficient tumors are also resistant to cisplatin and carboplatin, which are frequently used chemotherapies in EM cancer (Hewish et al., 2010). Furthermore, MMR-deficient tumors can be resistant to targeted therapies, because they acquire secondary mutations in genes that activate alternative or downstream signaling pathways (e.g., PIK3CA). Another possibility is that epigenetic silencing of MLH1 coincides with particular mutations, such as the BRAF V600E mutation (Donehower et al., 2013), which represents an established negative predictor of response to targeted anti-EGFR therapies in advanced CRC (Richman et al., 2009). Efforts to individualize the treatment of MMR-deficient tumors have focused on identifying synthetic lethal interactions within the MMR pathway. In particular, increased oxidative damage (by methotrexate exposure or PINK1 silencing [Martin et al., 2011]) and interference with the base excision repair (BER) pathway (by DNA polymerase γ or β inhibition [Martin et al., 2010]) can sensitize MMR-deficient tumors. Until now, these findings failed, however, to translate into clinically effective treatment options. Alternatively, as highlighted above, secondary mutations occurring because of MMR-deficiency may also critically determine therapeutic efficacy (Dorard et al., 2011). These secondary mutation spectra have, however, been poorly characterized, mainly because studies often focused at one or a few reporter loci, or exclusively on mutations at known hotspot sequences. More recently, the first whole-exome sequencing of MMR-deficient tumors was performed, highlighting the clearly distinct mutational landscape of these tumors (TCGA, 2012), whereas at the whole-genome level, Kim et al. (2013) revealed overrepresentation of MSI in euchromatic and intronic regions compared to heterochromatic and intergenic regions. To generate a more comprehensive picture of the mutation spectra arising in MMR-deficient tumors, and in particular, to interpret their clinical relevance with respect to diagnostically assessing MSI and therapeutically targeting MMR-deficient tumors, we sequenced another comprehensive set of MMR-deficient tumors. In particular, whole-genome and whole-exome sequencing was applied to 5 and 28 tumor–normal pairs, of which respectively 3 and 22 were MMR-deficient. Results Whole-genome sequencing of MMR-deficient tumors To select MMR-deficient tumors for whole-genome sequencing, standard diagnostic tests were used, including immunohistochemistry of MMR proteins (MLH1, MSH2, and MSH6), assessment of MSI using the extended Bethesda panel and methylation profiling of the MLH1 promoter. Three chemo-naive EM tumors, either deficient for MLH1, MSH2, or MSH6 and thus covering the full spectrum of MMR-deficiency, as well as two MMR-proficient EM tumors were selected (Table 1). Different sequencing technologies were leveraged to avoid potential technology biases in assessing mutation patterns in MMR-deficient tumor genomes, that is, Complete Genomics (CG) and Illumina short-read sequencing. We obtained high coverage sequencing data (30–120x) for tumor and matched normal samples (Table 1). Application of a standard annotation and filtering pipeline, as previously described (Reumers et al., 2011), revealed that each MMR-deficient tumor exhibited a clear hypermutator phenotype, containing on average 50 times more novel somatic mutations than MMR-proficient tumors (Figure 1A, Figure 1—source data 1, Figure 1—source data 2). Orthogonal technologies validated 98% of substitutions and 88% of indels in the three MMR-deficient tumors, while only 62% of substitutions and 11% of indels were validated in the two MMR-proficient tumors (Figure 1—source data 3). This difference in validation rates between MMR-deficient and MMR-proficient tumors is probably due to the fact that in normal genomes, as well as MMR-proficient tumor genomes, the number of true-positive indels is low in comparison to the number of false-positive indels. However, in MMR-deficient tumors, due to their specific hypermutator phenotype, the number of true-positive indels is vastly increased, thereby rendering the false positive fraction proportionally much smaller. Notably, all tumors were negative for POLE mutations (Kandoth et al., 2013; Palles et al., 2013). Table 1 Standard diagnostic tests to assess MMR-deficiency https://doi.org/10.7554/eLife.02725.003 TumorHistopathologyGradeStageCoverageIHCMSIMLH1 hyper-methylationTumorGerm-lineMLH1MSH2MSH6MMR− 1Endometrioid3IIIc87.181.1++−(*)+−MMR− 2Serous/clear cell3Ib24.821.9+−−−−MMR− 3Endometrioid2Ib28.530.0−++++MMR+ 1Endometrioid3I119.473.1+++−+MMR+ 2Serous3Ia79.277.0+++−− Tumors and matched germ-line were whole-genome sequenced using either Complete Genomics or Illumina sequencing technology. For each tumor, microsatellite instability (MSI) using the extended Bethesda panel, standard immunohistochemistry of MMR proteins (MLH1, MSH2, and MSH6), and methylation status of the MLH1 promoter are shown. * a weak positive nuclear staining in the minority of the tumor cells. Figure 1 with 3 supplements see all Download asset Open asset Somatic mutations in MMR-deficient tumors. (A) The average frequency of mutations, indels, and substitutions in MMR-deficient tumors vs MMR-proficient tumors, expressed as number of mutations per base (mpb). (B) The fraction of indels and substitutions observed in microsatellites, homopolymers (length over 5 bp), short homopolymers (length of 3–5 bp), and ‘not in repeat regions’ compared to their expected fraction in these regions. (C and D) Frequencies of substitutions (C) and indels (D) in MMR-deficient tumors stratified into exonic, intergenic, and intronic regions. (E) Indel frequencies corrected for homopolymer number, length, and base composition. Indel frequencies in MMR-deficient tumors represent estimates only, as orthogonal technologies revealed false-positive rates of 12%, while false-negative rates in CG and Illumina whole-genomes were estimated to be 27.7% and 0.5%, respectively, by Zook et al. (2014). In MMR-proficient tumors all detected somatic indels were independently validated. https://doi.org/10.7554/eLife.02725.004 Figure 1—source data 1 Sequence statistics of MMR-proficient and MMR-deficient whole genome sequenced tumour samples, and a list of somatic substitutions detected therein. https://doi.org/10.7554/eLife.02725.005 Download elife-02725-fig1-data1-v2.xlsx Figure 1—source data 2 List of somatic indels detected in the MMR-proficient and MMR-deficient, whole genome sequenced tumour samples. https://doi.org/10.7554/eLife.02725.006 Download elife-02725-fig1-data2-v2.xlsx Figure 1—source data 3 List and overview of validated somatic mutations, detected in the MMR-proficient and MMR-deficient whole genome sequenced tumour samples. https://doi.org/10.7554/eLife.02725.007 Download elife-02725-fig1-data3-v2.xlsx Somatic mutation patterns in MMR-deficient hypermutators Studies in model organisms and cell lines have shown that somatic mutations arising due to MMR-deficiency mostly involve indels affecting microsatellite sequences (di- to hexa-nucleotide repeats with a minimal length of six bases and at least two repeat units) and homopolymers (mononucleotide repeats with a minimal length of six bases) (Ellegren, 2004). We observed that indels were indeed more frequent than single basepair substitutions in all three MMR-deficient tumors (Figure 1A). Indels predominantly affected homopolymers (40-fold enrichment over expected by chance) and to a lesser extent also microsatellites (2.3-fold enrichment; Figure 1B, Figure 1—figure supplement 1). Substitutions were only slightly enriched in homopolymers and microsatellites (3- and 1.5-fold enrichment, respectively; Figure 1B). Mutations occurred as frequently in introns as in the rest of the genome, but were clearly less frequent in exons (excluding 5′ and 3′ untranslated regions [UTRs]). This decrease was caused by indels that were 91% less frequent in exons (Figure 1C,D). Correction for the number of homopolymers, the length of homopolymers or their basepair composition in exons versus other regions weakened this effect, but failed to completely alleviate it (Figure 1E, Figure 1—figure supplement 2). Since 92% of exonic indels resulted in frameshift mutations, which have a greater functional impact than substitutions (Montgomery et al., 2013), this suggests that exonic indels are prone to negative clonal selection during tumorigenesis. Somatic substitutions in MMR-deficient hypermutators There is extraordinary variation in the frequency and spectrum of somatic mutations affecting different cancers, shedding light on the underlying mutational processes and disease etiology of these tumors (Wheeler and Whang, 2013). When assessing somatic substitutions in MMR-deficient tumors, we observed that 74% of all substitutions represent transitions (i.e., purine-to-purine or pyrimidine-to-pyrimidine substitutions), which is similar to the patterns observed in the matched germ-line of these tumors (Figure 2A). This is surprising, since tumor genomes generally display patterns distinct from those found in the germ-line. Indeed, when extending these analyses to other hypermutators, that is, UV-light-induced melanoma (Pleasance et al., 2010), tobacco smoke-induced small cell lung adenocarcinoma (SCLC) (Pleasance et al., 2010), as well as breast tumors deficient for BRCA1 (Nik-Zainal et al., 2012) or EM tumors proficient for MMR, patterns were clearly dissimilar from the matched germ-line (Figure 2A). On the other hand, de novo germ-line substitutions identified through whole-genome sequencing of parent–offspring trios (Campbell et al., 2012; Kong et al., 2012), common genetic variation as catalogued by the 1000 Genomes Project (1 KG) (1000 Genomes Project Consortium, 2012), and substitutions that occurred in the human lineage during the divergence of humans and chimpanzees correlated strongly to the MMR-deficient tumor genome (Figure 2A). Given these remarkable parallels, we hypothesized that MMR-deficient genomes hypermutate in a way that mirrors the processes driving genetic variation on a population level, albeit somatically and on a shorter time scale. Figure 2 with 5 supplements see all Download asset Open asset Somatic substitution patterns in MMR-deficient tumors. (A) Somatic substitution patterns in whole-genome sequences of MMR-deficient endometrial tumors (MMR−), matched germ-line (peripheral white blood cell) DNA from MMR-deficient tumors (MMR-germ-line), de novo mutations as identified in parent-offspring trios (de novo), 1000 Genomes Project (1 KG), the human–chimpanzee divergence panel (Divergence), melanoma and small-cell lung cancer (SCLC), BRCA-deficient breast tumors (BRCA−), MMR-proficient endometrial tumors (MMR+). (B) Somatic substitution frequency per million dinucleotides and per million substitutions. The first row lists the base following the mutated base, the second row lists the base that was mutated, and the third row lists the new base. Gray boxes indicate transitions. Frequencies are depicted color-coded following a logarithmic distribution as shown by the gradient on the left. (C and D) Squared coefficients of correlation (R2) between dinucleotide substitution patterns (C) and between the number of intergenic substitutions per 1 Mb window (D). Substitutions in MMR-proficient and de novo data sets were too sparse for correlations at a 1 Mb scale. (E) Multivariate linear regression modeling of genomic features predicting substitutions frequencies per 1 Mb window in MMR-deficient tumors, and the outcome of the same multivariate linear regression modeling in the germ-line genetic variability panels. T-values resulting from the linear model are displayed as bar plots and indicate direction and significance of correlation (shaded grey box equals p > 0.05, Bonferroni-corrected per model). The de novo substitution frequency was too low to be modeled at this resolution. (F) Frequency of transitions (excluding G:C>A:T in CG) and transversions per 1 Mb window, binned per replication time. Frequencies are displayed relative to the earliest replicating bin. Linear regression analysis was performed to assess whether observed increases were significant and independent of other genomic features. All Bonferroni-corrected p-values were significant (p < 2.0E−5) except for transversions in MMR-deficient tumors, which were not significant (NS; p = 0.23). (G) Effect of homopolymer nucleotide composition (An, Tn, Cn, or Gn) on substitutions immediately flanking a homopolymer. For example, the nucleotide B next to the poly-A repeat 'NNB(A)nBNN' is mostly converted to an A (NNB(A)nANN) and not to a C, G, or T. The modest increase in A substitutions next to Cn homopolymers and T substitutions near Gn homopolymers is caused by C:G>T:A transitions in a CpG context. (H) Substitution frequency in and outside CpG islands, relative to genome-wide substitution frequencies. Data combined for all three MMR-deficient genomes are represented for (B, E–H), but individual MMR-deficient genomes display similar patterns (Figure 2—figure supplements 1–5). https://doi.org/10.7554/eLife.02725.011 To further assess the similarities between MMR-deficient mutation patterns and germ-line genetic variability, we analyzed small-scale and large-scale context-dependent effects on substitution patterns. At the small-scale level, when assessing the effect of flanking nucleotides on substitution frequencies, the patterns of all four sets of germ-line genetic variants were highly correlated to MMR-deficient tumors (average R2 = 0.77), but less to the four other cancer genomes (average R2 = 0.45; Figure 2B,C), providing further support for our hypothesis. On a large-scale context, the number of intergenic substitutions per 1 Mb in germ-line genetic variability databases was similarly highly correlated to those in MMR-deficient genomes (average R2 = 0.67), but not to those in other cancer genomes (average R2 = 0.42; Figure 2D). This suggests that also on a large scale, substitutions are comparably distributed in MMR-deficient tumor genomes as in germ-line genomes. At the large-scale level, nine genomic features are linked with genetic variability (Hodgkinson and Eyre-Walker, 2011). Each of these features correlated significantly with substitution frequencies in MMR-deficient tumors and germ-line genomes. Linear modeling revealed that six of these independently correlated with substitution rates in MMR-deficient tumors as well as with germ-line substitutions (Figure 2E). Overall, the types as well as the narrow and broad context-dependencies of substitutions thus appear to be largely shared between germ-line and MMR-deficient genomes, suggesting that a considerable fraction of human genetic diversity arises through mismatches escaping MMR. Since MMR-proficient tumors carried 50 times fewer substitutions and displayed more disparate substitution patterns than MMR-proficient tumors, the observed correlations can almost exclusively be attributed to the MMR-deficient phenotype of these tumors. As such, these correlations also provide novel insights into the functioning of the MMR system. First, replication timing correlated with transitions but not transversions in all three MMR-deficient tumors (Figure 2F). This contrasts with the increase in late S phase transversions observed in all other genomes studied here (Figure 2F), as well as in lymphoblastoid cell lines (Koren et al., 2012). The increase in MMR-proficient but not MMR-deficient cells suggests a reduced fidelity of DNA repair in late S phase, leading to an increase in transversions. Potential causes include a decreased MMR-activity in late S phase, or a longer window of time available for the repair of early vs late transversions in MMR-proficient cells (Hombauer et al., 2011). In contrast, DNA repair fidelity in MMR-deficient cells is invariably low and therefore not affected by replication time. Secondly, a positive association with simple repeat content was noted. Indeed, a 1.6-fold increase in substitutions at bases immediately flanking simple repeats was noted, with a threefold increase next to homopolymers and a 1.3-fold increase next to microsatellites (Figure These substitutions for the converted the base flanking the to the base the repeat (Figure are thus probably the of polymerase following a to the previously described and G:C>A:T transitions in CpG strongly on CpG but are correlated with the fraction of CpG (Figure 2E). of to T such transitions. the much increase in transitions observed in MMR-deficient compared to MMR-proficient tumors vs that MMR, described at the molecular et al., et al., 2012), is also in repair in et al., 2014). substitution frequencies correlated with CpG Indeed, of dinucleotide context, bases outside CpG were two times more to mutation than those CpG (Figure As CpG are generally DNA methylation thus to to the for this include the polymerase that DNA methylation may induce et al., 2012), and the repair of which is and thus on its et al., 2014). Somatic indels in MMR-deficient hypermutators We also somatic indel patterns in MMR-deficient tumors. As since the of indels was in homopolymers, a correlation between simple repeats and indel frequency was observed (Figure Indels were also predominantly 1 or 2 in length (Figure the minority of homopolymers of or bases an fraction of indels affected homopolymers Figure suggesting that homopolymers are less to accumulate indels. As observed in other MMR-deficient tumors and also in MMR-deficient et al., Kim et al., 2013), were more frequent than vs that DNA are more prone to than to a base during DNA Figure 3 with 1 supplement see all Download asset Open asset Somatic indel patterns in MMR-deficient tumors. (A) of genomic features in MMR-deficient tumors on indel frequency as by multivariate linear regression T-values resulting from the linear model are displayed for each genomic in the bar plots and indicate significance (shaded grey box equals p > 0.05, Bonferroni-corrected per and direction of the (B) of all indels or the number of (C) of homopolymers affected by an indel stratified per compared to the genome-wide fraction of homopolymers with that nucleotide of MMR-deficient tumors we selected MMR-deficient tumors, as well as four MMR-proficient tumors, from different (i.e., and these, six represented tumor cultures of low which we over cell because the due to their hypermutator phenotype are longer of the tumor (Figure data 2). of tumor and matched germ-line DNA at an average coverage of revealed that each MMR-deficient tumor somatic vs for MMR-proficient tumors Figure Figure data 1, Figure data 2). rates for substitutions and indels were respectively and analysis of all MMR-deficient tumors for the genes affected by either a somatic substitution or indel in the regions revealed in of cancer of or between tumors and cell cultures (Figure supplement 1). because of negative clonal selection and differences in homopolymer content in exons vs other genomic exonic substitutions indels (Figure Figure supplement similar to what we observed in the MMR-deficient whole-genomes (Figure 1C,D). a minority of these indels affected microsatellites, that homopolymers were frequently affected by indels. Figure with 5 supplements see all Download asset Open asset somatic indels. (A) The average mutation frequencies in the exons of MMR-deficient tumors and four MMR-proficient tumors. difference was observed between and in of the mutation frequencies, substitution and indel (Figure supplement (B) of homopolymers affected by an indel in of the homopolymer length stratified for 5′ and (C) The fraction of homopolymers in 5′ and that are affected by an indel in of the homopolymer (D) somatic indel frequencies in 5′ and of MMR-deficient tumors. (E) The enrichment of observed over expected frequencies of recurrent indels. were stratified by length of the affected homopolymer and for recurrent indels in and 5 or more of MMR-deficient tumors. Figure data 1 and statistics of MMR-deficient whole sequenced tumour samples, a list of somatic substitutions detected and results of validation of somatic substitutions. Download Figure data 2 A list of somatic indels detected in the MMR-deficient whole sequenced tumour samples, and results of their Download Figure data 3 Somatic substitutions and indels in homopolymers with their as identified by whole-exome and whole-genome sequencing. Download of homopolymers was affected by an indel in the MMR-deficient tumors that whole-genome or sequencing (i.e., of homopolymers were affected at least whereas were affected at least Figure supplement 3). Furthermore, and homopolymers were affected in or tumors (Figure data 3). In contrast, only substitutions were three of which were found in tumors (i.e., two substitutions affecting and were found in three and four tumors et al., whereas a substitution in affected three When homopolymer content of regions vs homopolymers were more frequent in than in regions (Figure these homopolymers were also more frequently affected (Figure the indel in regions was than in (Figure As a of this recurrent indels also occurred more frequently in than regions vs Figure data 3). however, recurrent indels were more frequently observed than expected on indel frequency in but not in homopolymers (Figure Figure supplement This suggests that features other than homopolymer length indel clonal selection of indels affecting short homopolymers, which are in represents a similar results were obtained when the analysis was only on the indicating that exonic mutations identified