Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks
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 .
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 ) . 10 . 7554/eLife . 02725 . 003Table 1 . Standard diagnostic tests to assess MMR-deficiencyDOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 003TumorHistopathologyGradeStageCoverageIHCMSIMLH1 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 . 10 . 7554/eLife . 02725 . 004Figure 1 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00410 . 7554/eLife . 02725 . 005Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00510 . 7554/eLife . 02725 . 006Figure 1—source data 2 . List of somatic indels detected in the MMR-proficient and MMR-deficient , whole genome sequenced tumour samples . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00610 . 7554/eLife . 02725 . 007Figure 1—source data 3 . List and overview of validated somatic mutations , detected in the MMR-proficient and MMR-deficient whole genome sequenced tumour samples . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00710 . 7554/eLife . 02725 . 008Figure 1—figure supplement 1 . The fraction of indels ( left panel ) and substitutions ( right panel ) observed in microsatellites , homopolymers , short homopolymers and in nonrepeat regions compared to their expected fraction in these regions . Data are shown for the individual MMR-deficient tumors . In all three tumors , substitutions predominantly affected non-repeat regions , while indels were mainly confined to homopolymers . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00810 . 7554/eLife . 02725 . 009Figure 1—figure supplement 2 . The relative indel frequency defined as the number of indels divided by the total bases of non-homopolymer regions in MMR-deficient tumors stratified into intergenic , exonic , 5′UTR , 3′UTR , and intronic regions is shown . Indel frequencies in homopolymers are shown in the left panel , whereas indel frequencies in non-homopolymer regions are shown in the right panel . The algorithm we used to correct for homopolymer content , composition , and length can be found in the ‘Materials and methods’ section under the header ‘Evidence of negative clonal selection’ . In homopolymer regions , there was a 16% decrease in indel frequency in exonic regions . In non-homopolymer regions , a clear decrease was also observed for exonic regions , confirming that the decrease in exonic indels is not only due to differences in homopolymer characteristics between exonic regions and the rest of the genome . This reveals apparent negative selection in exonic regions , independent of homopolmer content , composition or length of the homopolymers . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 00910 . 7554/eLife . 02725 . 010Figure 1—figure supplement 3 . Copy number status of the 5 whole-genomes assessed by Illumina Human-Omni1 and CytoSNP-12 chips . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 010 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 . 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 . 10 . 7554/eLife . 02725 . 011Figure 2 . 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 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01110 . 7554/eLife . 02725 . 012Figure 2—figure supplement 1 . Somatic substitution frequency per million dinucleotides and per million substitutions for the individual MMR-deficient genomes . The first row lists the base following the mutated base , the second row the base that was mutated , and the third row the new base . Transitions are indicated by grey boxes . Frequencies are depicted color-coded following a logarithmic distribution as shown by the gradient on the right . The average R2 between the MMR-deficient tumors is 0 . 75 . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01210 . 7554/eLife . 02725 . 013Figure 2—figure supplement 2 . Multivariate linear regression modeling of genome features predicting substitutions frequencies per 1 Mb window in the individual MMR-deficient genomes . T-values resulting from the linear model are displayed for each genome feature in the bar plots and indicate significance ( shaded grey box equals p > 0 . 05 , Bonferroni-corrected per model ) and direction of the correlation . High concordance between the individual tumors is observed . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01310 . 7554/eLife . 02725 . 014Figure 2—figure supplement 3 . Frequency of transitions ( excluding G:C>A:T in CG ) and transversions per 1 Mb window , binned per replication time , relative to the earliest replicating bin . Mutations are divided in 7 bins ( left to right bins represent early to late replication timing events ) . Linear regression analysis was performed to assess whether observed increases were significant and independent of other genomic features . Bonferroni-corrected p-values were significant ( p < 2 . 0E−5 ) for transitions and nonsignifciant for transversions . In none of the individual MMR-deficient genomes , transversions were significantly correlated to replication timing , whereas transitions correlated for each of the MMR-deficient genomes . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01410 . 7554/eLife . 02725 . 015Figure 2—figure supplement 4 . Effect of homopolymer nucleotide composition ( An , Tn , Cn , or Gn ) on substitutions immediately flanking a homopolymer in the individual MMR-deficient genomes . The slight increase in A substitutions next to Cn homopolymers and T substitutions near Gn homopolymers is exclusively caused by C:G>T:A transitions in a CpG context , indicating they are likely deaminations of methylated cytosines . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01510 . 7554/eLife . 02725 . 016Figure 2—figure supplement 5 . Frequency of transitions and tranvsersions in and outside of CpG Islands in the individual MMR-deficient genomes . The frequency of transitions and transversions inside and outside CpG islands was determined as the number of mutations divided by the total size of each of the features , and expressed relative to the general , genome-wide frequencies of transitions and transversions . Individual genomes display similar patterns . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 016 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 2G ) . These substitutions for the vast majority converted the base flanking the repeat , to the base constituting the repeat ( Figure 2G ) . They are thus probably the result of polymerase slippage events , following a mechanism akin to the previously described bacterial dislocation mutagenesis ( Kunkel and Soni , 1988 ) . Thirdly , G:C>A:T transitions in CpG sites strongly depend on CpG content , but are inversely correlated with the fraction of CpG islands ( Figure 2E ) . Spontaneous , replication-independent deaminations of methyl-C to T underlie such transitions . Here , the much larger increase in CG>TG transitions observed in MMR-deficient compared to MMR-proficient tumors ( 3449 vs 145 ) demonstrates that replication-independent MMR , recently described at the molecular level ( Shell et al . , 2007; Pena-Diaz et al . , 2012 ) , is also involved in deamination repair in vivo ( Chen et al . , 2014 ) . Finally , overall substitution frequencies correlated inversely with CpG islands . Indeed , irrespective of dinucleotide context , bases outside CpG islands were nearly two times more likely to undergo mutation than those inside CpG islands ( Figure 2H ) . As CpG islands are generally unmethylated , DNA methylation thus appears to contribute to the mutagenic process . Explanations for this observation include the polymerase stalling that DNA methylation may induce ( Song et al . , 2012 ) , and the repair of spontaneously deaminated methyl-Cs , which is error-prone and thus mutagenic on its own ( Chen et al . , 2014 ) . We also evaluated somatic indel patterns in MMR-deficient tumors . As expected , since the majority of indels was located in homopolymers , a strong correlation between simple repeats and indel frequency was observed ( Figure 3A ) . Indels were also predominantly 1 or 2 bps in length ( Figure 3B ) . Although the minority of homopolymers consists of C or G bases ( 7% ) , an even smaller fraction of indels affected C:G homopolymers ( 1 . 9%; Figure 3C ) , suggesting that C:G homopolymers are less likely to accumulate indels . As observed in other MMR-deficient tumors and also in MMR-deficient Caenorhabditis elegans ( Denver et al . , 2005; Kim et al . , 2013 ) , deletions were remarkably more frequent than insertions ( 81% vs 19% ) , confirming that DNA polymerases are more prone to remove than to add a base during DNA synthesis . 10 . 7554/eLife . 02725 . 017Figure 3 . Somatic indel patterns in MMR-deficient tumors . ( A ) Impact of genomic features in MMR-deficient tumors on indel frequency as assessed by multivariate linear regression modeling . T-values resulting from the linear model are displayed for each genomic feature in the bar plots and indicate significance ( shaded grey box equals p > 0 . 05 , Bonferroni-corrected per model ) and direction of the correlation . ( B ) Fraction of all indels inserting or deleting the indicated number of bases . ( C ) Fraction of homopolymers affected by an indel stratified per nucleotide , compared to the genome-wide fraction of homopolymers with that nucleotide content . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01710 . 7554/eLife . 02725 . 018Figure 3—figure supplement 1 . The distance between a somatic substitution and the nearest somatic indel ( top left ) , substitution ( top right ) , repeat ( bottom left ) , or homopolymer ( bottom right ) in the individual MMR-deficient genomes , and the expected distance based on 200 random models . The substitutions located nearby indels and substitutions were enriched respectively within a range of ∼30 bp and ∼200 bp , whereas substitutions near repeats were enriched only at the base immediately flanking the repeat . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 018 Next , we selected 13 additional MMR-deficient tumors , as well as four MMR-proficient tumors , collected from different tissues ( i . e . , endometrium , colon , and ovarium ) . Of these , six represented primary tumor cultures of low passage , which we preferred over cell lines , because the latter due to their hypermutator phenotype are no longer representative of the original tumor ( Figure 4—source data 2 ) . Exome-sequencing of tumor and matched germ-line DNA at an average coverage of 44x revealed that each MMR-deficient tumor contained ∼2015 somatic events vs 39 for MMR-proficient tumors ( 52-fold increase; Figure 4A , Figure 4—source data 1 , Figure 4—source data 2 ) . Validation rates for substitutions and indels were respectively 87% and 86% . Clustering analysis of all 13 MMR-deficient tumors for the genes affected by either a somatic substitution or indel in the coding regions revealed no obvious subgroups in terms of cancer of origin or between primary tumors and cell cultures ( Figure 4—figure supplement 1 ) . Presumably , because of negative clonal selection and differences in homopolymer content in exons vs other genomic regions , exonic substitutions outnumbered indels ( Figure 4A , Figure 4—figure supplement 2 ) , similar to what we observed in the MMR-deficient whole-genomes ( Figure 1C , D ) . Only a minority of these indels affected microsatellites , confirming that homopolymers were most frequently affected by indels . 10 . 7554/eLife . 02725 . 019Figure 4 . Recurrent somatic indels . ( A ) The average mutation frequencies in the exons of 13 MMR-deficient tumors and four MMR-proficient tumors . No obvious difference was observed between MLH1- , MSH2- , and MSH6- deficiency in terms of the mutation frequencies , substitution patterns , and indel compositions ( Figure 4—figure supplement 5 ) . ( B ) Fraction of homopolymers affected by an indel in function of the homopolymer length stratified for exons , 5′ and 3′UTRs . ( C ) The fraction of homopolymers in exons , 5′ and 3′UTRs that are affected by an indel in function of the homopolymer length . ( D ) Average somatic indel frequencies in exons , 5′ and 3′UTRs of 16 MMR-deficient tumors . ( E ) The enrichment of observed over expected frequencies of recurrent indels . Enrichments were stratified by length of the affected homopolymer and calculated for recurrent indels in 2 , 3 , 4 , and 5 or more out of 16 MMR-deficient tumors . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 01910 . 7554/eLife . 02725 . 020Figure 4—source data 1 . Sample info and sequence statistics of MMR-deficient whole exome sequenced tumour samples , a list of somatic substitutions detected therein and results of validation of somatic substitutions . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02010 . 7554/eLife . 02725 . 021Figure 4—source data 2 . A list of somatic indels detected in the MMR-deficient whole exome sequenced tumour samples , and results of their validation . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02110 . 7554/eLife . 02725 . 022Figure 4—source data 3 . Somatic substitutions and indels in homopolymers together with their recurrence rate as identified by whole-exome and whole-genome sequencing . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02210 . 7554/eLife . 02725 . 023Figure 4—figure supplement 1 . Clustering analysis of 13 MMR-deficient exomes for the genes affected by either a somatic substitution or indel in the coding regions . No obvious subgroups in terms of cancer of origin or between primary tumors and cell cultures were observed . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02310 . 7554/eLife . 02725 . 024Figure 4—figure supplement 2 . The fraction of indels ( left panel ) and substitutions ( right panel ) identified by whole-exome sequencing , as 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 . Indels mainly affected homopolymers ( 59 . 0% ) , whereas microsatellites and short homopolymers were affected at a frequency that was expected based on their genome-wide occurrence . In contrast , indels were depleted in non-repeat regions . Substitutions affected the exome independent of repeat composition . These distributions mirror our observations in the MMR-deficient tumors undergoing whole-genome sequencing . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02410 . 7554/eLife . 02725 . 025Figure 4—figure supplement 3 . Characteristics of the exonic homopolymers recurrently affected . For the 477 homopolymers affected in at least 2 out of 16 tumors , respectively 176 , 135 , 85 , and 81 consisted of A , T , G , or C stretches . Out of the 34 homopolymers affected in at least 6 out of 16 tumors , 15 , 15 , 1 , and 3 consisted of A , T , G , or C stretches , respectively . The length of recurrently affected homopolymers ( in at least 2 out of 16 tumors ) varied from 6 nucleotides to 25 nucleotides , but recurrence was biased towards homopolymers with length 7–9 nucleotides . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02510 . 7554/eLife . 02725 . 026Figure 4—figure supplement 4 . The observed and expected frequencies of indels recurrently affected in homopolymers ( in at least 2 out of 16 tumors ) stratified for homopolymer length and for those affecting coding exonic regions and the 3′UTR . The difference between observed and expected recurrent indels is high for short homopolymers , but non-existent for long homopolymers . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02610 . 7554/eLife . 02725 . 027Figure 4—figure supplement 5 . Mutation patterns obtained from MLH1-deficient , MSH2-deficient , and MSH6-deficient exomes . ( A–C ) Mutation frequencies . ( D ) Somatic substitution patterns . ( E–G ) Indel compositions . No obvious difference is observed . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 027 Remarkably , 1 . 6% of homopolymers was recurrently affected by an indel in the 16 MMR-deficient tumors that underwent whole-genome or exome sequencing ( i . e . , 2244 out of 29 , 663 homopolymers were affected at least once , whereas 477 were affected at least twice; Figure 4—figure supplement 3 ) . Furthermore , 34 and 10 homopolymers were affected in ≥6 or ≥8 tumors ( Figure 4—source data 3 ) . In contrast , only 55 substitutions were recurrent , three of which were found in ≥2 tumors ( i . e . , two substitutions affecting KRAS codon 12 and 13 were found in three and four tumors [Tie et al . , 2011] , whereas a substitution in ZNF648 affected three tumors ) . When comparing homopolymer content of coding regions vs UTRs , long homopolymers ( >10 bps ) were more frequent in UTRs than in coding regions ( Figure 4B ) . Because these long homopolymers were also more frequently affected ( Figure 4C ) , the overall indel rate in coding regions was lower than in UTRs ( Figure 4D ) . As a consequence of this difference , recurrent indels also occurred more frequently in UTRs than coding regions ( 31 , 438 vs 1337; Figure 4—source data 3 ) . Remarkably , however , recurrent indels were more frequently observed than expected based on indel frequency in short , but not in long homopolymers ( Figure 4E , Figure 4—figure supplement 4 ) . This suggests that features other than homopolymer length underlie indel recurrence rates . Positive clonal selection of indels affecting short homopolymers , which are predominant in coding regions , represents a possible explanation . Very similar results were obtained when the analysis was repeated only on the 13 whole-exomes , indicating that exonic mutations identified from whole-genome sequences did not introduce any bias . The extended Bethesda panel , which consists of eight microsatellite and two homopolymer markers , is currently used to diagnostically assess MSI ( Pinol et al . , 2005 ) . This panel was historically compiled from a limited set of markers known to be variable . Due to their length and variability , these markers are notoriously difficult to analyze and interpret . As a consequence , the Bethesda panel has reduced sensitivity to detect MSI . In an effort to improve MSI testing , we randomly selected 59 recurrent indels affecting ≥6 out of 16 tumors; 50 markers were in 5′ or 3′UTRs and 9 were in coding regions ( Figure 5—source data 1 ) . Furthermore , each of the markers was detected in both MMR-deficient EM and CRC . To facilitate high-throughput genotyping , the maximal length of affected homopolymers was restricted to 12 bps . First , we applied these 59 markers to a discovery set of 236 EM tumors for which MMR immunohistochemistry ( IHC ) data were available . This allowed us to determine three positive markers as the threshold with the best Matthew correlation coefficient to detect MMR-deficiency based on IHC and thus to define MSI ( Figure 5A , B ) . At this threshold , our markers detected 40 out of 41 tumors MMR-deficient on IHC ( sensitivity ∼98% ) , while only 1 out of 184 MMR-normal tumors on IHC were identified as MSI ( specificity > 99% ) . Notably , the latter patient had a familial history of cancer within the Lynch spectrum , suggesting that the tumor indeed exhibited MSI . Secondly , after having optimized the marker threshold , a head-to-head comparison against Bethesda panel was performed in 114 independent EM tumors as a validation . When observing discordances , we assessed MMR-deficiency using IHC to address which of both MSI panels was correct . Briefly , each MSI tumor on Bethesda ( >2 markers positive ) was also MSI with the 59-marker panel ( Figure 5C ) . However , 12 tumors were positive in the 59-marker panel , but negative in Bethesda . IHC on the nine discordant tumors for which a paraffin block was available confirmed that each of them was MMR-deficient either for MLH1 or MSH2 , indicating that the 59-marker panel has a higher sensitivity compared to Bethesda . 10 . 7554/eLife . 02725 . 028Figure 5 . The 56-marker panel for MSI testing . ( A ) Receiver–operator curve assessing the impact of the number of positive homopolymer markers ( out of 59 ) on the sensitivity and specificity of MSI testing , based on a panel of 236 EM tumors immunohistochemically characterized for their MMR status . ( B ) The Matthew Correlation Coefficient ( MCC ) of the ROC curve was calculated for each threshold , and a threshold of 3 resulted in the highest MCC-value ( MCC = 0 . 97 ) . ( C and D ) The extended Bethesda panel and the 59-marker panel were compared in an independent series of 114 unselected primary endometrial tumors ( C ) and 126 stage II or III CRC tumors ( D ) . Results were color-coded according to high microsatellite instability ( MSI-H; more than 1 markers positive ) , low microsatellite instability ( MSI-L; 1 marker positive ) , or microsatellite stable status ( MSS; 0 markers positive ) as determined with the extended Bethesda panel . For endometrial tumors , 71 tumors ( 62% ) were defined as MSS/MSI-L and 43 tumors ( 38% ) as MSI-H by the 59-marker panel . Out of these 43 MSI-H tumors , Bethesda identified 32 tumors as MSI-H ( >2 markers positive ) , 7 tumors as MSI-L , and 5 tumors as MSS . Vice versa , Bethesda did not identify any MSI-H tumor that was not identified by our panel . For colorectal tumors , there were 97 MSS tumors in our 59-marker panel that were concordantly called MSS or MSI-L by the Bethesda panel . The remaining 29 samples were detected as MSI in the 59-marker panel . 28 of these were also called MSI-H by the Bethesda panel , whereas one was called MSS by the Bethesda panel . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02810 . 7554/eLife . 02725 . 029Figure 5—source data 1 . Recurrent indels selected for the 59-marker MSI panel and the results of a logistic regression analysis to detect differences between MSI-H and MSI-L/MSS tumors . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 02910 . 7554/eLife . 02725 . 030Figure 5—source data 2 . Clinical information , MMR-mutation status and sequencing statistics for ovarian tumors and leukemias . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 030 Likewise , we assessed MSI in 126 stage II or III CRC tumors . Each of the 28 MSI tumors on Bethesda was also positive with our 59-marker panel . In contrast , one tumor was MSI-positive in the 59-marker panel but not in the Bethesda panel ( Figure 5D ) . This tumor contained a V600E BRAF mutation and was MLH1 hypermethylated , indicating that it was MMR-deficient and that our panel was also more sensitive for CRC ( Deng et al . , 2004 ) . Finally , we also assessed whether our 59-marker panel can detect MSI in other cancer types . In a limited set of ovarian tumors and leukemias , we indeed correctly identified MSI in each of the samples tested ( Figure 5—source data 2 ) . Since we observed clear signs of clonal indel selection in MMR-deficient tumors , we assessed whether specific pathways were enriched for indels . We focused on frameshift indels in exons and exon/intron boundaries as they represent loss-of-function mutations ( Ham et al . , 2006 ) , and thus have a less ambiguous functional impact than indels in UTRs . On average , each MMR-deficient tumor contained 472 such indels , 59 of which were recurrent indels . Pathway analyses using IPA of all genes affected by a somatic indel , excluding the core MMR genes , ranked the ‘Role of BRCA1 in DNA damage response’ as the top enriched pathway . IPA analysis of genes affected by recurrent indels moreover revealed that the ‘Double-strand break repair by homologous recombination’ pathway ( DSBR by HR ) ranked top ( Table 2 ) . We also performed pathway analyses using the more advanced GenomeMuSiC , which takes background mutation rates into account and assigns weights depending on the number of tumors and genes affected in a given pathway . GenomeMuSiC analyses based on either the independently assembled Reactome or BioCarta pathway databases , ranked respectively the ‘ATR/BRCA pathway and the DNA repair’ pathway first , with the more specific ‘Homologous recombination repair’ pathway ranking third in the latter ( Table 2 ) . Based on an expert curated DNA repair database ( DNARepairDB ) , ‘Homologous recombination’ represented the only DNA repair pathway that was significantly enriched in indels . Since each pathway database differed with respect to the genes included , we finally compiled a literature-based set of genes with proven involvement in DSBR by HR , allowing us to more accurately estimate that each MMR-deficient tumor on average contained 3 . 3 ± 0 . 4 indels in the ‘DSBR by HR’ pathway ( Table 2 , Table 2—source data 1 ) . Notably , none of the top-ranking pathways for any of the databases contained significantly more homopolymers in their genes than expected . 10 . 7554/eLife . 02725 . 031Table 2 . Pathways most significantly affected by exonic indelsDOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 03110 . 7554/eLife . 02725 . 032Table 2—source data 1 . Results of pathway enrichments , custom definition of the DSBR by HR pathway and the allelic frequencies of mutations in HR genes . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 032DatabasePathwayRankFDRAffected samples ( n = 16 ) Mutations per sample*DSBR by HR ( custom definition ) n . a . n . a . 163 . 25BioCarta ( ranking by GenomeMusic ) *ATR/BRCA pathway11 . 0E−16153 . 50ATM pathway25 . 9E−11152 . 69G2 pathway37 . 2E−08152 . 81IL10 pathway42 . 2E−05121 . 75CARM1 and regulation of the Estrogen Receptor pathway52 . 2E−05143 . 19DNA Repair DB ( ranking by GenomeMusic ) *Homologous recombination pathway11 . 3E−04131 . 56Base excision repair pathway29 . 0E−02100 . 75Non-homologous end joining pathway31 . 7E−0190 . 69Nucleotide excision repair pathway48 . 3E−0170 . 50Reactome ( ranking by GenomeMusic ) DNA repair12 . 5E−11156 . 69Double strand break repair27 . 2E−08152 . 94*Homologous recombination repair31 . 9E−07152 . 31G2/M checkpoints42 . 3E−07153 . 50Cell cycle checkpoints54 . 5E−05154 . 75Base excision repair158 . 3E−03100 . 94Non-homologous end joining591 . 0E+0080 . 63Nucleotide excision repair615 . 9E−01101 . 50IPA ( ranking by IPA ) *DNA double-strand break repair by homologous recombination14 . 7E−03151 . 56Ovarian cancer signaling4 . 7E−03165 . 75Role of NFAT in cardiac hypertrophy36 . 8E−03143 . 88Cell cycle: G2/M DNA damage checkpoint regulation41 . 3E−02152 . 88PPARα/RXRα activation51 . 4E−02154 . 63DNA double-strand break repair by non-homologous end joining601 . 7E−01141 . 50The five top ranking pathways are listed , as well as all annotated pathways relevant for DNA repair . The custom definition used throughout this manuscript was added for illustrative purposes . n . a . = not applicable . *The DSBR by HR pathway . In an effort to replicate these findings , we analyzed mutation data of 27 CRC and 65 EM tumors with MSI sequenced by The Cancer Genome Atlas ( TCGA , 2012; Kandoth et al . , 2013 ) . Although most of these tumors were sequenced at low coverage depth , we identified 2183 and 3138 mutated genes from respectively the CRC and EM tumor data sets . IPA analysis confirmed that the Role of BRCA1 in DNA damage response was again amongst the top enriched pathways for each of the data sets . The corresponding p-values were 9 . 06E−3 and 2 . 97E−4 , although only the latter survived multiple testing correction ( p = 0 . 022; Table 2—source data 1 ) . As raw data sets were not accessible , the more sensitive GenomeMuSiC could not be used . Homozygous mutations affecting genes in the DSBR by HR pathway cause DSB repair defects reminiscent of BRCA1 or BRCA2 loss , a phenotypic feature dubbed BRCAness ( McCabe et al . , 2006 ) . Having established that MMR-deficient tumors are enriched in heterozygous frameshift mutations in the DSBR by HR pathway , we investigated the functional impact of these events . First , we confirmed that indels affecting the DSBR by HR pathway were located in the major tumor subclone ( Table 2 , Table 2—source data 1 ) . Then , we analyzed HR in seven MMR-deficient and four MMR-proficient patient-derived primary tumor cultures . We exposed these cultures to the PARP inhibitor olaparib , which induces DSBs upon DNA replication through single-strand break repair inhibition , and to mitomycin C , which induces DSBs through DNA cross-links and replication fork collapse ( Bunting et al . , 2012 ) . We then quantified the relative number of cells with γH2AX- and RAD51-positive foci , respectively , as a measure of induced DSBs and ongoing HR . Exposure to olaparib or mitomycin C triggered an increase in γH2AX-foci in all tumor cultures , regardless of MMR status . In contrast , although RAD51 foci formation was evident in MMR-deficient and MMR-proficient cultures , the increase was far less pronounced in MMR-deficient cultures ( Figure 6A , B ) , and this for both olaparib ( p = 0 . 021 ) and mitomycin C ( p = 0 . 006 ) exposure . The reduction in RAD51 foci could not be ascribed to differences in RAD51 protein expression or differences in cell cycle between MMR-deficient and -proficient cells , as these were similar between both sets of cultures , under both treated and untreated conditions ( Figure 6—figure supplements 1–3 ) . Since RAD51 foci are completely absent upon PARP inhibition in cells with homozygous loss of BRCA1 , but not affected in heterozygous mutation carriers ( Farmer et al . , 2005 ) , these ex vivo data suggest that the accumulation of indels in MMR-deficient tumors gradually impairs the DSBR by HR pathway to a level that is intermediate to that of cells heterozygous- and homozygous-deficient for BRCA1 . 10 . 7554/eLife . 02725 . 033Figure 6 . Reduced DSBR by HR activity in MMR-deficient cells . ( A ) Representative confocal images of MMR-deficient and MMR-proficient primary tumor cells exposed for 24 hr to vehicle , 26 μM olaparib , or 300 nM mitomycin C stained for the homologous repair marker RAD51 ( green ) , the DNA damage marker γH2AX ( red ) , and counterstained with DAPI ( blue ) . The bar is 10 µm wide . ( B ) Quantification of cells containing >5 RAD51 or γH2AX foci . Averages are shown for MMR-deficient and MMR-proficient primary tumor cultures after 24 hr of treatment with vehicle , 26 μM olaparib or 300 nM mitomycin C . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 03310 . 7554/eLife . 02725 . 034Figure 6—figure supplement 1 . Cell cycle distribution in untreated MMR-deficient and MMR-proficient cell cultures . No difference was observed in G1 , S , or G2/M phase frequency between 7 MMR-deficient and 4 MMR-proficient cultures ( p = 0 . 45 , 0 . 30 or 0 . 94 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 03410 . 7554/eLife . 02725 . 035Figure 6—figure supplement 2 . MMR-deficient tumor cultures were challenged with olaparib ( 26 μM ) , camptothecin ( 30 nM ) , or mitomycin C ( 300 nM ) for 24 hr , pulsed with BrdU for 2 hr and analyzed for cell cycle by propidium iodide staining ( DNA content analysis ) using flow cytometry . The bar plot shows the fraction of unlabeled ( arrested ) cells in S and G2/M , normalized to the G1 fraction; bars indicate SEM; data represent the results from 7 cultures . All experiments were repeated twice . DNA damage provoked by exposure to camptothecin consistently increased stalled ( BrdU-negative ) cells in S phase ( average 13-fold increase; p = 5 . 23E−5 ) . Mitomycin C caused an increase of stalled cells in S phase ( 3 . 08-fold; p = 5 . 8E−3 ) and in G2/M phase ( 3 . 12-fold; p = 2 . 2E−7 ) . Olaparib induced , as expected , an increase in stalled cells in S and G2/M ( respectively , a 3 . 35 and a 2 . 54-fold increase; p = 2 . 1E−3 and 5 . 2E−4 ) . Overall , this indicates that MMR-deficient cultures did not exhibit any loss of G2/M cell cycle checkpoints or DNA damage signaling . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 03510 . 7554/eLife . 02725 . 036Figure 6—figure supplement 3 . Example of a 2 hr BrdU pulse-labeled MMR-deficient cell culture , demonstrating S-phase stalling and G2/M stalling upon mitomycin C exposure , S-phase stalling upon camptothecin exposure and S-phase stalling and G2/M stalling upon olaparib exposure . Cell cycle phases in unlabeled ( stalled ) fractions were determined as described by Watson et al . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 036 As MMR-deficient tumors are compromised in their DSBR by HR activity , we wondered whether these tumors , similar to BRCA1-deficient tumors ( Farmer et al . , 2005 ) , are more sensitive to agents that induce DSBs . First , since PARP inhibitors are already used in clinical practice , all seven MMR-deficient and four MMR-proficient cultures were dose-dependently exposed to olaparib . This revealed that MMR-deficient cultures exhibited a dose-dependent decrease in proliferation upon exposure to olaparib , whereas MMR-proficient cultures were only affected at higher concentrations . Likewise , cell cytotoxicity assays revealed a dose-dependent sensitivity of MMR-deficient cells to olaparib that was more pronounced than in MMR-proficient cells ( 50% growth inhibition [GI50] ) was reached at 26 µM vs 129 µM , respectively , p = 0 . 0064 ( Figure 7A , B , Figure 7—figure supplement 1 ) . Other DSB-inducing compounds such as mitomycin C or ionizing radiation similarly proved more detrimental for MMR-deficient than MMR-proficient cells ( Figure 7B ) . In contrast , cytotoxicities of other chemotherapeutic compounds such as paclitaxel were comparable between both groups . 10 . 7554/eLife . 02725 . 037Figure 7 . MMR-deficient cells are sensitive to PARP inhibition . ( A ) Dosimetry experiments assessing the effect of decreasing concentrations of olaparib on in vitro cell proliferation relative to the corresponding untreated cultures as measured by sulforhodamine B assays . ( B ) Cytotoxicity of olaparib , mitomycin C , ionizing radiation and paclitaxel as measured by sulforhodamine B assays . Displayed are the average concentrations ( μM ) or dose ( Grey , Gy ) that inhibit 50% of the normal growth . p-values are 0 . 0077 , 0 . 040 , and 0 . 038 for olaparib , mitomycin C , and ionizing radiation , while p-value is not significant ( NS ) for paclitaxel . ( C ) Effect of knock-down of BRCA1 , BRCA2 , and ATR mRNA on olaparib sensitivity of the MMR-proficient , HR-proficient MCF7 cell line . Cells were transfected with the indicated siRNA concentration ( X axis ) , and after 24 hr incubated with 26 µM olaparib or vehicle . Another 48 hr later , cell viability was assessed using the sulforhodamine B assay . The siRNA concentration corresponding to a growth inhibition of 50% was subsequently assessed for the level of knock-down induced . The resulting values are indicated on the plots and are expressed as % . Values plotted were normalized to vehicle-treated cells transfected with a scrambled siRNA of matching concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 03710 . 7554/eLife . 02725 . 038Figure 7—figure supplement 1 . Cell proliferation of MMR-deficient cultures was measured in real-time using the xCELLigence RTCA DP system ( for up to 48 hr after treatment ) . Values are normalized to the vehicle-treated control . Error bars represent SEM . The average cell proliferation of 7 MMR-deficient cells ( A ) and 4 MMR-proficient cells ( B ) with increasing concentrations of olaparib ( 1 μM , 3 μM , 10 μM ) is shown . Overall , MMR-deficient cultures were characterized by a dose-dependent decrease in proliferation , whereas MMR-proficient cells did not response to olaparib . DOI: http://dx . doi . org/10 . 7554/eLife . 02725 . 038 Finally , in order to more accurately measure the level of HR-deficiency in MMR-deficient tumors , we assessed the level of knock-down of BRCA1 , BRCA2 , and ATR needed to achieve an olaparib sensitivity similar to that observed in MMR-deficient cells , that is , a GI50 of 26 µM . BRCA1 , BRCA2 , or ATR expression was dose-dependently reduced using siRNAs in the MMR- and HR-proficient cell line , MCF7 . A growth inhibition of 50% was reached in MCF7 cells when applying 5 . 9 nM ATR , 0 . 88 nM BRCA1 or 0 . 41 nM BRCA2 siRNA , corresponding respectively to a reduction in expression of 69 . 5 ± 1 . 1% , 76 . 1 ± 4 . 4% , and 80 . 0 ± 2 . 4% ( Figure 7C ) . These data thus suggest that the loss of DSBR by HR activity in MMR-deficient tumors corresponds to a loss of about 75–80% BRCA1 or BRCA2 expression .
Here , we surveyed whole-genomes of MMR-deficient tumors to provide a comprehensive picture of the mutations associated with human MMR-deficiency . With respect to somatic substitutions , we observed that the majority represented transitions and not transversions , and that adjacent nucleotides and various genomic features had an important context-dependent effect on determining which nucleotides were affected . Remarkably , the observed substitution pattern , in particular how it was impacted by small and large-scale contexts , was very similar to that in the germ-line at different time scales: for germ-line substitutions as they currently arise ( de novo ) , as they have accumulated in the human population or as they served as a substrate for human-chimpanzee divergence ( Hodgkinson and Eyre-Walker , 2011 ) . Our observations thus suggest that , similar to bacterial populations and other lower organisms ( Saint-Ruf and Matic , 2006 ) , incomplete mismatch repair in humans contributes significantly to genetic variability and probably also to natural selection through genetic adaptation . Additionally , our data provide fundamental insights into the function of the MMR machinery . We observed , for instance , a higher number of substitutions in methylated CpG sequences , implicating MMR in the repair of methylated cytosine deamination and demonstrating that MMR disconnected from the replication fork is also critical to maintain genomic integrity . At the whole-genome level , ∼80% of somatic mutations represented indels . Although indel detection using high-throughput sequencing is burdened with high false-positive rates , 88 . 0% of the indels identified here validated favorably using orthogonal technologies . When focusing on the clinical relevance of indel mutation patterns to diagnose MSI , we observed that indels specifically affected homopolymer stretches , which is relevant as the extended Bethesda panel consists of eight microsatellite and only two homopolymer markers and possibly therefore has only limited sensitivity relative to IHC ( ∼75% for both EM and CRC tumors [Hampel et al . , 2005 , 2006 , 2008] ) . Our 59-marker panel consisting only of markers in homopolymers was clearly more sensitive than Bethesda , yielding sensitivity rates of 87% relative to IHC . This was not due to the fact that we genotyped more markers than Bethesda , as restricting our panel to 10 markers still resulted in a sensitivity rate of 85% ( data not shown ) . Furthermore , since our panel was based on recurrent mutations present in both CRC and EM , and since 50 out of 59 markers were located in UTRs , which are less likely to drive clonal selection and thus to represent tissue-specific events , it could be used to detect MSI in cancers affecting various tissues . Finally , since all markers were located in homopolymers ≤12 bps in length , they are , in contrast to the 25 or 26 bps markers from Bethesda , compatible with various low- to high-throughput genotyping technologies , thereby greatly facilitating their clinical adoption . For instance , we were able to multiplex all 59 markers in just five PCR amplification reactions compatible with Sequenom MassArray genotyping . Pathway analyses on all genes affected by exonic indels further revealed that the DSBR by HR pathway was enriched for somatic indels . Although mutations in genes involved in this pathway , such as MRE11A or RAD50 , have previously been reported in MMR-deficient tumors , these studies focused on specific mutations in individual genes rather than on pathways , and for this reason could establish that only a fraction of MMR-deficient tumors was affected by mutations in these genes ( Miquel et al . , 2007 ) . In contrast , our study identified that every MMR-deficient tumors was affected by on average 3 . 3 somatic indels in the DSBR by HR pathway . Furthermore , although it is well established that cells deficient in BRCA1 , BRCA2 , Fanconi anemia , or other HR-related genes are hypersensitive to DSB inducers ( Murai et al . , 2012 ) , as for instance , synthetic lethality in BRCA1- or BRCA2-deficient tumors through PARP inhibition is already approved as therapy in breast and ovarian cancer ( Metzger-Filho et al . , 2012 ) , data demonstrating sensitivity of MMR-deficient cells to DSB inducers have not been conclusive ( Takahashi et al . , 2011; Vilar et al . , 2011; Park et al . , 2013 ) . For instance , although there are some reports highlighting the sensitivity of MSH3-deficient cell lines to DSB inducers , this appeared to occur through a non-canonical MMR pathway , as MLH1 was not involved in this process ( Takahashi et al . , 2011; Park et al . , 2013 ) . Furthermore , the only clinical study set-up so far to explore efficacy of PARP inhibitors as a single-agent therapy in previously treated patients with metastatic CRC stratified by MSI status , was unfortunately delayed due to patient accrual issues . Our hypothesis-free discovery that DSBR by HR is the top pathway affected by heterozygous loss-of-function mutations in MMR-deficient tumors , both in our own data set and TCGA , also suggests that mutations in DSBR by HR genes converge in an oligogenic model , wherein the number of indels dose-dependently decreases DSBR by HR activity , thereby rendering them gradually more sensitive to DSB inducers . As a result of this double-hit , our ex vivo culture experiments are , however , difficult to compare to experiments relying on genotype-matched cells that have a single hit in the MMR or HR pathway . In addition , since MMR and DSB by HR pathway activities are not characterized in a clinical setting , it is difficult to relate our data to clinical studies assessing the outcome of therapeutics such as cisplatin or 5-fluorouracil , which have potential opposing activities on MMR- and HR-deficient tumors . Clinical studies are therefore needed to assess whether DSB inducers , such as PARP inhibitors , are indeed also effective in MSI tumors . In particular , since on average 3 . 3 heterozygous loss-of-function mutations only partially inactivate the DSB repair by HR pathway ( ∼80% inactivation ) , it remains to be seen whether , compared to BRCA1 or BRCA2-deficient tumors , in which the HR pathway is completely inactivated , clinically relevant benefits are also achievable in MSI tumors . Possibly , only those MMR-deficient tumors containing large numbers of indels ( ≥5 ) in the DSBR by HR pathway will show a significant response . Nevertheless , there is a great clinical need for novel treatment options in MSI tumors . Indeed , although stage II or III CRC tumors with MSI are characterized by a modestly improved prognosis , MSI tumors in the advanced setting are generally associated with a more peritoneal metastasis and a worse overall survival independent of the chemotherapy regimen ( Smith et al . , 2013; Yoon et al . , 2013 ) . Our observations thus clearly warrant novel clinical studies assessing the therapeutic efficacy of DSB inducers in MMR-deficient tumors .
To assess MLH1- , MSH2- , and MSH6-deficiency immunohistochemistry using monoclonal antibodies against MLH1 ( clone ES05; DAKO , Heverlee , Belgium ) , MSH2 ( clone G219-1129; BD Pharmagen , Erembodegem , Belgium ) , and MSH6 ( clone EP49; Epitomics , Burlingame , USA ) were applied . Absence of nuclear staining in tumor cells and normal staining in the surrounding normal tissue were considered as MMR-deficient . Methylation of the MLH1 promoter was determined using the SALSA MS-MLPA KIT ( MRC-Holland , Amsterdam , The Netherlands ) . PCR reaction fragments covering the Deng C and Deng D regions were separated by capillary gel electrophoresis ( ABI 3130; Applied Biosystems , Ghent , Belgium ) and quantified using the Genemarker ( v1 . 91 ) software ( Softgenetics ) . MSI status was detected by the extended Bethesda panel using capillary gel electrophoresis , as described previously ( Dietmaier et al . , 1997; Boland et al . , 1998 ) . We selected 17 endometrial , three colorectal , and two ovarian tumor–normal pairs for either whole-genome or whole-exome sequencing . Samples were all chemo-naive . DNA was derived from fresh frozen , primary tumors . Matched normal DNA for these 22 samples was extracted from peripheral white blood cells . Five tumor–normal pairs were selected for whole-genome sequencing . Paired-end sequencing was performed using the Complete Genomics service ( CG , Mountain View , California , USA ) as described in Drmanac et al . ( 2010 ) or by Illumina HiSeq2000 . For CG sequencing , reads were initially mapped to the reference genome ( hg18 ) using Complete Genomics' CGAtools . Between 207 and 338 Gb of sequencing data were obtained , resulting in a haploid coverage between 73× and 119× . Approximately , 2 . 7 × 109 bases were called in each genome , representing ∼95% of the total genome and ∼97% of the exome . Substitutions and indels were called by the variant caller in the CGAtools . On average , 3 , 132 , 715 substitutions and 357 , 153 indels were detected in each genome . The CGAtool ( v1 . 0 . 3 . 9 ) calldiff method was used to detect somatic mutations in the tumor–normal pairs . For Illumina sequencing , 2 × 100 bp paired-end sequencing was performed , yielding 25–30x coverage per sample . Burrows-Wheeler Alignment ( BWA ) was used to align the raw reads to the reference genome ( hg19 ) ( Li and Durbin , 2010 ) . PCR duplicates were removed with Picard MarkDuplicates ( v1 . 32 ) . Base recalibration , local realignment around indels and single nucleotide variant calling were performed using the GenomeAnalysisToolKit ( GATK v1 . 0 . 4487 ) ( McKenna et al . , 2010 ) . Small indels were detected using Dindel ( v1 . 01 ) ( Albers et al . , 2011 ) . Substitutions and indels with quality score >Q30 were considered . On average , 3 , 977 , 086 substitutions and 837 , 915 indels were detected in each genome . Somatic mutations were detected by means of intersectBed command of BEDTools ( v2 . 12 . 0 ) ( Quinlan and Hall , 2010 ) . Raw data for all whole-genomes are available under restricted access in the European Genome-Phenome Archive ( EGA ) with accession number EGAS00001000182 . Sequence data were annotated using ANNOVAR ( v2013Jun21 ) and the UCSC RefGene annotation track . Germ-line substitutions and indels were eliminated from the list of somatic mutations using the following publicly available datasets: ( i ) common SNPs in dbSNP ( v132 ) with a minor allele frequency of >1% , ( ii ) substitutions identified in the November 2010 release of the 1000 Genomes Project , ( iii ) the Axiom Genotype Data Set containing common SNPs from 1261 HapMap3 individuals in 11 populations , and ( iv ) variant data identified in 46 HapMap individuals ( CG diversity panel ) . Somatic mutations were validated using Sequenom MassARRAY genotyping , as previously described ( Reumers et al . , 2011 ) . Details of validation experiments are shown in Figure 1—source data 3 . A quality score method to enrich for true somatic mutations by defining a threshold that differentiates false-positive and true-positive variants based on Sequenom validation data was applied to CG genomes and increased the validation rate for substitutions from 93 . 5% , 71 . 4% , and 55 . 6% to 97 . 7% , 100% , and 73 . 3% for MMR− 1 , MMR+ 1 , and MMR+ , 2 respectively . Detailed data of all somatic mutations are in Figure 1—source data 1 and Figure 1—source data 2 . Copy number status of the sequenced tumors was determined by Illumina CytoSNP-12 chips and analyzed using the ASCAT algorithm ( Van Loo et al . , 2010 ) . Copy number status of the five whole-genomes was shown as Figure 1—figure supplement 3 . The genome was annotated into the following functional genomic regions: ( coding ) exonic regions ( 1 . 12% ) , intronic regions ( 34 . 01% ) , 3′ untranslated regions ( 3′UTR , 0 . 78% ) , 5′ untranslated regions ( 5’UTR , 0 . 14% ) , noncoding RNA ( ncRNA , 2 . 81% ) , upstream genic regions ( defined as 1 kb before the start of the gene , 0 . 58% ) , downstream genic regions ( defined as 1 kb after the end of the gene , 0 . 58% ) , and intergenic regions ( 59 . 98% ) . Overall mutation frequencies were defined as the number of somatic mutations per base ( mpb ) in a given genomic region . To assess negative selection in the exome , we checked whether ( i ) there was a lower mutation frequency in the exome relative to the whole-genome , and whether ( ii ) the frequency of somatic mutations was more prominently decreased in the exome . As homopolymers in exomes have characteristics that differ from those in the rest of the genome in terms of number , base composition and length , we corrected indel frequencies for these confounding factors . We calculated the frequency of affected homopolymers for each genomic location ( t: exonic , 5′UTR , 3′UTR , intronic , intergenic , or genomic ) , for each type of homopolymer ( AT or CG composition ) and each homopolymer length ( 6 , 7 , 8 , etc[l] ) . ATFreqtl = ATaffntl . Next , we calculate the relative increase of observed frequencies relative to the frequency observed at the genome-wide level: ATrFreqtl = ATFreqtl/ATFreqgenomel . The frequency ATrFreqtl was normalized for the number of homopolymers of a given length l , for each genomic location t and for homopolymer composition ( ATwrFreqtl = ATrFreqtl × ATntl/∑ ATntl ) , and further normalized for the number of AT ( or GC ) homopolymers for each genomic location and homopolymer length ( ATnwrFreqtl = ATwrFreqtl × ATntl/ ( ATntl + CGntl ) ) . All the weighted frequencies are then summed for every genomic location ( cFreqt = ∑ ATnwrFreqtl + ∑ CGnwrFreqtl ) and divided by the overall summed genomic frequency ( rFreq = cFreqt/cFreqgenomic ) . The following datasets were used: ( i ) the 1000 Genomes Project containing common variants with a minor allellic frequency >10% , ( ii ) all germ-line variants identified in the 3 MMR-deficient tumors sequenced in this study , ( iii ) de novo mutations from 83 trios as published by Campbell et al . ( 2012 ) and Kong et al . ( 2012 ) , and ( iv ) a human-chimp divergence set of substitutions as previously described ( Stamatoyannopoulos et al . , 2009 ) . Somatic mutations identified in other tumor whole-genomes were: ( i ) BRCA-deficient breast cancer tumors as published by Nik-Zainal et al . ( 2012 ) , ( ii ) MMR-proficient endometrial tumors sequenced in this study , ( iii ) melanoma genomes as published by Pleasance et al . ( 2010 ) , and ( iv ) small cell lung cancer ( SCLC ) as published by Pleasance et al . ( 2010 ) . The distance to telomere was defined as the distance from the middle of the 1 Mb window to the beginning or the end of the chromosome whichever was the shortest . Replication time was considered as published by Chen et al . ( 2010 ) . Simple repeats represented the number of homopolymer and microsatellite bases . GC% was calculated as ( G+C ) / ( A+T+G+C ) , CpG content as the number of CG dinucleotide bases , CpG islands as the number of bases belonging to CpG islands , gene content as the number of bases belonging to each genomic region . DNase hypersensitivity ( DNAseI size ) and nuclear lamina binding sites were downloaded from UCSC and the number of bases per site was counted for both . We sequenced 11 tumor–normal pairs , 6 primary cell cultures ( PC ) and their match normal DNA samples . Detailed clinical information is shown in Figure 4—source data 1 . Exomes were captured using Illumina's TruSeq Exome Enrichment Kit . The TruSeq capture regions encompass 62 Mb , consisting of 94 . 4% , 83 . 9% , and 91 . 9% of the exonic , 5′UTR and 3′UTR regions respectively . 2 × 75 bp paired-end sequencing reactions were used for all EM tumors , while 2 × 100 bp paired-end sequencing was performed on CRC tumors and PC samples . Analysis , annotation , and validation were performed similarly as for whole-genome sequencing . On average , the coverage was 44 . 5× and 95 . 1% of bases were called in the captured regions , yielding 51 , 782 substitutions and 30 , 290 indels per sample . Raw data are available under restricted access in EGA under accession number EGAS00001000182 . Details of validated somatic mutations are available in Figure 4—source data 1 and Figure 4—source data 2 . The 13 MMR-deficient whole-exomes and whole-exome data extracted from 3 MMR-deficient whole-genomes were screened for recurrent mutations . Random selection and validation of 24 indels occurring in 6 or more samples revealed a validation rate of 100% . Given the high validation rate for somatic indels per se , and the even higher rate for recurrent indels , we considered all recurrent indels as true-positives . Subsequent analyses were limited to indels recurrently affecting homopolymer regions , that is , 29 , 663 Illumina TruSeq-captured exonic homopolymers . Details of recurrent mutations in these homopolymers are available in Figure 4—source data 3 . We also screened 5430 and 60 , 942 homopolymers located in the exome-captured 5′ and 3′ UTRs for recurrent indels . Details of these recurrent indels are in Figure 4—source data 3 . Recurrent indels meeting the following criteria were considered for a targeted Sequenom panel assessing MSI: ( i ) occurring in 6 or more samples , ( ii ) detected in both EM and CRC exomes , ( iii ) the maximal length of affected homopolymer <12 bp . After extensive optimization experiments , 59 markers were chosen . Detailed information about each indel is given in Figure 5—source data 1 . 236 EM tumors used to establish MSI thresholds were drawn from the Australian National Endometrial Cancer Study ( ANECS ) . IHC analyses of these tumors were independently performed at the Molecular Cancer Epidemiology Laboratory in Brisbane , Australia as described ( Tan et al . , 2013 ) . 11 out of 236 tumors were excluded for the 59-marker panel due to their low tumor percentage ( ≤10% ) . By varying the marker threshold , we calculated the number of true-positives and false-positives identified by our MSI panel relative to the IHC data . A ROC curve was constructed based on these values . The Matthew Correlation Coefficient of the ROC curve was calculated for each threshold . Tumors were considered MSI when they had three markers positive . We did not distinguish between MSI-low and microsatellite stable ( MSS ) , as this is currently not clinically relevant . All tumors with less than three positive markers were thus considered MSS/MSI-L . For the Bethesda panel , we defined three categories as follows: microsatellite stable ( MSS , 0 out of 10 markers ) , low microsatellite instability ( MSI-L , 1–2 out of 10 markers ) , and high microsatellite instability ( MSI-H , 3 or more out of 10 markers ) . Two sets of data ( 114 EM tumors and 97 CRC tumors ) were used for the comparison . Details of these sample sets are given in Figure 5—source data 1 . The 59-marker panel was applied to ovarian tumors and leukemia . Four samples with proven MSI status were selected , including one ovarian tumor ( OV ) and three leukemia cell lines ( DND41 , CCRF-CEM , and SUPT1 ) . The MSI-H OV tumor , two MSS OV tumors , and their matched normal samples , as well as three MSI-H leukemia cell lines and a MSS leukemia cell line ( RPMI-8402 ) were exome-sequenced . Detailed information for all samples can be found in Figure 5—source data 2 . Raw data are available in EGA under the accession number EGAS00001000182 . Two pathway tools ( IPA and GenomeMuSiC ) and three pathway databases ( IPA , BioCarta , and Reactome [Haw et al . , 2011] ) were used . We first selected all genes with somatic exonic indels , and then extended our mutation calling to indels occurring 25 bp up or down-stream of each exon . Mutation calling and filtering for the later set of mutations was done as described above . In total , 1989 additional indels in exon/intron boundaries were detected . These were combined with the previously described indels in exonic regions , which—after the removal of indels in MMR genes—yielded 7546 indels in 4116 genes . As a validation , we selected 27 CRC and 65 EM tumors with MSI sequenced by The Cancer Genome Atlas ( TCGA , 2012; Kandoth et al . , 2013 ) . We selected genes recurrently affected not only by frameshift indels but also by non-synonymous substitutions . There were 2183 and 3138 genes from the CRC and EM tumor data sets , respectively . Detailed results of pathway analyses are given in Table 2—source data 1 . 11 primary endometrial and ovarian tumor cell cultures were established from tumors of patients undergoing surgery at the Division of Gynecologic Oncology , UZ Leuven ( Belgium ) . Tissue was washed with PBS supplemented with penicillin/streptomycin and fungizone , digested with collagenases type IV ( 1 mg/ml; Roche , Vilvoorde , Belgium ) and DNAse I ( 0 . 1 mg/ml; Roche ) in RPMI+ medium . Single cell suspensions were prepared by filtration through a 70-µm filter . Red blood cells were lysed using ammonium chloride ( Stem Cell Technologies , Grenoble , France ) . Single cells were plated into a 25-cm ( Parsons et al . , 2012 ) culture flask . After 1–3 weeks , when cells reached 60–70% confluency , fibroblasts were removed using mouse anti-human CD90 ( Clone AS02; Dianova , Hamburg , Germany ) bound to Mouse Pan IgG Dynabeads ( Life Technologies , Erembodegem , Belgium ) . Cell cultures were subsequently passaged at 70–90% confluency and banked at −80°C . Primary tumor cell cultures were grown in RPMI Medium 1640 supplemented with 20% fetal bovine serum ( FBS ) , 2 mM L-Glutamine , 100U/ml penicillin , 100 μg/ml streptomycin , 1 μg/ml fungizone , and 10 μg/ml gentamicin ( Life Technologies ) up to 25 passages . Cells were seeded in 8-well Lab-tek Permanox Chamber slides ( Nunc , Zellik , Belgium ) , treated for 24 hr , fixed in 4% paraformaldehyde for 15 min at room temperature , and ice-cold methanol for 5 min . Primary antibodies recognizing γH2AX ( JBW301 , Millipore , Overijse , Belgium ) and RAD51 ( PC130 , Merck , Darmstadt , Germany ) followed by secondary antibodies conjugated to Alexa Fluor 647 and 488 ( Life Technologies ) were used . Images were acquired using an A1R Eclipse Ti inverted confocal microscope ( Nikon , Brussels , Belgium ) and processed using Fiji software , with compound or vehicle-treated cells being processed identically . Nuclei with >5 foci were scored as positive , and at least 200 nuclei were counted per condition by two independent individuals , blinded to the genotypes . Cells were treated for 24 hr with 26 µM olaparib , 0 . 3 µM mitomycin C , 0 . 03 µM camptothecin or carrier , and incubated for 90 min with BrdU ( 10 µM ) before harvesting . Cells were resuspended in ice-cold PBS and ice-cold ethanol was slowly added to 70% . Cells were fixed for 5 min at room temperature , treated with 2 M HCl for 30 min and stained with FITC-conjugated anti-BrdU antibody ( BD ) . Cells were washed , resuspended in PI/RNase staining buffer ( BD ) , and analyzed on a BD Biosciences FACSVerse flow cytometer . Cell cycle distributions were modeled using FlowJo software , and the fraction of cells in S-phase , G2/M and G1 determined as described by Watson et al . ( 1987 ) . 5 , 000 cells/well were seeded in 96-well plates . After 24 hr , cells were treated in quadruplicate , incubated for 48 hr at 37°C and analyzed using the In Vitro Toxicology Assay Kit , Sulforhodamine B-based ( Sigma , Diegem , Belgium ) as per the manufacturer's instructions . Growth inhibition was calculated as described ( Vichai and Kirtikara , 2006 ) . siRNA ON-TARGETplus SMART pools ( Thermo ) were diluted in Optimem I reduced serum medium using Lipofectamine RNAiMAX ( Life technologies ) to reverse transfect MCF7 cells For cytotoxicity screening , transfections were in 96-well format and medium was changed 14 hr after transfection . Cells were treated with olaparib ( 26 μM ) and after 48 hr processed for cytotoxicity screening . Simultaneously , siRNA transfections in 12-well plates were done to quantify knockdown . Total RNA was extracted using the RNeasy Mini kit ( Qiagen , Venlo , The Netherlands ) and reverse transcribed using the SuperScript III reverse transcription system ( Life technologies ) . Quantitative RT-PCR ( qRT-PCR ) with ACTB an internal control was performed using TaqMan gene expression assay probes and 5 μl TaqMan Fast Universal PCR master mix ( Life technologies ) . Reactions were amplified in a Roche LightCycler 480 using the following cycles: 50°C ( 2 min ) , 95°C ( 30 s ) , and 40 cycles of 95°C ( 3 s ) , 60°C ( 30 s ) . Mouse anti-phospho-Histone H2A . X ( Ser139 ) monoclonal antibody ( clone JBW301 ) was from Millipore Corporation , Billerica , MA , USA . Rabbit anti-Rad51 ( PC130 ) polyclonal antibody was from Calbiochem/Merck , Darmstadt , Germany . Rabbit anti-ACTB ( #4967 ) polyclonal antibody was from Cell Signalling , Danvers , MA , USA . FITC-conjugated anti-BrdU antibody ( 347583 ) was from Becton–Dickinson , San Jose , CA , USA . Alexa Fluor 647 goat anti-mouse IgG ( A-21235 ) and Alexa Fluor 488 goat anti-rabbit IgG ( A-11034 ) were from Life technologies , Carlsbad , CA , USA . Olaparib ( AZD-2281 , batch JSAR104 ) was purchased from JS Research Chemicals Trading , Schleswig Holstein , Germany . Cis-platinum ( II ) diammine dichloride ( P4394 ) , paclitaxel ( T7402 ) , mitomycin C ( M4287 ) , ( S ) - ( + ) -camptothecin ( C9911 ) and carmustine ( C0400 ) were purchased from Sigma-Aldrich , St . Louis , MO , USA , and prepared and stored according to the manufacturer's recommendations . siRNA ON-TARGETplus SMART pools were purchased from Thermo Scientific Dharmacon , Chicago , IL , USA: Non-targeting ( D-001810-10-05 ) ; ATM ( L-003201-00-0005 ) ; ATR ( L-003202-00-0005 ) ; BRCA1 ( L-003461-00-0005 ) ; and BRCA2 ( L-003462-00-0005 ) . TaqMan gene expression assays ( Life technologies , Carlsbad , CA , USA ) used in this study were as follows ATM: Hs01112355_g1; ATR: Hs00992123_m1; BRCA1: Hs01556193_m1; BRCA2: Hs00609073_m1; ACTB: Hs99999903_m1 . Normal goat serum ( 005-000-121 ) was from Jackson Immunoresearch Labs , West Grove , PA USA .
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 .
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 .