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Title: Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks}
Article:
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.
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.
Discussion:
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.
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 .