Mismatch Repair Profiles in Human Cancer
Dr Alfred Grech, Dr Stephen West & Dr Ramon Tonna
DNA mismatch repair (MMR) is a highly conserved biological pathway. Mismatch repair proteins excise and rectify DNA mismatches that result from the action of DNA polymerase during cell division. Commonly such DNA mismatches occur in microsatellites, which are repetitive DNA sequences in our genome. Thus, DNA MMR is decisively essential in maintaining DNA replication fidelity, avoiding the occurrence of mutations and giving stability to the genome. Clinically, MMR protein status is being increasingly used in the management of human cancer.
Robin Holliday in 1964 proposed the notion that mismatched base pairs arise in cells and that such errors prompt their own repair during genetic recombination.1 In 1975, Wildenberg and Meselson2 showed that E. coli could correct DNA with mismatched nucleotides. Subsequently, Marinus3 and Radmanet al.4 did pivotal studies putting E. coli as a model for the MMR pathway. Their work demonstrated that the mutator genes MutS5, MutL6, MutH7, UvrD7 together with DNA adenine methylase8, which were already recognized, were essential for the MMR process.
These works on the MMR pathway in E. Coli pioneered other extensive studies and today the pathway is well characterized. Briefly, the MMR process in E. Coli has the following main stages: (i) MutS binds to the mismatch, (ii) MutS recruits MutL and MutH, (iii) DNA strand containing the mismatched nucleotide is excised, and then (iv) the excision gap is resynthesized by the DNA polymerase.
The main function of the MMR system is that it corrects mismatch nucleotides that are generated by the action of DNA polymerase during DNA replication.9 Indeed, the MMR system increases the fidelity during replication by 100- to 1000-fold.10,11Besides preventing accumulation of mutations, MMR also plays in cell cycle checkpoint and apoptosis activation in the ‘MMR-dependent DNA damage response’.12
LOSS OF MMR FUNCTION, MICROSATELLITE INSTABILITY (MSI) AND TUMORIGENESIS
Like practically all eukaryotic species, the human genome is full of tandemly repetitive DNA sequences. Microsatellites also called short tandem repeats (STRs) are typically repeated 5 to 50 times. The most commonly agreed number of nucleotides that define them is 1 to 6. Thus such repeats can be dinucleotide repeats, trinuleotide repeats, up to hexanucleotide repeats. During DNA replication in the S phase of the cell cycle, DNA polymerase tends to ‘slip’ (DNA polymerase slippage) in these repetitive sequences, causing mismatch of nucleotides, which are repaired by the MMR protein machinery coded by their respective genes, namely MSH2, MLH1, MSH6 and PMS2.
Cells that are ‘MMR deficient’ have what is called a ‘mutator phenotype’ with an increased tendency for spontaneous mutations.13,14 These mutations can affect the whole genome but especially affect microsatellites. This creates ‘microsatellite instability’(MSI) and favours tumorigenesis.15,16
The mutations are insertions or deletions. If these mutations affect microsatellites of coding genes they result in ‘frameshift mutations’ and result in the coding of ‘truncated’ proteins that have impaired function or no function at all.
The link between MMR deficiency and tumorigenesis has been extensively studied in Lynch syndrome, where autosomal dominant inherited (germline) mutations in the main MMR genes predispose to various cancer, mainly colorectal cancer (CRC, here called Hereditary Non Polyposis Colon Cancer,HNPCC) and endometrial cancer (EC).17 In fact, 3% of all CRC and 2% of all EC are Lynch-syndrome associated.17 Other cancers associated with Lynch syndrome include pancreatic ductal adenocarcinoma (PDAC),18 gastric, esophageal19 and upper urinary tract cancers.
TESTING FOR MMR AND MSI PROFILES
In clinical oncology, testing for MMR gene deficiency (MLH1, PMS2, MSH2 and MSH6) on tumour tissue is carried out. The tests can include MMR immunohistochemistry (IHC) testing, PCR-based MSIanalysis, and DNA sequencing analysis. This is becoming crucial in order toguide treatment and predict prognosis. Always, test results are to be interpreted based also on clinical findings and family history.
MMR Immunohistochemistry Testing
MMR IHC staining of tumour tissue is done using the standard ‘streptavidin–biotin–peroxidase’ procedure. Here one is looking for MMR IHC antibodies, that mainly include those of MLH1, MSH2, MSH6 and PMS2. Tumour cells are scored as negative (MMR deficient) if they show total absence of nuclear staining, whilst adjacent tissue elements cells are scored as benign (MMR proficient) if they show nuclear staining.
PCR-based MSI Analysis
PCR-based MSI analysis is often done in combination with MMR IHC. The National Cancer Institute (NCI) recommends the following ‘microsatellite marker panels’, BAT-25 and BAT-26 (with mononucleotide repeats) and D5S346, D2S123 and D17S250 (with dinucleotide repeats).20 Another useful kit contains five mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). Xicola et al.21state that mononucleotide markers have a higher level of sensitivity and specificity in detecting the MSI-H phenotype.
Three phenotypes are defined:
- Microsatellite Stable (MSS): none of the markers show instability
- Microsatellite-Low (MSI-L): one of the markers show instability
- Microsatellite-High (MSI-H): two or more of the markers show instability.
Phenotypes MSS and MSI-L are usually grouped as a single subset because rarely tumours with such phenotypes are MMR protein deficient.
Next Generation Sequencing
Next-generation sequencing (NGS) panels have been devised to detect mismatch repair deficient (MMR-D) and MSI-H based on mutational phenotype in various tumours.22–25 Indeed, several panels and kits are available that allow high parallel sequencing of MMR genes. Moreover, NGS used for genome-wide analyses is possible and have shown that hypermutation (more than 10 somatic mutations per megabase) are more prevalent than previously thought, in adult cancer, reaching approximately 17% of cancers. This inflates the use of immunotherapy, since the latter is effective in cancers with an increased mutational burden.24
MMR AND MSI STATUS IN THERANOSTICS
Translational studies that analyse the history, genomics, epigenetics and pathology of MMR-D tumours are being used in theranostics, the latter being an emerging field of medicine that uniquely combines drugs and/or techniques to simultaneously or sequentially help in the diagnosis, prognosis and treatment of medical conditions, here cancer.
Identifying patients who possibly have Lynch syndrome is essential as they and their family members need to be monitored. As already mentioned, Lynch syndrome is associated with various cancer types. Screening for MMR and MSI status in these associated cancer types can diagnose this syndrome. The molecular signature of Lynch syndrome is microsatellite instability from germline mutation in the DNA sequence that code for the MMR proteins. More commonly, tumours with MSI profiles are sporadic and are associated with epigenetic inactivation of the MMR genes.26
Currently there is valuable evidence supporting the use of the MMR and MSI status in the prognosis of colorectal tumours. However, less is known in extra-colonic tumours. Nevertheless, MMR status and MSI are being investigated worldwide in their use in the clinical prognosis for survival in many other tumours.
A case in point is that of PDAC. Specifically, patients who have PDAC that is MMR-D have a better prognosis and thus a better prolonged survival time.27,28 In fact, Nakata et al.27 report that patients with MSI-H PDAC have a survival time of 62 months, whilst those with MSI-L have 10 months.
In the MAGIC trial,29 the consequence of MMR-D and MSI in curatively resected gastric cancer treated with peri-operative chemotherapy was evaluated. The trial showed that patients with MMR-D and MSI-H profiles had a better prognosis when treated with surgery alone, whilst those treated with chemotherapy peri-operatively had a differentially negative effect. The authors thus propose a prognostic stratification of patients based on their MSI and MMR profiles made on pre-operative biopsies.
Immunotherapy is leading to a significant change in cancer treatment. Indeed, immunotherapy has spurred cancer research because it greatly improves the efficacy of treatment and overall survival in certain patients with various cancer types. Having said that, only a small proportion of patients respond to immunotherapy, and thus specific biomarkers need to be found to stratify those that are sensitive from those that are not. Currently, the key prognostic biomarkers for immunotherapy efficacy are ‘Programmed Death-1’ (PD-1) expression and defective mismatch repair genes, causing the MSI-H phenotype.
The MSI-H phenotype leads to an accumulation of somatic mutations in tumour cells causing a high mutational burden, increased expression of neoantigens and profuse tumour-infiltrating lymphocytes. All these changes are associated with increased response to immune checkpoint inhibitors (ICIs). Thus, MSI status is emerging as a pivotal predictor of response to strategies based on immunotherapy. In keeping with this, the US Food and Drug Administrationfast-tracked the approval in 2014 for the use of ICIs for refractory MMR-D or MSI-H tumours, in both children and adults.
MSI status may also predict sensitivity or resistance of certain cancers to certain chemotherapies. For example, Pembrolizumab(PD-1 inhibitor) has been indicated for the treatment of unresectable or metastatic tumours that are MSI-H or MMR-D.
PDAC is a refractory cancer. Surgery is the only choice for the 15% to 20% of cases that are operable at diagnosis.18 After surgery, the recurrence rate stands at 80% to 85%, and many patients after resection die to their disease.30,31 A subset of PDAC patients are MMR-D and may benefit from a targeted approach using the FDA approved pembrolizumab.32–35
In a similar case scenario, this time involving colorectal cancers that are MSI-H or MMR-D, pembrolizumab can be prescribed if cancer has advanced after treatment with fluoropyrimidine, oxaliplatin, and irinotecan.36
More papers are coming out showing that MMR-D solid tumours respond to ICI.
The repercussions of MMR and MSI profiles in cancer continue to develop. It is becoming clear that these profiles are important in the diagnosis, prognosis and therapeutics of MSI-H cancers. MMR and MSI profile analyses are justified to screen for Lynch syndrome and also to stratify certain patients for their response to chemotherapy. More research across all cancer types will provide deeper understanding into MSI tumorigenesis and help further in developing better personalized therapeutic strategies.
- Holliday R. A mechanism for gene conversion in fungi. Genetical Research 2007;89(5-6):285-307.
- Wildenberg J, Meselson M. Mismatch repair in heteroduplex DNA. Proceedings of the National Academy of Sciences 1975;72(6):2202-6.
- Marinus MG. Adenine methylation of Okazaki fragments in Escherichia coli. J Bacteriol 1976;128(3):853-4.
- Radman M, Wagner RE, Glickman BW, et al. DNA methylation, mismatch correction and genetic stability. Progress in Environmental Mutagenesis 1980:121-30.
- Siegel EC, Bryson V. Mutator gene of Escherichia coli B. J Bacteriol 1967;94(1):38-47.
- Goldstein A, Smoot JS. A strain of Escherichia coli with an unusually high rate of auxotrophic mutation. J Bacteriol 1955;70(5):588-95.
- Hill RF. Location of genes controlling excision repair of UV damage and mutator activity in Escherichia coli WP2. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 1970;9(3):341-4.
- Marinus MG. Location of DNA methylation genes on the Escherichia coli K-12 genetic map. Molecular &General Genetics 1973;127(1):47-55.
- Friedberg EC, Walker GC, Siede W,et al. DNA Repair and Mutagenesis. The Quarterly Review of Biology 2006;81(3):273.
- Iyer RR, Pluciennik A, Burdett V, et al. DNA mismatch repair: functions and mechanisms. Chemical Reviews 2006;106(2):302-23.
- Jascur T, Boland CR. Structure and function of the components of the human DNA mismatch repair system. International Journal of Cancer 2006;119(9):2030-5.
- Gupta D, Heinen CD. The mismatch repair-dependent DNA damage response: Mechanisms and implications. DNA Repair 2019;78:60-9.
- Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363(6429):558-61.
- Loeb LA. Mutator phenotype may be required for multistage carcinogenesis. Cancer Research 1991;51(12):3075-9.
- Shibata D, Peinado MA, Ionov Y, et al. Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation. Nature Genetics 1994;6(3):273-81.
- Zhang H, Richards B, Wilson T, et al. Apoptosis induced by overexpression of hMSH2 or hMLH1. Cancer Research 1999;59(13):3021-7.
- Pellat A, Netter J, Perkins G, et al. Lynch syndrome: What is new?Bulletin Du Cancer 2019;106(7-8):647-55.
- Hu ZI, Shia J, Stadler ZK, et al. Evaluating Mismatch Repair Deficiency in Pancreatic Adenocarcinoma: Challenges and Recommendations. Clinical Cancer Research 2018;24(6):1326-36.
- Vrána D, Matzenauer M, Neoral Č, et al. From Tumor Immunology to Immunotherapy in Gastric and Esophageal Cancer. International Journal of Molecular Sciences 2018;20(1).
- Richman S. Deficient mismatch repair: Read all about it (Review). International Journal of Oncology 2015;47(4):1189-202.
- Xicola RM, Llor X, Pons E, et al. Performance of different microsatellite marker panels for detection of mismatch repair-deficient colorectal tumors. Journal of the National Cancer Institute 2007;99(3):244-52.
- Nowak JA, Yurgelun MB, Bruce JL, et al. Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing. The Journal of Molecular Diagnostics 2017;19(1):84-91.
- Vanderwalde A, Spetzler D, Xiao N, et al. Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients. Cancer Medicine2018;7(3):746-56.
- Yamamoto H, Imai K. An updated review of microsatellite instability in the era of next-generation sequencing and precision medicine. Seminars in Oncology 2019;46(3):261-70.
- Hempelmann JA, Lockwood CM, Konnick EQ, et al. Microsatellite instability in prostate cancer by PCR or next-generation sequencing. Journal for Immunotherapy of Cancer 2018;6(1):29.
- Baretti M, Le DT. DNA mismatch repair in cancer. Pharmacology &Therapeutics 2018;189:45-62.
- Nakata B, Wang YQ, Yashiro M, et al. Prognostic Value of Microsatellite Instability in Resectable Pancreatic Cancer. Clinical Cancer Research 2002;8(8):2536-40.
- Yamamoto H, Itoh F, Nakamura H, et al. Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability. Cancer Research 2001;61(7):3139-44.
- Smyth EC, Wotherspoon A, Peckitt C, et al. Mismatch Repair Deficiency, Microsatellite Instability, and Survival: An Exploratory Analysis of the Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) Trial. JAMA Oncology 2017;3(9):1197-203.
- Groot VP, Rezaee N, Wu W, et al. Patterns, Timing, and Predictors of Recurrence Following Pancreatectomy for Pancreatic Ductal Adenocarcinoma. Annals of Surgery 2018;267(5):936-45.
- Allen PJ, Kuk D, Castillo CF, et al. Multi-institutional Validation Study of the American Joint Commission on Cancer (8th Edition) Changes for T and N Staging in Patients With Pancreatic Adenocarcinoma. Annals of Surgery 2017;265(1):185-91.
- Connor AA, Denroche RE, Jang GH, et al. Association of Distinct Mutational Signatures With Correlates of Increased Immune Activity in Pancreatic Ductal Adenocarcinoma. JAMA Oncology 2017;3(6):774-83.
- Bailey P, Chang DK, Nones K, et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 2016;531(7592):47-52.
- Witkiewicz AK, McMillan EA, Balaji U, et al. Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets. Nature Communications 2015;6(1):6744.
- Humphris JL, Patch AM, Nones K, et al. Hypermutation In Pancreatic Cancer. Gastroenterology 2017;152(1):68-74.e2.
- Marcus L, Lemery SJ, Keegan P, et al. FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors. Clin Cancer Res 2019;25(13):3753-3758.