2.3.4 Microsatellite Instability
Microsatellites (MS) are tandem repeats of short DNA sequences, abundant throughout the human genome.Owing to their high mutation rates, MS have been widely used as polymorphic markers in population genetics and forensics.As shown in Figure 2.4, microsatellite instability (MSI) is a hypermutator phenotype that occurs in tumor with impaired DNA mismatch repair (MMR) and is characterized by widespread length polymorphisms of MS repeats due to DNA polymerase slippage1 as well as by elevated frequency of single-nucleotide variants (SNVs).MSI in sporadic cases is caused by inactivation of MMR genes (for example, MLH1, MSH2, MSH3, MSH6 and PMS2) through somatic mutations, with increased risk of cancer for those with inherited germline mutations [71].
Figure 2.4 Di fferent immune microenvironment in microsatellite instability-high (MSI) hypermutated tumours a nd i n micr osatellite stable (MSS) tumo urs wit h lo w-mutational rat e.(A) In the presence of deficient mismatch repair (MMR), DNA replications errors go undetected and unrepaired,leading to a tumour with high mutational burden.Hyper-mutated cancer cells produce several neo-antigens, which stimulate T-cell activation and tumour infiltration by immune cells.To counteract this vigorous immune response, tumour cell exposes checkpoint molecules, e.g., PD-L1, to inhibit anti-tumour activity.(B) In the presence of functional MMR system, replication errors occur rarely with lower mutational rate and, as a consequence, limited production of neo-antigens.For this reason, in MSS tumour, the amount of T-cell infiltration and checkpoint molecules exhibition is low.The peculiar immune microenvironment of MSI tumours is thought to explain why they are ideal target for therapy with immune-checkpoint inhibitors.MHC major histocompatibility complex, TCR T-cell receptor.(Please scan the QR code on the Preface to get original color figures.)
Source: Ratti M, Lampis A, Hahne J C, Passalacqua R, Valeri N.“Microsatellite Instability in Gastric Cancer:Molecular Bases, Clinical Perspectives, and New Treatment Approaches”.CellandMolecularLifeScience,2018, 75(22):4151-4162.
MSI also occurs by hypermethylation of the MLH1 promoter (for example, associated with the somatic BRAF V600E mutation), epigenetic inactivation of MSH2, or downregulation of MMR genes by microRNAs5.MSI events within coding regions can alter the reading frame, leading to truncated, functionally-impaired proteins6.Interestingly, recent studies have hypothesized that alterations in the mismatch repair (MMR) system may predict clinical benefit for treatment with immune-checkpoint inhibitors, due to a positive correlation between MSI-H and PD-L1 expression.In this review, the current evidences about microsatellite instability-high (MSI-H) gastric cancer(GC) are summarized, with a special focus on pathological characteristics, predictive and prognostic values, and future perspectives for clinical approaches of MSI-H GC subgroup [71].
Microsatellites are DNA sequences with a length ranging from one to six repetitions of nucleotides(usually between 10 and 60 times).These DNA motifs are scattered throughout coding and non-coding regions of the genome, highly polymorphic among population but stable in each individual.The MMR system consists of several proteins, which include the products of hMLH1,hMSH2, hMSH6, and hPMS2 genes, which are responsible for surveillance of correct DNA replication.The MMR system targets and corrects replication errors when detected.The heterodimeric protein complexes hMSH2/hMSH6 and hMSH2/hMSH3 are responsible for the initial detection of replication errors.The subsequent recruitment of the complex formed by hMLH1 and hPMS2 removes the mismatched nucleotide or fragment and allows DNA re-synthesis.Inactivation of MMR proteins can be caused by mutations in the coding region, promoter methylation, or chromosomic rearrangements that lead to loss of heterozygosity.Microsatellite unstable GC can be observed in sporadic GC and in the setting of Lynch syndrome.Lynch syndrome is caused by autosomal dominant mutations in the MMR genes—mainly hMLH1 and hMSH2 and less frequently hPMS2 and hMSH6.Moreover, a constitutional 3′-end deletion of EPCAM, which is immediately upstream of the MSH2 gene, may cause Lynch syndrome through epigenetic silencing of MSH2.Patients affected by Lynch syndrome present an increased predisposition to develop colorectal cancer and endometrial cancer, but also to ovarian and gastric cancer occurring at a younger age (11.3-fold in the 30s and 5.5-fold in the 40s).Increased risk for developing pancreatic, bladder and breast cancer, and most possibly also prostate cancer has been related with Lynch syndrome carrier.Patients with MSH6 mutations appear to be particularly at risk of gastrointestinal and endometrial cancers, whereas carriers of an MSH2 gene mutation have the highest cancer risks across the spectrum, especially for the development of urinary tract cancer.In the sporadic setting, more than 50% of MSI GCs contain an epigenetic hypermethylation of hMLH1 promoter, whereas mutations in hMLH1 and hMSH2 have been reported in 12%-15% of this GC subgroup.Gene expression inactivation by alternative unknown genetic or epigenetic alterations have been hypothesized to be responsible for all of the remaining cases of microsatellite unstable GC.The functional loss of MMR proteins results in a highly mutated phenotype with a large number of frameshift and missense mutations in key oncogenes and tumor suppressor genes.Mutations in genes responsible for cell cycle regulation and apoptosis or for genomic integrity maintenance have been also associated with MSI-H GC.Moreover, increased expression of mitotic pathways components, such as AURKA A/B, E2F, FOXM1, PLK1, and targets of MYC activation, has been described and confirmed on a transcriptomic level in MSI-H tumors.Indeed, inactivation of MMR genes is not, by itself, a transforming event and additional genetic changes are needed to determine tumor progression.It is well established that MSI cancers are associated with 100- to 1000-fold increased mutation rates compared to microsatellite stable (MSS)tumors.The repetitive sequences of microsatellites are particularly prone to replication errors, and therefore, they can be used as a marker for an intact or defective MMR system [72, 73].
The increasing knowledge about the prognostic and predictive role of MSI-H vs MSS in several cancer subtypes has led to a larger number of patients routinely tested for this molecular feature.Gastrointestinal and non-gastrointestinal cancers with high prevalence of MSI-H (≥ 10%) are summarized.For an accurate determination of MSI status and the subsequent therapeutic decision,sensitive, fast, and precise techniques are necessary.Currently, several different methods are validated and in use to detect an MMR deficient tumor:
(1) PCR amplification of microsatellite sequences;
(2) Immunohistochemistry (IHC) staining for expression of MMR proteins;(https://www.daowen.com)
(3) Next-generation sequencing (NGS) for detection of MSI.
PCR amplification with specific primers for microsatellite repeats results in a distinctive amplification profile.By comparing the allelic position of the microsatellite locus in tumor and normal tissue, MSI can be assessed as “shift” in the pherogram of one or more microsatellites.To reach high specificity and sensitivity and also to ensure reproducibility and standardization between different laboratories, The National Cancer Institute (USA) recommends the so-called Bethesda Panel as reference for diagnostic testing.This panel is composed of five microsatellite markers specific for two mononucleotide loci (BAT-25 and BAT-26) and three dinucleotide loci (D2S123,D5S346, and D17S250).These regions are amplified in parallel using fluorescent PCR and their sizes are evaluated by subsequent capillary electrophoresis.Using this method, three different stati can be established based on different allelic size patterns in the cancer tissue compared to the normal one.The MSI-high (MSI-H) status is given by a shift in size in at least two of the five microsatellite loci; MSI-low (MSI-L) is given by a shift in size in one locus out of five and microsatellite stable (MSS) with any shift in cancer tissue compared to the normal one.The dinucleotide markers were demonstrated to be less sensitive and specific than mononucleotide for the detection of tumors with mismatch repair deficiencies (Figure 2.5).Furthermore, mononucleotide markers are more commonly quasi-monomorphic, potentially obviating the need to test the corresponding normal DNA.To overcome the limitations of Bethesda system due to the presence of dinucleotide markers, a commercial available panel has been developed by Promega Corporation.This is commonly employed in the diagnostic practice and replaced dinucleotides of the Bethesda Panel with mononucleotide markers helping to resolve cases of MSI-low into either MSI-H or MSS by comparison of tumor and the surrounding normal tissue [74, 75].
Figure 2.5 Repr esentative capil lary electr ophoresis (pherogram) o f the pr omega MSI analysis system.The upper part of the figure shows microsatellite stability (MSS) in normal tissue, without shifted alleles.The lower part is representative of tumor microsatellite instability-high (MSI) in all loci,with evident alleles shifting.Green: peaks of mononucleotides NR-21, BAT-25, and MONO-27.Blue:peak of BAT-26.Black: peak of NR-24.(Please scan the QR code on the Preface to get original color figures.)
Source: Ratti M, Lampis A, Hahne J C, Passalacqua R, Valeri N.“Microsatellite Instability in Gastric Cancer:Molecular Bases, Clinical Perspectives, and New Treatment Approaches”.CellularandMolecularLifeSciences, 2018, 11, 75(22):4151-4162.
Immunohistochemistry staining allows detection of expression or total absence of MMR proteins and relative scoring is possible.This method shows comparable performance characteristics and high concordance rate (＞ 90%) with MSI detection with PCR.The loss of expression of a single protein or of a heterodimeric couple of the MMR complex suggests the presence of MMRD; thus, it is an indirect evidence of MSI.On protein level, hMLH1 and hMSH2 are stable without their respective dimeric partners’ hPMS2 and hMSH6, whereas these latter components are rarely stable without their counterparts.Therefore, tumors with mutated hMLH1 or hMSH2 genes usually show loss of the respective functional dimer; conversely, mutations of hPMS2 or hMSH6 genes generate loss of only the affected protein.Hence, IHC allows the determination which of the MMR genes is defective and supports the decision about further genetic analysis.It must be taken into account that IHC provides misleading information for those rare cases of missense mutations in hMLH1 or hMSH6 genes, resulting in translated proteins with normally antibody affinity but missing enzymatic activity.In these cases, only PCR-based MSI testing can help to determine whether there are true functional MMR proteins through these mutations [76, 77].
Many studies have attempted to evaluate and compare the best and cost-effective method in defining the MMR status between IHC and PCR.Moreover, it has been evidenced in many reports the high correlation between IHC results and PCR-based tests in determining the phenotypic trait of the tumor.In a recent study, a discrepancy between MMRD and MSI assessment was found.Nevertheless, the overall concordance between immunohistochemical analysis of MMR protein expression and MSI was high.The authors explained the imperfect correlation with interobserver variability in immunohistochemical analysis assessment, heterogeneity of biomarker expression in gastric cancer, and the presence of normally translated but non-functional MMR proteins in the setting of a missense MLH1 mutation, or rare genomic defects that result in MSI-H status with intact MMRD function, e.g., polymerase DNA ε1 mutation [77, 78].
One of the advantages of IHC technique consists in its wide integration in routine testing in molecular and diagnostic pathology laboratories and in its ability in identifying which gene should be investigated for further molecular analyses in case of suspected hereditary cancer syndromes.Moreover, when IHC is used only the tumor tissue is required, whereas both normal and tumor samples are required for MSI testing with PCR.Molecular testing with PCR detects MSI directly as a result of MMRD.In these 5%-11% of MSI malignancies that do not exhibit MMR protein loss, usually due to retained antigenicity in an otherwise non-functional protein, IHC may underestimate MSI-H cases.In this situation, PCR-based test helps defining the correct diagnosis [77, 78].