Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.


Introduction
Microsatellites (MSs) are repeated sequences of 1-6 nucleotides in the human genome. MSs are highly polymorphic; in particular, their distribution and their dimensions are variable [1,2]. MSs are widely used both as markers in studies of genetic disease and as predictive biomarkers in various cancer types [3][4][5]. MSs are often subject to the incorporation or deletion of repeated units made by DNA polymerase during DNA replication; the mismatch repair system (MMR) is generally able to correct these errors [6]. The MMR is a pivotal system for maintaining genome integrity, consisting of several proteins, such as MLH1, MLH3, MSH2, MSH6, MSH3, PMS2, PMS1 and Exo1. The DNA errors are detected by MSH2/MSH6 and MSH2/MSH3 heterodimers; subsequently, the MLH1/PMS2 complex nicks DNA at sites of mismatch in order to begin strand repair [7]. MLH1 and MSH2 are the primary partners of their heterodimer, while PMS2 and MSH6 are the respective secondary partners. Consequently, the loss of the primary partners results in the loss of the entire

Methods of MSI Detection
The MSI implication in cancer leads researchers to develop several techniques for its  the heterogeneous staining of one or more MMRPs needs an additional molecular assay (original magnification 10×); and the loss of one or more MMRPs needs a molecular assay (original magnification 10×). The molecular analysis performed through PCR and/or NGS leads to a conclusive result in cases with IHC equivocal staining. MMRP: mismatch repair proteins; IHC: the heterogeneous staining of one or more MMRPs needs an additional molecular assay (original magnification 10×); and the loss of one or more MMRPs needs a molecular assay (original magnification 10×). The molecular analysis performed through PCR and/or NGS leads to a conclusive result in cases with IHC equivocal staining. MMRP: mismatch repair proteins; IHC: immunohistochemistry; PCR: polymerase chain reaction; NGS: new-generation sequencing; MSS: microsatellite stability; MSI-L: microsatellite instability-low; MSI-H: microsatellite instability-high. The stars indicate the PMS2 heterogeneous staining (center) and the loss of MLH1 and PMS2 (right).

MSI in Colorectal Cancer
The Cancer Genome Atlas Network (TCGA) has included the MSI status in the molecular classification of CRC. Particularly, the TCGA classified CRC in different molecular subsets, including the chromosome instability phenotypes (CIN), accounting for 70% of cases, the MSI phenotype, accounting for 20%, and the CpG island methylation phenotype (CIMP), accounting for 10% [61] (Figure 1). Although some studies showed that some MSI CRC cases harbouring also CIMP and CIN overlap with different molecular phenotypes, TCGA classified MSI CRC as a distinct molecular phenotype [62,63]. Approximately 3% to 5% of all CRCs have dMMR associated with LS. This syndrome, known as hereditary non-polyposis colorectal carcinoma (HNPCC), is an autosomal dominant syndrome with germline mutations of the MMR genes [64][65][66]. Beyond the CRCs associated with LS, approximately 70-95% of MSI CRCs are sporadic [67]. The relationship between CRC MSI and some clinical features, such as primary right-sided tumors and older age, has been demonstrated [68]. The MSI status is an early event in the development of CRC; it has never been identified in polyps [69]. On the contrary, MSI has also been observed in the normal colonic mucosa of patients with LS [70][71][72]. Several studies suggest that MSI CRCs at advanced stages show a poor prognosis compared to MSS CRC [73,74]. The clinical  studies KEYNOTE-016 and KEYNOTE-164 demonstrated the efficacy of pembrolizumab,  with durable responses in patients with advanced MSI-H/dMMR or mCRC treated after chemotherapy [75,76]. In KEYNOTE-177, a phase 3 study, patients with unresectable or metastatic MSI/dMMR CRC were treated with pembrolizumab as the first-line treatment without receiving prior therapy; this showed clinically significant improvements in survival compared to chemotherapy. In 2020, the FDA approved pembrolizumab as the first-line treatment for advanced or metastatic CRC with MSI-H/dMMR [77]. Furthermore, CheckMate 142 demonstrated the efficacy of nivolumab (an anti-PD1 antibody) used alone or in combination with ipilimumab (an anti-CTLA-4 antibody) in mCRC MSI-H/dMMR patients previously treated with chemotherapy. In 2018, the FDA approved nivolumab either alone or in combination with ipilimumab [78,79] (Table 1). The predictive value of MSI in CRC requires the optimization of testing in clinical practice. As already mentioned, IHC is the most used method; however, it showed several discrepancies with PCR and NGS results. Several studies have compared the different assays for MSI detection in CRC series [41,50,[80][81][82] ( Table 2). The comparative analysis between different methods showed several discrepancies; in particular, some of the dMMR-IHC cases were MSS-PCR (ranging from 1% to 33% of cases) [82][83][84]. In contrast, only 5% of pMMR-IHC cases analyzed in the literature showed MSI by PCR [41,50,80,81,85]. Generally, the PCR results are in agreement with the NGS results, showing a discrepancy of up to 3% [58,83,86,87]. To date, no recommendations have been proposed regarding the gold standard molecular test for MSI analysis. The NGS is the most sensitive method for MSI detection. Moreover, the NGS represents the best molecular approach since it allows simultaneously the detection of both MSI and a wide panel of other mutations. However, PCR can represent a valid molecular test for MSI detection in daily clinical practice since it is economical and widespread in the laboratories compared to NGS. Finally, MSI analysis by NGS could be useful in selected cases showing discordant results between IHC and PCR.

MSI in Gastric Cancer
The TCGA has included MSI status in the molecular classification of GC. Particularly, the TCGA classified GC in different molecular subsets, including the chromosome instability phenotypes (CIN), accounting for 50% of cases, the genomically stable (GS) tumors, accounting for 19%, the MSI phenotype, accounting for 22%, and the Epstein-Barr virus (EBV)-positive tumors, accounting for 9% ( Figure 1) [88]. Although rare cases simultaneously carrying MSI and EBV have been identified, the TCGA considers MSI as a molecular profile distinct from the others [89]. Similarly, the Asian Cancer Research Group (ACRG) classified GC in MSS and MSI [90]. Many studies have found MSI in precancerous lesions and an increase in MSI frequency in GC cases is observed [91,92]. MSI GCs usually occur in older female patients, with an intestinal subtype at an early stage (I/II) and with better differentiation and distal position, and no lymph node involvement [44,91]. Ottini et al. observed that the MLH1 promoter methylation is frequently in sporadic GC, while MLH1 and MSH2 mutations are rare in MSI GCs [93]. MSI occurs both in sporadic GCs and in GCs associated with LS [94,95]. A positive correlation was observed between the presence of Helicobacter pylori (H. pylori) and MSI. The active bacterial infection is present more frequently in GC MSI patients, suggesting that H. pylori may affect the MMR system during the gradual progression of gastric carcinogenesis [52]. Approximately 85% of the MSI GC cases concomitantly showed a KRAS mutation. On the contrary, no association between MSI GC and a mutated BRAF was reported [96]. MSI and human epidermal growth factor receptor 2 (HER2) amplification are reported to be mutually exclusive, suggesting that HER2 amplification could be a negative predictive marker for immunotherapy in GC patients [97]. The MSI status is not used as an exclusive prognostic biomarker, since the prognosis of GC patients is affected by other clinical and pathological features, including the age, stage, grade and chemotherapy treatment [98]. To date, controversial data about the prognostic role of MSI in GC have been reported. Several studies have shown that GCs carrying MSI have a good prognosis [91,99,100]. However, other studies showed MSI not having a prognostic role; in particular, MSI GCs do not have a better survival time than MSI-L/MSS patients. [101,102]. The metastatic MSI GCs have shown long-lasting positive results after receiving treatment with anti-PD-1 antibodies (durvalumab, pembrolizumab and nivolumab), as a single therapy or in combination with anti-CTLA4 antibodies (ipilimumab and tremelimumab) [103]. Pembrolizumab was investigated among patients with advanced MSI gastric or gastroesophageal junction cancer who were enrolled in different studies, such as KEYNOTE-059, KEYNOTE-061 and KEYNOTE-062. The results indicate that pembrolizumab or pembrolizumab plus chemotherapy provided durable antitumor activity compared to chemotherapy alone in patients with an MSI gastric junction or gastroesophageal cancer. Other clinical trials have suggested drug combinations, such as durvalumab and tremelimumab or nivolumab and ipilimumab in phase 2, for the treatment of advanced MSI GC [104][105][106] (Table 3). The comparative analysis between these methods showed some discrepancies, particularly as some dMMR-IHC cases were MSS by PCR (ranging from 1% to 38%) [51,107], while pMMR-IHC cases were MSI by molecular analysis (ranging from 1% to 7,4%) [52,108,109]. The FDA accelerated approval of pembrolizumab in 2017 to treat MSI patients in solid tumors, including GC, who have progressed following prior treatment [27]. Few data have been reported regarding the MSI testing in GC; all the comparison data between IHC and PCR are summarized in Table 4.

MSI in Gynecologic Cancers
Several studies have shown the presence of MSI from 2 to 20% in ovarian cancer (OC) (Figure 1), particularly in clear cell carcinomas and endometrioid cancers [110][111][112]. In clear cell carcinoma, the expression of CD8, PD-1 and tumor-infiltrating lymphocytes (TIL) was higher in MSI tumors than in those of MSS. In contrast, in endometrioid carcinomas, no correlation between the MSI status and TIL was reported [111,113]. MSI OCs are characterized by advanced stage, poor differentiation, grade and poor prognosis [114]. MSI OCs are frequently associated with LS [115]. However, the correlation between MSI and LS is not always verified since the LS was observed in both MSI OC and MSS OC [116]. MSI detection in OC has not yet been extensively studied and no adequate method is currently defined. The suitability and specificity of the Bethesda panel are still today an open issue in this cancer type [117]. There are few studies that have compared IHC and PCR for detecting MSI in OC (Table 5).
A concordance was shown between the IHC analysis and PCR [113]. Some discordant results have been observed; in particular, dMMR-IHC cases were MSS by PCR in a wide subset accounting from 1% to 80% [118][119][120]. Although MSI is still a poorly understood marker in OC patients, several studies showed its potential predictive role for ICI in this cancer ( Table 6). The TCGA has included the MSI status in the molecular classification of endometrial cancer (EC). Particularly, the TCGA classified EC in different molecular subsets, including the no specific molecular profile (NSMP), accounting for 38% of cases, the MSI phenotype, accounting for 30%, the copy number high/mutant TP53 (CNH), accounting for 25%, and the POLE/ultramutated (POLE), accounting for 7% (Figure 1) [121][122][123].
Although some studies showed that some MSI EC cases also harbouring other mutations overlapped with different molecular phenotypes, the TCGA classified MSI EC as a distinct molecular phenotype [124]. LS in the EC was observed with a frequency of 3% [125].  Regarding morphological features, MSI ECs are frequently characterized by the presence of peritumoral lymphocytes and TILs [126]. In approximately 95% of ECs, the MSI is due to the hypermethylation of the MLH1 promoter [127]. MSI ECs showed in about 10-53% of cases the simultaneous presence of other gene alterations, particularly the mutations of the following genes: RPL22 (46%) [128][129][130], PTEN (34%) [128,131], KRAS (35%) [128], ATR (15%) [132], CHK1 (29%) [133], Caspase5 (10%) [131,134] and BAX (27%) [129,134,135]. Furthermore, the PTEN gene mutations, frequently observed in sporadic EC (25-83%), showed a correlation with MSI with a frequency of 60% [136]. Moreover, MSI ECs are associated with a higher neoantigen load and increased TIL, CD3-positive, CD8-positive and PD-L-1 positive compared to MSS/pMMR EC [137]. The role of MSI in ECs is until now unclear; most of the studies have concluded that the MSI status has no prognostic role [138][139][140]. Previous studies have shown that the combination of pembrolizumab and lenvatinib can be used for second-line treatment in patients with EC who are not dMMR/MSI-H, and studies are ongoing for the treatment of patients with advanced EC MSI [141,142]. In the USA, both pembrolizumab and dostarlimab are approved as monotherapy for the treatment of MSI EC patients. In the EU, dostarlimab is the only therapy approved for patients with EC dMMR/MSI who have failed platinum therapy [143] (Table 7). Several studies have compared the different assays for MSI detection in EC series (Table 8).   The comparative analysis between these methods showed some discrepancies; in particular, approximately 1-68% of dMMR-IHC cases were MSS by PCR/NGS [83,86,[144][145][146], and about 5% of pMMR-IHC cases were MSI-H by molecular analysis [147,148].

MSI in Other Malignancies
MSI has been found in several cancer types, including non-small cell lung cancer, melanoma, breast cancer, urothelial cancer, pancreatic ductal adenocarcinoma and brain cancer. The expansion of MSI clinical trials into other cancers may elucidate the prognostic and predictive value of MSI for non-colorectal. The frequency of MSI reported in non-small cell lung cancer (NSCLC) is very heterogeneous, ranging from 0.8% to 40% [149][150][151][152]. The role and clinical implications of MSI are still unclear in NSCLC. Carpagnano et al. suggested that MSI is associated with a poor prognosis in NSCLC [153].
Moreover, recent findings suggested that MSI in NSCLC is associated with an increased response to immunotherapy [154]. Several clinical trials showed the efficacy of pembrolizumab for the treatment of previously treated NSCLC patients carrying MSI [155][156][157] (Table 9). MSI is extremely rare in breast cancer, showing a frequency of 0.04-3% [158,159]. However, MSI was observed in triple negative breast cancer (TNBC), with a high frequency accounting for between approximately 0.2% and 18,6% [160][161][162]. The role of MSI in BC is still uncertain. Some studies have shown that MSI does not have a significant impact on patient survival [149]. Conversely, other studies have reported a positive prognostic value of MSI in TNBC [160]. A significant correlation was observed between MSI and the negative expression of estrogen and progesterone receptors, indicating a possible relationship between genetic changes in microsatellite regions and hormonal deregulation in the progression of BC [163]. In BC, MSI showed a correlation with the advanced clinical stage, the tumor size and the high grade [164]. The KEYNOTE-028 and KEYNOTE-012 studies demonstrated the efficacy of pembrolizumab in BC previously treated with CDK4/6 inhibitors. [158,165]. The pancreatic ductal adenocarcinoma (PDAC) showed MSI in about 1-2% [166,167]. PDACs carrying MSI are generally associated with LS [168]. LS-associated pancreatic tumors, ranging from 0.5% to 3.9%, are generally associated with medullary histology showing prominent lymphocyte infiltration [167,169]. PDAC is an aggressive type of human cancer; moreover, most patients are resistant to chemotherapy. Thus, immunotherapy could be a possible therapeutic option [170]. Previous studies showed that pMMR PDCA patients treated with conventional chemotherapy have a survival advantage compared to dMMR patients, suggesting that MSI can be considered a potential predictor of treatment sensitivity [171][172][173]. KEYNOTE-016 evaluated the efficacy of pembrolizumab in eight cases of MSI PDAC, showing a complete response in two patients and a partial response in three patients [27,174]. These preliminary results suggested that anti-PD-L1 antibody treatment could be effective in patients with MSI PDAC. MSI was reported in approximately 10-15% of thyroid carcinomas (TC) [175][176][177]; moreover, a correlation between MSI and clinical-pathological features was observed. Particularly, MSI TCs were generally papillary or anaplastic and poorly differentiated histotypes. A recent study showed that MSI TC patients have a good prognosis compared to MSS TC [177]. Although several clinical trials are being evaluated for the efficacy of pembrolizumab, nivolumab and atezolizumab in MSI TC, until now no immunological therapy has been approved for MSI TC [178,179]. The ICI targeting the PD-1/PD-L1 pathway may be a treatment option for MSI TC; however, further studies are needed [177]. The frequency of MSI brain tumors is very low, ranging from 2% to 3% of cases [27,116]. Brain tumors are rarely associated with LS (0.5-3.7% of all cases). No information is available regarding the specific molecular or phenotypic characteristics of LS-related brain tumors [167]. The MSI phenotype was observed in about 25% of glioblastomas (GBM) [180]. MSI GBM showed a low level of PD-L1 expression, suggesting it is not a good predictive marker for ICI targeting the PD1/PD-L1 pathway [181]. MSI has been shown to occur in malignant melanomas, with a frequency of between 2 and 30% [182][183][184]. Kubecek et al. suggested that MSI might be a predictive marker in malignant melanoma [185]. Although the KEYNOTE-016 study showed the efficacy of pembrolizumab in MSI melanomas that have progressed after previous treatment, the treatment for MSI melanoma is not yet used in clinical practice [27].
MSI has been identified in approximately 1-28% of urothelial carcinomas (UC) [186][187][188]. The upper tract urothelial carcinomas (UTUCs) also occur in about 5% of patients with LS [189]. The heterodimer MSH2/MSH6 is frequently lost in the UTUC in about 50-86% of cases [186,190,191]. Several studies reported that UTUCs MSI showed unique morphological features compared to MSS UTUC, including increased intratumoral lymphocytes, the lack of nuclear pleomorphism and the presence of pushing edges [167,190,192]. The KEYNOTE-158 study demonstrated the benefit of pembrolizumab treatment in patients carrying MSI-H/dMMR including UCs [174]. The prostate cancers (PCs) showed MSI status in 1,2-12% of cases. MSI PCs represent a clinically aggressive phenotype [193]. The germline alterations in the MMR genes are less common in localized PC, suggesting an increase in mutations in patients with metastatic spread [194]. In the KEYNOTE-016 study, MSI PCs responded better to ICI [27]. Although MSI can be considered a biomarker capable of responding to immunotherapy in prostate cancer, ICIs are not yet used in clinical practice for the treatment of metastatic PCs [195].

Conclusions
The MMR system is a key repair mechanism for maintaining sequence fidelity and stability. The inactivation of the MMR system due to the germinal, somatic or epigenetic prevents error correction and, thus, promotes microsatellite instability. MSI has been detected in various cancers with a prognostic and/or predictive role; in particular, it is clinically important for predicting a response to immunotherapy. MSI represents the FDA's first cancer type-agnostic biomarker approved for selection to the treatment with pembrolizumab of patients with any advanced solid cancer, regardless of the histology. The results reported underline the pivotal role of MSI in the choice of drug treatment in patients, especially in the cases of those who no longer respond to chemotherapy. To date, MSI detection aimed at selecting patients for ICI treatment could be performed through both IHC and PCR/NGS. Although the two tests are equated in clinical practice for the therapeutic choice, several controversial results have been reported. In this view, the optimization of MSI detection in clinical practice is currently needed in order to implement an adequate selection of patients eligible for ICI treatment of dMMR tumors. A feasible diagnostic algorithm for MSI testing could include IHC as prescreening requiring an additional molecular test for cases with an equivocal IHC of one or more MMR proteins, and also to confirm dMMR cases in order to exclude false IHC results.

Conflicts of Interest:
The authors declare no conflict of interest.