Elevated Microsatellite Alterations At Selected Tetranucleotide Repeats Research Articles

Compound heterozygous MSH3 germline variants and associated tumor somatic DNA mismatch repair dysfunction

We describe here an individual from a fourth family with germline compound heterozygous MSH3 germline variants and its observed biological consequences. The patient was initially diagnosed with invasive moderately-differentiated adenocarcinoma of the colon at the age of 43. Germline multigene panel testing revealed a pathogenic variant MSH3 c.2436-1 G > A and a variant of (initial) uncertain significance MSH3 c.3265 A > T (p.Lys1089*). Germline genetic testing of family members confirm the variants are in trans with the c.2436-1 G > A variant of paternal and the c.3265 A > T variant of maternal origin. Tumor DNA exhibits low levels of microsatellite instability and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). Tissue immunohistochemical staining for MSH3 demonstrated variant MSH3 protein is present in the cytoplasm and cell membrane but not in the nucleus of normal and tumor epithelial cells. Furthermore, variant MSH3 is accompanied by loss of nuclear MSH6 and a reduced level of nuclear MSH2 in some tumor cells, suggesting that the variant MSH3 protein may inhibit binding of MSH6 to MSH2.

Analysis of microsatellite aberrations and loss of heterozygosity in follicular lymphoma, diffuse large B-cell lymphoma, and high-grade B-cell lymphoma patients

Background . The study of genetic predictors of non-Hodgkin’s lymphomas prognosis is one of the most relevant areas of oncohematology. It is extremely interesting to search for integral markers that reflect the most important stages of tumor pathogenesis. DNA repair system plays one of the key roles in genomic instability. Aberrations of microsatellite repeats such as microsatellite instability (MSI), in particular elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is characteristic for mismatch repair system and loss of heterozygosity (LOH) is an integral feature of genomic instability. Objective . Analysis of MSI, EMAST, LOH significance in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and high-grade B-cell lymphoma (HGBL) patients. Materials and methods . The study was performed by multiplex PCR and fragment analysis with diagnostic panels COrDIS Plus and COrDIS MSI in 85 FL patients, 32 DLBCL patients, and 37 HGBL patients. Results . The frequency of LOH in the general FL group was 40/81 (49.4 %), MSI – 10/82 (12.2 %), EMAST – 15/81 (18.5 %). The frequency of LOH in the HGBL group was 21/31 (67.8 %), MSI – 11/37 (29.7 %), EMAST – 13/31 (41.9 %). The frequency of LOH in the DLBCL group was 18/29 (62.0 %), MSI – 5/32 (15.6 %), EMAST – 14/32 (43.8 %). When considering the morphological types of FL, it was noted that a higher frequency of genetic aberrations was characteristic of lymphomas with a more aggressive morphology (p <0.05). LOH identifies FL and HGBL patients with an unfavorable prognosis. The EMAST analysis allows identifying additional patients in the LOH+ cohort with early events and low EFS. Conclusion . LOH and EMAST have a prognostic value for FL and HGBL. No associations of LOH and EMAST with the survival were observed in DLBCL. Changes in mononucleotide repeats in FL, DLBCL and HGBL did not correspond to the MSI-H characteristic of solid tumors. For this reason, the clinical consequences of MSI-H in solid neoplasms, in particular the efficacy of immune checkpoint inhibitors, in lymphomas cannot be expected to be replicated solely on the basis of microsatellite aberrations detection.

High expression of ZEB1 is associated with EMAST & metastasis in colorectal cancer patients.

Transforming growth factor-beta (TGF-β) signalling pathway has been reported to be involved in metastasis and at the same time has been considered compellingly an important mediator of epithelial-to-mesenchymal transition (EMT). Besides, EMT process is maintained by zinc-finger E-box-binding homeobox 1 (ZEB1) gene which is induced by TGF-β pathway. TGF-β has been shown to be associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) phenomenon, which is one of the prognostic biomarkers of colorectal cancer (CRC). This study was conducted to determine the link among ZEB1-induced TGF-β, EMAST status and metastasis. The expression level of ZEB1 was evaluated using quantitative reverse transcription (qRT) real-time PCR in 122 formalin fixed paraffin-embedded tissues of CRC sample with known EMAST status and TGF-β/Smad-dependent pathways. The association among ZEB1 expression, TGF-β signalling pathway, EMAST status and metastatic behaviour was examined. ZEB1 gene expression level was higher in tumour tissues as compared to normal samples (P<0.045). In addition, ZEB1 positive expression level was associated significantly with metastasis (P=0.05), EMAST+ status (P=0.052) and activated TGF-β signalling pathway (P=0.002). Our results validated significant association between activated TGF-β signalling pathway and EMAST+ phenotype with higher expression of ZEB1 and higher level of metastasis.

Lack of correlation between MSH3 immunohistochemistry and microsatellite analysis for the detection of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in colorectal cancers

Immunohistochemical evaluation of mismatch repair protein (MMR) expression is an important screening tool in diagnostic pathology, where it is routinely used to identify subsets of colorectal cancers (CRCs) with either inherited or sporadic forms of microsatellite instability (MSI). MSH3 is not included in current MMR panels, although aberrant MSH3 expression is reported to occur in 40-60% of CRCs and is associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and a worse prognosis. In this study, we applied MSH3 immunohistochemistry and tetranucleotide MSI analysis to a cohort of 250 unselected CRCs to evaluate the potential use of the methods in routine practice. Partial, complete, and focal loss of nuclear MSH3 and its cytoplasmic mislocalization were evident in 67% of tumors, whereas MSI was evident in two to six of a panel of six tetranucleotide repeats in 46% of cases. However, concordance between MSH3 immunohistochemistry and tetranucleotide MSI results was only 61%, indicating the unsuitability of this combination of tests in routine pathology practice. MSH3 immunostaining was compromised in areas of tissue crush and autolysis, which are common in biopsy and surgical samples, potentially mitigating against its routine use. Although tetranucleotide MSI is clearly evident in a subset of CRCs, further development of validated sets of tetranucleotide repeats and either MSH3 or other immunohistochemical markers will be required to include EMAST testing in the routine evaluation of CRCs in clinical practice.

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers.

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.

Is elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)-negative/MSI-high colorectal cancer a distinct subtype of the disease?

Microsatellite instability (MSI) plays a prognostic and predictive role in colorectal cancer (CRC). Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a novel type of MSI, was recently identified. A retrospective analysis of a prospective cohort database was performed. Patients who attempted curative surgery for MSI-high (MSI-H) CRC and had available testing results of EMAST were included for analysis. The difference in clinical characteristics, immunohistochemistry profile, and 3-year recurrence-free and overall survival between EMAST-negative and EMAST-positive tumors was measured. EMAST status was successfully evaluated in 86 cases among patients who received EMAST testing, and only 16.3% (14/86) of these patients were EMAST-negative/MSI-H. Patients with EMAST-negative tumors were younger; their tumors exhibited well differentiation, less venous invasion, and greater mutS homolog 3 expression. There was no distant metastasis or cancer-specific death among EMAST-negative patients. Yet no statistically significant difference was found between the two groups in 3-year overall or recurrence-free survival. Patients with EMAST-negative/MSI-H CRC seem to have different clinicopathological characteristics. Future large-scale studies could clarify the role of EMAST genotype as a sub-classifier of MSI-H CRC.

The Clinicopathological Features and Genetic Mutations in Gastric Cancer Patients According to EMAST and MSI Status.

Background: There has been no report regarding the clinicopathological features and genetic mutations regarding elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in gastric cancer (GC). Methods: The correlation among EMAST status, microsatellite instability (MSI) status, mutations of common GC-related genes and 16 DNA repair-associated genes, and the clinicopathological features were analyzed. Results: Among the 360 GC patients enrolled, there were 76 (21.1%) with EMAST+ tumors and 284 with EMAST− tumors, and 59 (16.4%) were MSI-high (MSI-H) tumors, and 301 were microsatellite stable (MSS) tumors. Patients with EMAST+ tumors exhibited an earlier pathological T category and had more genetic mutations in the PI3K/AKT pathway, ARID1A and DNA repair-associated genes than those with EMAST− tumors. Patients with MSI-H tumors have more genetic mutations in the PI3K/AKT pathway and DNA repair-associated genes than those with MSS tumors. In the subgroup analysis for MSI-H GC, EMAST+ tumors were associated with earlier pathological T and N categories, earlier TNM stages, higher frequency of DNA-repair-associated genetic mutations, and a better survival rate than EMAST− tumors. Conclusions: PI3K/AKT pathway mutations may play an important role in EMAST+ and/or MSI-H GC. EMAST+/MSI-H tumors seem to represent a different subtype of gastric cancer from EMAST−/MSI-H tumors.

Application of msh3 immunohistochemistry and tetranucleotide microsatellite analysis to identify Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) in colorectal tumours

Background and aims: Reduced function of the DNA mismatch repair enzyme MSH3 in tumour cells results in elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). In contrast to microsatellite instability (MSI) caused by MLH1/PMS2/MSH2/MSH6 defects, EMAST in CRCs is a marker of poor prognosis. This study examined the prevalence of EMAST-positive CRCs in a Western Australian population and critically evaluated immunohistochemical and molecular detection methods. Methods and results: MSH3 immunostaining of 250 archival CRCs identified aberrant MSH3 expression (focal or widespread reduction of nuclear MSH3, cytoplasmic MSH3 mis-localisation) in 66% cases. Tetranucleotide MSI (EMAST) was detected in 46% cases, a proportion consistent with limited previous reports. Discordance between MSH3 immunostaining and molecular detection of tetranucleotide MSI resulted from over-detection of abnormal MSH3 immunostaining, which is focal and confounded by common tissue damage (crush, autolysis). Summary and conclusions: Aberrant MSH3 function in CRCs results from elevated inflammatory signalling, a potentially modifiable risk factor that may be targeted to reduce or prevent disease relapse. A combination of MSH3 immunostaining and molecular methods can be used to identify EMAST-positive CRCs, however further antibody development and establishment of consistent tetranucleotide panels will improve the reliability and reproducibility of its detection in routine practice.

Tetranucleotide Microsatellite Mutational Behavior Assessed in Real Time: Implications for Future Microsatellite Panels.

Background & AimsFifty percent of colorectal cancers show elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and are associated with inflammation, metastasis, and poor patient outcome. EMAST results from interleukin 6–induced nuclear-to-cytosolic displacement of the DNA mismatch repair protein Mutated S Homolog 3, allowing frameshifts of dinucleotide and tetranucleotide but not mononucleotide microsatellites. Unlike mononucleotide frameshifts that universally shorten in length, we previously observed expansion and contraction frameshifts at tetranucleotide sequences. Here, we developed cell models to assess tetranucleotide frameshifts in real time.MethodsWe constructed plasmids containing native (AAAG)18 and altered-length ([AAAG]15 and [AAAG]12) human D9S242 locus that placed enhanced green fluorescent protein +1 bp/-1 bp out-of-frame for protein translation and stably transfected into DNA mismatch repair–deficient cells for clonal selection. We used flow cytometry to detect enhanced green fluorescent protein–positive cells to measure mutational behavior.ResultsFrameshift mutation rates were 31.6 to 71.1 × 10-4 mutations/cell/generation and correlated with microsatellite length (r2 = 0.986, P = .0375). Longer repeats showed modestly higher deletion over insertion rates, with both equivalent for shorter repeats. Accumulation of more deletion frameshifts contributed to a distinct mutational bias for each length (overall: 77.8% deletions vs 22.2% insertions), likely owing to continual deletional mutation of insertions. Approximately 78.9% of observed frameshifts were 1 AAAG repeat, 16.1% were 2 repeats, and 5.1% were 3 or more repeats, consistent with a slipped strand mispairing mutation model.ConclusionsTetranucleotide frameshifts show a deletion bias and undergo more than 1 deletion event via intermediates, with insertions converted into deletions. Tetranucleotide markers added to traditional microsatellite instability panels will be able to determine both EMAST and classic microsatellite instability, but needs to be assessed by multiple markers to account for mutational behavior and intermediates.

Combined Microsatellite Instability and Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) Might Be a More Promising Immune Biomarker in Colorectal Cancer.

The form of microsatellite instability (MSI) affecting tetranucleotide repeats known as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has emerged as a new potential biomarker in multiple cancers. In colorectal cancer (CRC), the correlation between EMAST and MSI mutations remain inconclusive. We evaluated 1,505 patients with CRC using five EMAST markers (D20S82, D20S85, D8S321, D9S242, and MYCL1) and the Bethesda panel of MSI markers. Most commonly, mutations involved in CRCs were identified by MassArray Assay, and DNA repair genes were analyzed by next-generation sequencing. Clinical characteristics and prognostic relevance were correlated with EMAST and MSI. Tumors that were EMAST positive and MSI high (MSI-H) were detected in 159 (10.6%) and 154 (10.2%) of 1,505 patients with CRC. Patients were divided into four groups according to EMAST and MSI status (EMAST-positive and MSI-H, EMAST-positive and microsatellite-stable [MSS], EMAST-negative and MSI-H, and EMAST-negative and MSS). The EMAST-positive and MSI-H group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Furthermore, compared with only EMAST-positive tumors or only MSI-H tumors, tumors that were both EMAST-positive and MSI-H had a higher frequency of MLH1, MSH3, MSH6, PMS2, and EXO1 gene mutations. Finally, the presence of EMAST-positive and MSI-H tumors was a good prognostic indicator in CRC. High mutations in several DNA repair genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a unique molecular subtype of colorectal cancer (CRC). The current study demonstrated that the EMAST-positive and MSI-high (MSI-H) group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Most importantly, high mutations in DNA repair genes and MSI-related genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy compared with MSI-H tumors alone.

MSI-L/EMAST is a predictive biomarker for metastasis in colorectal cancer patients.

Microsatellite instability (MSI) is a prognostic marker in colorectal cancer (CRC). The biological significance of MSI-low (MSI-L) phenotype and its differences with microsatellite stable (MSS) phenotype remains unclear. The aim of this study is indicating the role of mononucleotide repeat in identifying MSI-L and revealing the association of MSI-L with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and oncologic outcome in CRC patients. MSI and EMAST status were analyzed using three quasimonomorphic panel (BAT-25, BAT-26, and NR-27) and five tetranucleotide repeats (D20S82, D20S85, D9S242, D8S321, and MYCL1), respectively, by capillary electrophoresis method without the need to fluorescent primers. The associations of MSI status with clinicopathological features, EMAST status, metastasis, and overall survival (OS) were investigated. Among 159 CRC patient 22.0% were MSI-H, 40.3% were MSS, 37.7% were MSI-L, and 41.5% showed EMAST + phenotype. MSI-L were associated with advanced stages, EMAST+ tumors and worse OS ( p ≤ 0.001). Metastasis was relatively common in MSI-L/EMAST + CRCs and BAT-25 were the most unstable marker in these tumors. MSI-L tumors have different clinicopathological features from MSS and MSI-H tumors. The MSI-L phenotype is a worse prognostic biomarker in CRC and when accompanied by EMAST could be a predictor for metastasis.

Prevalence of elevated microsatellite alterations at selected tetranucleotide repeats in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) prognosis remains poor even after complete resection owing to no valuable biomarkers for recurrence and chemosensitivity. Tumors not expressing MSH3 show elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). EMAST reportedly occurs in several tumors. In colorectal cancer (CRC), EMAST was reportedly correlated with 5-fluorouracil (5-FU) sensitivity. However, EMAST prevalence in PDAC and its significance as a prognostic biomarker are unknown. This study aimed to investigate EMAST prevalence in PDAC and the associations between EMAST and pathological factors, EMAST and prognosis, and EMAST and MSH3 expression via immunohistochemistry (IHC). We assessed 40 PDAC patients undergoing surgery. Genomic DNA was extracted from tumors and normal tissues. EMAST and microsatellite instability-high (MSI-H) were analyzed using five polymorphic tetranucleotide markers and five mononucleotide markers, respectively. Tumor sections were stained for MSH3, and staining intensity was evaluated via the Histoscore (H-score). Eighteen of 40 (45%) PDAC patients were EMAST-positive; however, none were MSI-H-positive. Clinicopathological characteristics including overall survival (OS) and recurrence-free survival (RFS) were not significantly different between EMAST-positive and EMAST-negative patients (P = 0.45, 0.98 respectively). IHC was performed to evaluate MSH3 protein expression levels for the PDAC tissue specimens. H-scores of EMAST-positive patients ranged from 0 to 300 (median, 40) and those of EMAST-negative patients ranged from 0 to 300 (median, 170). MSH3 protein was not significantly downregulated in EMAST-positive patients (P = 0.07). This study is a preliminary study and the number of cases investigated was small, and thus, study of a larger cohort will reveal the clinical implication of EMAST.

174P - Microsatellite instability-high (MSI-H) colorectal cancers with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) signature represent a target population for immune checkpoint and DNA damaging therapies

174P - Microsatellite instability-high (MSI-H) colorectal cancers with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) signature represent a target population for immune checkpoint and DNA damaging therapies

531P - The clinical features and genomic landscape of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in patients with colorectal cancer

531P - The clinical features and genomic landscape of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in patients with colorectal cancer

A new method for discovering EMAST sequences in animal models of cancer

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) occur in up to 60% of colorectal cancers and may associate with aggressive and advanced disease in patients. Although EMAST occurs in many cancer types, current understanding is limited due to the lack of an animal model. Reported here is the design and implementation of an algorithm for detecting EMAST repeats in mice. This algorithm incorporates properties of known human EMAST sequences to identify repeat sequences in animal genomes and was able to identify EMAST-like sequences in the mouse. Seven of the identified repeats were analyzed further in a colon cancer mouse model and six of the seven displayed EMAST instability characteristic of that seen in human colorectal cancers. In conclusion, the algorithm developed successfully identified EMAST repeats in an animal genome and, for the first time, EMAST has been shown to occur in a mouse model of colon cancer.

Abstract 421: Improved detection and identification of microsatellite instability features in colorectal cancer: Implications for immunotherapy

Abstract Colorectal cancers with loss of DNA mismatch repair display microsatellite instability (MSI). These tumors have a better prognosis and an improved response rate to immune check point therapies compared to non-MSI tumors. MSI-positive tumors demonstrate a surprising range of somatic alterations in repetitive sequence tracts. For example, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that may be related to functional loss of MSH3. Current MSI tests are based on PCR and capillary electrophoresis. This conventional approach is suboptimal because i) only a small number of microsatellite markers are interrogated per assay, and ii) sensitivity is insufficient for low allelic fraction alleles in heterogeneous tumors. Next generation sequencing permits the inclusion of more microsatellite markers, but these NGS method still rely on PCR methods, which is the major source of artifacts that obscure the true somatic alterations. In this study, we analyzed a series of colorectal cancers using a new sequencing technology that sequences in parallel more than 200 microsatellite regions - we evaluate mono-, di- , tri- and tetranucleotide repeats to provide a broader spectrum of MSI events. This deep sequencing technology provides greater than 2,000X coverage, uses in vitro CRISPR-Cas9 segmentation, and eliminates the need for PCR amplification. Because every read corresponded to a single DNA molecule, we eliminated the artifacts introduced by PCR that obscured true MSI events. Moreover, we improved the sensitivity of detecting minor allele fractions to as low as 0.1% by measuring microsatellite haplotypes. Included in our analysis was new method for somatic copy number changes in 82 cancer genes, by which we can infer chromosomal instability (CIN) status. Among our cases, 10 cancers had MSI when considering both mononucleotide and tetranucleotide repeats. Interestingly, diagnostic testing of two of these tumors were false negatives and were demonstrated to be MSI-high. There were three major types among the 10 MSI cancers: MSI-high, EMAST, and the cancers having both. However, such types were distinguishable only by microsatellites with intermediate length (10 - 20 bp). MSI-high tumors (n = 3) showed no sign of CIN while all the EMAST tumors (n = 4) also showed CIN phenotype. Our results suggest that the current gold standard MSI diagnostic test may have issues with both false negatives and false positives, indicating that NGS approaches are better suited for evaluating MSI status of tumors and consideration of immunotherapy. Citation Format: Giwon Shin, HoJoon Lee, Susan M. Grimes, Matthew A. Kubit, Hanlee P. Ji. Improved detection and identification of microsatellite instability features in colorectal cancer: Implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 421.

The clinical features and genomic landscape of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in patients with colorectal cancer.

e15591Background: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of microsatellite instability (MSI) at tetranucleotide repeats and has emerged as a...

Comprehensive genomic profiling of colorectal cancers with and without microsatellite instability (MSI) and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST).

e15620Background: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) represents a different type of genomic instability in colon cancer (CRC) in contrast to mono-, and ...

Deciphering Elevated Microsatellite Alterations at Selected Tetra/Pentanucleotide Repeats, Microsatellite Instability, and Loss of Heterozygosity in Colorectal Cancers

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) are common in colorectal cancers (CRCs). The association between EMAST and classic mono/dinucleotide microsatellite instability (MSI) is unknown. We assessed the stability of 13 tetranucleotide and three pentanucleotide repeat markers in tumor and normal tissue from 22 MSI-high and 107 microsatellite-stable CRC samples. When present, instability was observed at tetra/pentanucleotide repeats and was defined as elevated microsatellite alterations at selected tetra/pentanucleotide repeats-high (EMASTP-H; ≥30% instability), -low (EMASTP-L; <30% instability), or -stable (EMASTP-S). EMASTP instability, including high and low, was observed in 50 of 123 CRCs (41%), including all MSI-high tumors and 28 of 101 microsatellite-stable tumors (28%). MSI-high CRCs were more likely to be EMASTP-H compared with microsatellite-stable tumors with EMASTP instability. Tetranucleotide markers VWA and D13S317 were the two most frequently altered loci. Loss of heterozygosity was more common in EMASTP-L/S than in EMASTP-H CRCs. Frequencies of loss of heterozygosity at three loci were different between EMASTP-L and EMASTP-S tumors. In addition, right-sided tumor site, large tumor size, high tumor grade, and the presence of Crohn-like reaction were significantly associated with EMASTP-H CRCs. However, there were no differences in clinicopathologic features between EMASTP-L and EMASTP-S tumors. In summary, more CRCs exhibited genomic instability as EMASTP than as MSI. EMASTP instability may prove to be an important prognostic/therapeutic indicator in CRCs.

Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) and Microsatellite Instability in Patients with Colorectal Cancer and Its Clinical Features.

Recently, a different type of microsatellite instability (MSI) instability designated 'elevated microsatellite alterations at selected tetranucleotide repeats' (EMAST) has been reported in several neoplasms, but its clinical implications remain unclear. We aimed to determine the relationships among EMAST, MSI and clinicopathologic characteristics, including oncologic outcomes, in colorectal cancer (CRC). We evaluated 100 sporadic CRC cases subjected to surgery using five markers (MYCL1, D9S242, D20S85, D8S321, and D20S82) for EMAST and the Bethesda panel for MSI status. Immunohistochemical detection of hMSH3, c-erbB2, EGFR and thymidylate synthase was performed. Clinical characteristics and prognostic relevance were assessed. We identified 22 EMAST-positive tumors (22.0%) and 32 MSI-high (MSI-H) tumors (32.0%). EMAST was more frequent in colon cancer than rectal cancer (p=0.033), and associated with MSI-H phenotype (p<0.001), low expression of hMSH3 (p=0.004), and overexpression of thymidylate synthase (p=0.006). Among the 38 MSI-L tumors, only one (4.5%) showed EMAST. Long-term oncologic results in terms of overall and disease-free survival were similar between EMAST and non-EMAST tumors. EMAST is more closely related to MSI-H than MSI-L or MSS status. The clinical and molecular characteristics of EMAST were distinct in terms of tumor location, thymidylate synthase expression, MSI status and hMSH3 expression. Our preliminary findings support the utility of EMAST as a new potential classifier in CRC.