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2023-01-03 12:32:11

TNM stage remains the key determinant of patient prognosis after surgical resection of colorectal cancer (CRC), and informs treatment decisions. However, there is considerable stage-independent variability in clinical outcome that is likely due to molecular heterogeneity. This variability underscores the need for robust prognostic and predictive biomarkers to guide therapeutic decision-making including the use of adjuvant chemotherapy. Although the majority of CRCs develop via a chromosomal instability pathway, approximately 12-15% have deficient DNA mismatch repair (dMMR) which is characterized in the tumor by microsatellite instability (MSI). Tumors with the dMMR/MSI develop from a germline mutation in an MMR gene (MLH1, MSH2, MSH6, PMS2), i.e., Lynch syndrome, or more commonly from epigenetic inactivation of MLH1 MMR gene. CRCs with dMMR/MSI status have a distinct phenotype that includes predilection for the proximal colon, poor differentiation, and abundant tumor infiltrating lymphocytes. Consistent data indicate that these tumors have a better stage-adjusted survival compared to proficient MMR or microsatellite stable (MSS) tumors, and may respond differently to 5-fluorouracil-based adjuvant chemotherapy. To increase the identification of dMMR/MSI patients in clinical practice that includes those with Lynch Syndrome, it is recommended that all resected CRCs to be analyzed for MMR status. Available data indicate that patients with stage II dMMR CRCs have an excellent prognosis and do not benefit from 5-FU-based adjuvant chemotherapy which supports their recommended management by surgery alone. In contrast, the benefit of standard adjuvant chemotherapy with the FOLFOX regiment in stage III dMMR CRC patients awaits further study and therefore, all patients should be treated with standard adjuvant FOLFOX.

TNM stage remains the gold standard for informing patient prognosis and guiding management after resection for non-metastatic colorectal cancer (CRC). Despite the same disease stage, however, CRC patients exhibit considerable variability in clinical outcome that is likely related to molecular tumor heterogeneity. Therefore, the molecular classification of CRC may identify patient subgroups at varying risk of recurrence and death and for who personalized approaches to therapy may beneficial. The majority of CRCs develop via the chromosomal instability pathway (CIN), whereas 12-15% arise from the microsatellite instability (MSI) pathway that is a consequence of deficient (d) DNA mismatch repair (MMR). Deficient MMR can develop from an inherited germline mutation in a MMR gene (MLH1, MSH2, MSH6, PMS2) i.e., Lynch Syndrome, or more commonly due to epigenetic inactivation of the MLH1 gene and the CpG island methylator phenotype (CIMP). These sporadic dMMR tumors carry somatic mutations in the BRAF oncogene in approximately half of cases. Studies have shown that dMMR tumors have phenotypic features including poor differentiation, proximal colon location, and abundant tumor-infiltrating lymphocytes. Furthermore, dMMR tumors have been consistently associated with a better stage-adjusted survival compared to proficient MMR (pMMR) tumors.

Among early stage CRCs, the survival advantage of dMMR status appears to be greater among stage II compared to stage III patients. In patients with stage II colon cancers and dMMR, studies demonstrate a lack of benefit of adjuvant 5-FU-based chemotherapy. Among patients with stage III disease, the predictive impact of MMR status for adjuvant chemotherapy remains controversial. Multiple prior studies have demonstrating a lack of benefit for 5-fluorouracil (FU) as adjuvant chemotherapy, although only limited data exist for patients with stage III dMMR CRCs treated with standard the adjuvant FOLFOX regimen. In contrast to 5-FU, in vitro data indicate that dMMR/MSI CRC cell lines display sensitivity to oxaliplatin and accordingly, this agent may provide benefit in patients with dMMR CRCs.

The Cancer Genome Atlas (TCGA) Research Network has revealed a comprehensive characterization of the genomes of 224 cancerous colorectal tumors and normal pairs (1). Among CRCs studied, 16% of were found to be hypermutated, and 77% of these tumors displayed high frequency microsatellite instability (MSI-H) that was generally associated with hypermethylation and MLH1 gene. The remaining hypermutated tumors were primarily characterized by having mutations in somatic MMR pathways and in polymerase epsilon (POLE)[1].

The DNA MMR system repairs base-base mispairs introduced into microsatellites during DNA synthesis to maintain genomic stability (2). Microsatellites are short, tandemly-repeated sequences that occur throughout the genome and are used as markers of deficient (d) MMR. The DNA MMR system is composed of 4 MMR genes and their encoded proteins (MLH1, MSH2, MSH6, PMS2). Inactivation of MLH1 and MSH2 account for over 90% of dMMR cases. Deficiency of MMR results in production of a truncated, nonfunctional protein or loss of a protein that causes MSI. Therefore, dMMR is frequently analyzed by testing for loss of an MMR protein or for MSI using a PCR-based assay.

MSI testing can be performed on fresh, frozen or paraffin-embedded tumor tissue using a PCR-based assay for detection of instability (3, 4).The National Cancer Institute Workshop agreed on five microsatellite markers necessary to determine MSI (5) that include two mononucleotide – BAT25/26 and three dinucleotide markers – D2S123, D5S346, and D17S250. Interpretation of the profiles requires a comparison with normal DNA from each patient. An alternative molecular method based exclusively on quasi-monomorphic mononucleotide markers was developed to avoid the analysis of matching normal DNA. This method has been proven to be more specific and sensitive than the original NCI panel (5).

On the basis of the MSI status, CRCs can be classified into three groups: MSI-H, if two or more of the five microsatellite markers show instability; MSI-L (low-frequency MSI), if only one of five markers shows instability; and microsatellite stable (MSS) if none of the markers show instability (6).

Another advantage of IHC testing is that loss of a specific mismatch gene product (MLH1, MSH2, MSH6, and PMS2) can direct germline testing to that specific gene, and assists in the identification of patients with LS (4).

MSI testing and IHC are complimentary, and loss of MMR protein expression by IHC has been shown to be highly concordant with DNA-based MSI testing with a good sensitivity (>90%) and a excellent specificity (100%) (4).

In CRCs with loss of MLH1 protein expression, testing for a mutation in the BRAF oncogene is the most cost-effective approach to confirm a sporadic case and generally exclude LS which support the use of this strategy for LS screening (8). Patients with non-mutated BRAF would then have germline testing for a mutation in the presumed altered MLH1 gene (Figure 1).

MSH2 and MSH6 proteins are often lost concurrently. Isolated loss of MSH2 or MSH6 proteins on IHC testing has high specificity for a germline mutation of these genes leading to the diagnosis of LS (Figure 1). Also, loss of the MSH2 protein can be caused by germline mutation in the EPCAM gene rather than MSH2 gene.

Tumors displaying loss of an MMR protein can be collectively referred to as dMMR and are considered to be MSI-H, whereas those with intact MMR proteins can be classified as pMMR are are expected to be microsatellite stable (MSS) or MSI-low (MSI-L).➢

LS accounts for approximately 3-4% of all CRCs and one third of all dMMR/MSI-associated CRCs. LS is inherited in an autosomal dominant manner and results from a germline loss-of-function mutation (9) that occurs more commonly in MLH1 or MSH2, and infrequently in MSH6 or PMS2 (10). A germline mutation in an MMR gene followed by a second hit (somatic event) to the wild-type copy is needed to produce LS, and can occur due to point mutation, loss of heterozygosity or methylation.

CRCs from LS patients are significantly less likely to carry KRAS mutations compared to pMMR/MSS cancers and importantly, BRAFV600E mutations are lacking in these patients. Among dMMR/MSI CRCs, BRAFV600E mutation testing can be performed to distinguish LS cases from sporadic dMMR tumors (13) (Figure 1).

Bethesda criteria were revised in 2004 to guide selection of patients for LS testing (14). The guideline indicated that tumors should be tested for MSI in the following clinical situations:

Families that meet the Amsterdam Criteria (15) but who lack a germline mutation in an MMR gene and an MSI-H tumor, have been termed familial CRC, type × (16).

Sporadic MSI-H CRCs show loss of MLH1 that generally occurs in a background of the CpG island methylator phenotype (CIMP) (17, 18). CIMP represents dense promoter hypermethylation of cancer-specific genes. CIMP-related silencing of the MLH1 gene is responsible for about 80% of cases in which MLH1/PMS2 protein expression are lost (7).

Patients with MSI-H sporadic CRCs share most of the clinicopathological features with LS cases, yet have distinct epidemiological features including older age at diagnosis, predominance of female gender and increased rate of cigarette smoking (21).

CRC with dMMR is more frequent in stage II (almost 20%) compared to stage III (12%), and are relatively uncommon among metastatic tumors (4%) (22). This highlights the importance of MSI testing in early stage disease where patients can be potentially cured by surgery alone or combined with adjuvant chemotherapy.

A meta-analysis from 32 studies with 1,277 MSI/dMMR cases included 7,642 patients with stage I-IV CRC. A better prognosis was found for patients with MSI/dMMR than those with MSS, MSI-L/pMMR tumors (28) among patients that were untreated or treated with 5-fluorouracil (5-FU)-based adjuvant chemotherapy. The hazard ratio (HR) for overall survival (OS) was 0.65 [95 % confidence interval (CI), 0.59-0.71] in favor of dMMR CRC patients. Results were confirmed when the analyses was restricted to patients with stage II or III CRC participating in clinical trials (28).

In a retrospective analysis of the CALGB 89803 trial where patients with stage III colon cancer were randomly assigned to weekly bolus 5-FU/leucovorin (LV) or weekly bolus irinotecan, 5-FU, and LV (IFL), IFL-treated patients with dMMR/MSI-H tumors showed improved 5-year DFS as compared to pMMR tumors (HR=0.76; 95% CI, 0.64 to 0.88 vs. 0.59; 95% CI, 0.53 to 0.64; P = .03), which was not observed among patients treated with 5-FU/LV (63). However, data from the PETACC-3 study (64) failed to show a benefit for irinotecan in patients with dMMR colon cancers.

4. Lindor NM, Burgart LJ, Leontovich O, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol.2002;20:1043–8. [PubMed]

This study showed that BRAF mutation was not prognostic for recurrence-free survival but was for overall survival, particularly in patients with MSI-L and MSI-S tumors.

Adjuvant study found that MSI-H was significantly associated with RFS in stage II and III colon cancer patients treated with 5-FU/LV alone or combined with irinotecan. However, the relationship with OS was only significant for stage II patients.

msi lcd panel testing free sample

Universal testing of microsatellite instability (MSI) is recommended for colorectal cancer (CRC) and endometrial cancer (EC) to screen for Lynch syndrome and to aid in assessing prognosis and optimal treatment. We compared the performance of Idylla MSI test to immunohistochemistry (IHC) of mismatch repair (MMR) proteins in consecutive series of 100 CRC and 108 EC samples, as well as in retrospective series of 28 CRC and 33 EC specimens with known deficient MMR protein expression. The concordance between the Idylla test and IHC was 100% in all CRC samples (n=128) but lower in EC samples (87.2%; n=141). In the EC samples, sensitivity of Idylla test was 72.7% and specificity 100%. EC MSI/dMMR agreement was 85.4% for MLH1, 87.5% for MSH2, and only 35.3% for MSH6. When we analyzed 14 EC samples that were discrepant, i.e., dMMR using IHC and microsatellite stable using Idylla, with microsatellite markers BAT25 and BAT26, we found four cases to be replication error (RER) positive. All RER positive cases were deficient for MSH6 protein expression. We also re-analyzed EC samples with variable tumor cellularity to determine the limit of detection of the Idylla test and found that a 30% or higher tumor cellularity is required. We conclude that Idylla MSI test offers a sensitive and specific method for CRC diagnostics but is less sensitive in EC samples especially in the case of MSH6 deficiency.

Microsatellites are short repetitive DNA sequences that are prone to replication errors (RER). Microsatellite instability (MSI) is caused by deficient mismatch repair (dMMR) system, leading to hypermutation phenomenon and cancer susceptibility [1, 2]. Approximately 15% of colorectal cancers (CRC) and 30% of endometrial cancers (EC) arise from MSI pathway [3]. Most MSI cancers are sporadic and account for approximately 90% of the MSI EC cases and 80% of the MSI CRC cases, which most often develops through acquired MLH1 promoter hypermethylation [4, 5]. Lynch syndrome (LS), on the other hand, is a hereditary form of MSI most often caused by germline mutation in one of the MMR genes (MLH1, MSH2, MSH6, or PMS2) [2]. To this end, universal testing of MSI is recommended for CRC and EC patients to screen for LS and to aid in assessing prognosis and determining optimal treatment and follow-up [6–8].

Immunohistochemistry (IHC) technique is used in many pathology laboratories as a standard method to detect the loss of MMR protein expression to screen dMMR cases. The other standard method is PCR-based microsatellite test that consists of variable number and kinds of microsatellite markers, including at least mononucleotide markers BAT25 and BAT26, and is an alternative method for IHC MMR testing, especially in case of doubtful IHC results. [6] The sensitivity to detect MSI in dMMR CRC tumors using PCR-based microsatellite tests has been reported to be 89% for MLH1/MSH2 deficient but only 77% for MSH6 deficient cases [9], and in EC, the sensitivity has been estimated to range from 41 to 100% and the specificity from 69 to 89% [10].

The requirement of extensive hands-on time and trained personnel for above-mentioned IHC and PCR analysis, along with increasing demand of MSI testing, makes fast and automated molecular methods attractive alternatives. One novel way to assess the MSI status is Idylla MSI test, which analyzes a panel of seven monomorphic microsatellite biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2) using fluorescent-labeled molecular beacons combined with PCR amplification. Idylla test is an automatic system performing all the necessary steps from a formalin-fixed paraffin-embedded (FFPE) tissue flake to MSI status information, including DNA extraction, amplification, and data analysis in 150 min [11].

The aim of this study was to evaluate the diagnostic performance of the Idylla MSI test as compared to the routine MMR IHC in CRC and EC samples. Analysis was performed in both consecutive sample series (CRC n=100 and EC n=108) and in retrospective series (CRC n=28 and EC n=33) with known dMMR IHC result. In addition, we scrutinized the minimum tumor cellularity requirement for the Idylla MSI test to detect the MSI status in the EC samples.

We analyzed a prospective and consecutive series of 100 CRC samples (CRC Set I, of which one was an appendix adenocarcinoma and one a colon descendens adenoma) from patients who underwent surgical resection at Helsinki University Hospital (HUH) between February and April 2019. These patients were routinely screened for MMR proteins MLH1, MSH2, MSH6, and PMS2 in real-life diagnostic setting using IHC and experimentally tested in blinded manner for MSI using Idylla MSI test at the Meilahti Pathology Department, Helsinki, Finland. We also collected a consecutive series of 108 EC samples (EC Set I) from patients operated at HUH between February 2018 and March 2020, which were analyzed in blinded manner using Idylla MSI test. In addition to the consecutive EC Set I, we collected a historical EC series (EC Set II; n=33) with known dMMR protein IHC result from patients operated at HUH between January 2007 and February 2012 and reanalyzed the samples using IHC and Idylla. A retrospective CRC Set II consisted of 28 samples with known IHC result of dMMR for MSH2, MSH6, or PMS2 and operated at HUH between October 2017 and September 2020, which had not been included to the CRC Set I. This study was approved by the Ethics Committee of the Helsinki University Central Hospital.

The minimum tumor cell percentage instructed by the manufacturer for Idylla MSI test is ≥ 20% for CRC samples, and for the EC samples, we used ≥ 30% proportion of tumor cells. To increase the tumor cell percentage at or above the detection limit, macrodissection was performed for the FFPE tissue blocks of 4/100 CRC Set I samples, 71/108 EC Set I samples, 26/33 EC Set II samples, and 19/28 CRC Set II samples. After macrodissection, the tumor cell percentages were 20–90% for CRC samples and 30–90% for EC samples, as estimated from the HE slides by an experienced pathologist (AR). Manufacturer’s protocol to perform the Idylla test requires a total tissue area between 25 and 300 mm2 with section thickness of 10 μm. For Idylla analysis, one or two 10-μm tissue sections were cut from the FFPE tissue blocks with a Leica SM2000R microtome (Leica Microsystems GmbH, Wetzlar, Germany) using aseptic conditions.

FFPE tissue samples were tested using automated Idylla MSITM Test (Biocartis NV, Mechelen, Belgium) that has been CE-IVD validated for CRC samples. The tissue handling and analysis were performed according to the manufacturer’s protocol. The Idylla test result is considered valid if at least five out of the seven biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2) are fully analyzed. The presence of at least two mutant biomarkers give rise to the judgment of MSI phenotype, whereas the presence of zero or one mutant biomarker indicates MSS phenotype.

To scrutinize the limit of detection of the Idylla MSI test, we analyzed samples with variable tumor cell percentages. For that, we selected eight representative cases from the EC set I samples, which had been dMMR using IHC and MSI by the Idylla test. We then macrodissected these samples to achieve altogether 21 separate samples with tumor cellularity varying from 10 to 70%, as estimated by AR, that were analyzed with the Idylla platform.

Discrepant samples between IHC and Idylla test were further analyzed with RER test using BAT25 and BAT26 mononucleotide repeats, in which 10-μm FFPE tissue flakes were cut from whenever 50% or more tumor cells [12] could be retrieved and DNA was extracted from the deparaffinized flakes by using a Maxwell® CSC Blood DNA Kit (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. MSI status was then assessed using the two mononucleotide-repeat markers using fluorescently labeled PCR. Detection of allelic variation in both BAT25 and BAT26 was considered as a positive result representing MSI.

The MMR IHC was considered as the golden standard reference test against which the overall agreement (concordance), sensitivity and specificity, and the positive predictive value and negative predictive value were calculated. To quantify the degree of agreement between IHC and the Idylla test, two-tailed Fisher’s exact test was used (GraphPad QuickCalcs: https://www.graphpad.com/quickcalcs/contingency2/). In order to compare the mutated biomarker spectrums between MSI CRC cases and MSI EC cases detected by the Idylla test, the unpaired t-test was used (GraphPad QuickCalcs: https://www.graphpad.com/quickcalcs/ttest2/). P-value less than 0.05 was considered as statistically significant, and results are shown as means±SD.

One hundred consecutive CRC samples (CRC Set I) were analyzed in prospective, blinded, and real-life diagnostic setting by IHC of MMR proteins (MLH1, MSH2, MSH6, and PMS2) and Idylla MSI test. Of these patients, 44 were males and 56 females with a median age of 76 years (range from 36 to 96), and the tumors localized to the right colon (n=54), the left colon (n=33), and the rectum (n=11). CRC tumor cell percentages estimated for the Idylla analysis varied between 20 and 90%, and none of the Idylla test results were invalid. IHC and Idylla results showed a 100% agreement and were dMMR/MSI in 32 and pMMR/MSS in 68 of the cases (Table (Table1;1; two-tailed Fisher’s exact test, P < 0.0001). Based on IHC results, dMMR resulted from loss of MLH1 protein (and concomitant loss of PMS2 protein) expression in 31 cases and an isolated loss of MSH6 in one case.

dMMR, deficient MMR protein expression, MMR mismatch repair, MSI microsatellite instable, MSS microsatellite stable, pMMR proficient MMR protein expression

Since sensitivity of MSI detection might depend on the type of MMR gene affected, we collected 28 retrospective CRC samples (Set II) with known dMMR IHC staining pattern. Based on IHC results, there was loss of MSH2 (and concomitant loss of MSH6) protein expression in 18, an isolated loss of MSH6 protein expression in 5, and an isolated loss of PMS2 protein expression in 5. CRC tumor cell percentages estimated for the Idylla analysis varied between 20 and 80%, and none of the Idylla test results were invalid. Again, IHC and Idylla results showed a perfect 100% agreement. When the MSI cases of CRC Sets I and II were combined (n=60), Idylla test detected three to seven mutated biomarkers in 59/60 samples and two mutated biomarkers in one sample, whereas all the MSS samples had seven wild-type biomarkers. On average, 82.1±12.9% of the seven unstable sites were mutated in the MSI CRC cases. Two markers, ACVR2A and MRE11, were most commonly mutated (56 samples, 93.3%), and SEC31A was least commonly mutated (35 samples, 58.3%) (Fig. (Fig.11).

Comparison of the mutated biomarker spectrum between MSI CRC cases and MSI EC cases detected by the Idylla MSI test. Two-tailed unpaired t-test, P < 0.05

Patients of the consecutive EC sample series (Set I, n=108) had median age of 71 years (range from 34 to 90). Tumor cell percentage estimated for the Idylla analysis varied between 30 and 90%. None of the Idylla test results were invalid, and 27/108 were MSI and 81/108 MSS. IHC, in turn, showed dMMR protein expression in 33/108 samples and pMMR protein expression in 75/108 samples. We thus identified 6/108 (5.6%) discrepant cases between IHC (all dMMR) and the Idylla test (all MSS), the overall agreement being 94.4% (102/108) (Table (Table2;2; two-tailed Fisher’s exact test, P < 0.0001). Loss of MLH1 protein (and concomitant loss of PMS2 protein) was detected in 27/33, loss of MSH2 (and concomitant loss of MSH6) protein expression in 4/33, an isolated loss of MSH6 protein expression in 1/33, and combined loss of MLH1, PMS2, and MSH6 expression in 1/33 samples. In the latter case, however, the loss of MSH6 protein expression was not homogeneous, which may suggest a somatic mutation in the microsatellites of MSH6 gene due to the MLH1 deficiency [13]. Using IHC analysis as a reference, sensitivity of Idylla test was 81.8% and specificity 100%, while positive predictive value (PPV) was 100% and negative predictive value (NPV) 92.6%. With IHC, four of these discrepant cases were in the MLH1 deficient sample group (4/28, 14.3%), one was in the MSH2 deficient group (1/4, 25%), and one was the only MSH6 deficient sample (1/1, 100%) (Table (Table33).

dMMR deficient MMR protein expression, MMR mismatch repair, MSI microsatellite instable, MSS microsatellite stable, pMMR proficient MMR protein expression

Since there is no IVD-CE claim for the EC tissue samples, we next studied the limit of detection for the Idylla MSI test. For that, eight concordant dMMR IHC and Idylla MSI EC Set I samples were selected and macrodissected in order to obtain 21 samples with tumor cell percentage varying from 10 to 70%. The MSI result was obtained in 2/8 (25%) samples with tumor cell percentage < 20% (10–15%), in 2/3 (66.7%) samples with tumor cell percentage 25%, and in 10/10 (100%) samples with tumor cell percentage ≥ 30% (30–70%), suggesting that at least for these samples the 30% tumor cell cut off was optimal (Supplementary Table S1).

To further validate Idylla MSI test in EC, we collected EC Set II (n=33) from cases with known dMMR protein IHC diagnosis. EC patients included into this retrospective dMMR EC sample series had median age of 60 years (range from 43 to 81). First, we restained the samples with the current MMR IHC protocol to verify the dMMR status. IHC showed loss of MLH1 protein (and concomitant loss of PMS2 protein) in 13, loss of MSH2 (and concomitant loss of MSH6) protein expression in 4, and an isolated loss of MSH6 protein expression in 16. Tumor cell percentage estimated for Idylla analysis varied between 30 and 90%. The Idylla test scored all the results valid and they were MSI in 21/33 and MSS in 12/33 of the dMMR cases. We here identified 12/33 (36.3%) discrepant cases between Idylla (all MSS) and IHC (all dMMR), the overall percent agreement between the tests being 63.6% (21/33). With IHC, 10 of these discrepant cases were of the MSH6 deficient sample group (10/16, 62.5%), two were of the MLH1 deficient sample group (2/13, 15.4%), and none were of the MSH2 deficient samples (0/4) (Table (Table44).

MSI/dMMR agreement for each dMMR protein group calculated from the combined EC Set I and EC Set II is presented in Table Table5.5. In this combined EC series (n=48), Idylla test detected three to seven mutated biomarkers in 41/48 samples and two mutated biomarkers in 7/48 samples, whereas one MSS sample had one mutated biomarker and the rest of them had seven wild-type biomarkers. On average, 61.0±12.5% of the seven unstable sites were mutated in the MSI EC cases (Fig. (Fig.1),1), which was significantly lower than that of the CRC cases (82.1 ± 12.9%; P < 0.05). Of the mutated markers, ACVR2A and RYR3 were mutated in 20 and 24 samples (41.7% and 50.0%), SEC31A and SULF2 in 28 samples (58.3%), and the rest of the markers were mutated in 34 to 36 cases (70.8 to 75.0%). In combined EC Set I and Set II, mutated biomarkers detected by Idylla MSI test varied depending on the MMR protein deficiency. Especially this applied to the MSH6 deficient cases, which less frequently showed ACVR2A and BTBD7 mutations (both 16.7%), but most often had mutated DIDO01 (83.3%) (Fig. (Fig.2a).2a). In MSH6 deficient CRC samples, similar divergences were not observed (Fig. (Fig.2b2b).

Mutated biomarker spectrums for each dMMR IHC protein in MSI EC and CRC cases detected by the Idylla MSI test. a Combined MSI cases from EC Sets I and II (n=48) and b combined MSI cases from CRC Sets I and II (n=60)

In the EC Set I, we identified six and in EC Set II 12 discrepant cases demonstrating dMMR IHC result and MSS using the Idylla test, which were first re-evaluated (tumor cell percentage) and retested with the Idylla MSI test using new paraffin slices. The result of re-testing was MSS in all 18 cases. We also re-evaluated IHC staining patterns and identified one case (B107) with heterogeneous loss of MLH1 (and concomitant PMS2) (Supplementary Fig. S1). Subclonal loss of MMR protein expression and heterogeneous dMMR IHC staining has been reported to occur in up to 7% of ECs, and exclusively in glandular endometrioid component [14], as in the case of B107. It is also noteworthy that this heterogeneous loss of MLH1 is suggestive for sporadic rather than germline deficiency. As a third method to evaluate the MSI phenotype, the discrepant cases (from EC Set I to Set II) were further tested for RER status using BAT25 and BAT26 microsatellite markers (Table (Table6).6). For this analysis, two samples (B14 and C26) did not meet the required 50% tumor cell content [12], and two (B107 and C9) did not have enough tissue available. Of the 14 samples that were subjected to RER analysis, four were shown to be positive for both BAT25 and BAT26, indicating MSI, and rest of them were negative (Table (Table6).6). These four RER positive cases had been shown to be deficient for MSH6 protein expression.

CaseAge (years)Tumor cells (%)Tissue area (mm2)Macro-dissectionIHC (loss of MMR proteins)Idylla MSI analysisNumber of mutated biomarkersRER analysis (BAT25 and BAT26)B146930300YesMSH2MSS0/7NDa

dMMR deficient MMR protein expression, IHC immunohistochemistry, MMR mismatch repair, MSI microsatellite instable, MSS microsatellite stable, ND not determined, RER replication error

In this study, we evaluated the diagnostic performance of Idylla MSI test in detecting MSI in CRC (n=128) and EC (n=141) samples. The Idylla test showed 100% concordance with the MMR IHC results in the CRC cohort but only 87.2% concordance in the EC cohort. None of the Idylla analysis failed due to technical errors or sample-related problems. Strength of the study includes real-life prospective blinded design of the 100 consecutive CRC cases. We were not able to perform identical study protocol for the EC cases, which were analyzed in two cohorts, i.e., a consecutive and blinded Set I and a separate historical Set II with known dMMR IHC status. As compared to IHC, sensitivity of the Idylla test was 72.7% and specificity 100% in EC samples. Eighteen discrepant EC cases between IHC and Idylla test were detected, and we were able to subject 14 of these to RER analysis using two mononucleotide markers BAT25 and BAT26. Four cases were identified that were repeatedly MSS in the Idylla analysis but were found to be MSI when using the BAT25 and BAT26 markers. All these cases were deficient for expression of MSH6 protein. Weakness of this study was that we used only two mononucleotide microsatellite markers instead of five, which may have been more sensitive in detecting MSI [6].

One explanation for discrepancies between IHC and DNA-based assay could depend on the amount of tumor cells included to the FFPE flakes. To this end, we scrutinized the minimum tumor cellularity requirement for Idylla MSI test in the EC samples. Our data suggest that at least 30% EC cell percentage is required for the Idylla analysis. Further, low tumor cell number does not explain the discrepant cases that we found between IHC and the Idylla test, since all four RER-positive (BAT25 and BAT26 positive) cases had tumor cell percentages between 50 and 80%. It should be pointed out that we also included MSH6 losses to the CRC cohort (n=6) that were all correctly identified as MSI using the Idylla test. Our results are supported by previous findings showing that MSI in EC demonstrates a higher frequency of minimal (1–2 nucleotide) microsatellite shifts especially in the case of MSH6 loss, which is a challenge for DNA-based MSI assays [9, 15, 16]. Importantly, detection of MSH6 deficiency is in parts crucial, since females carrying a pathological MSH6 mutation are reported to be at especially high risk of endometrial cancer compared with other LS-related cancers [17].

Previous reports have also found a very high concordance (97.6–100%; n=42–105) of the Idylla MSI test with reference tests in retrospective CRC samples [18–20]. In a larger sample set, Zwaenepoel et al. reported the Idylla test to show 98.7% overall agreement using historical and partially dMMR-enriched IHC data in 330 CRC samples [21]. In this report, one case was MSS in Idylla analysis, whereas IHC was dMMR and Promega MSI analysis was MSI and interestingly tumor mutation burden was very high, whereas three IHC dMMR cases showed MSS in DNA-based assays and low tumor mutation burden. In addition, a multi-center real-life global study including 44 clinical centers and 1301 CRC samples showed the concordance level between the Idylla test and IHC to be as high as 96.4%, with Idylla having lower failure rates [11].

Excellent accuracy of the Idylla MSI test in CRC samples has encouraged to study the diagnostic performance of this novel assay in other solid tumors as well. Farmkiss et al. compared retrospectively IHC and the Idylla test across 50 biopsies of gastric adenocarcinoma, scoring concordant results in 48 samples with a 95.7% sensitivity and 100% specificity [22]. Pécriaux et al. have evaluated the Idylla test in a panel of solid tumors, including 15 EC samples using extracted DNA, and reported sensitivity of 89% and specificity of 100% to detect MSI status in EC samples as compared to IHC [23]. It is important to note that all nine MSI cases in this report were MLH1 deficient. Gilson et al. reported similarly with 15 EC samples a 100% specificity and sensitivity with the Idylla test as compared to previously used PCR-based Promega MSI analysis, but if compared to IHC, the sensitivity seems to be 80–90% and specificity 100% [24]. Our study agrees with these reports on the excellent specificity of the Idylla MSI test in EC diagnostics, but the sensitivity when compared to IHC was clearly lower in our study (72.7%), which to our knowledge is the largest EC study thus far (n=141).

Our study showed significant differences in the mutated biomarker spectrum between CRC and EC MSI cases detected by the Idylla MSI test. First, on average 82.1% of the seven unstable sites were mutated in MSI CRC, whereas only 61.0% of the sites were mutated in MSI EC cases. This is in line with the previous publication reporting lower proportion of unstable markers per tumor in EC as compared to CRC, even in the tumors originating from the same LS patient [25]. Second, our results implicate that mutated biomarkers ACVR2A and RYR3 are more frequently mutated in CRC when compared to EC, whereas SEC31A seems to be mutated by the same frequency in both CRC and EC MSI samples. Previously, Kim et al. have showed that ACVR2A is indeed a gene harboring frameshift microsatellite instability specifically in CRC and less often in EC genomes [26]. They also demonstrated SEC31A to be a gene with no extreme specificity to harbor frameshift MSI in either CRC or EC genomes. Further, the multicenter Idylla MSI test study of CRC reported ACVR2A to be the most often mutated biomarker (94.0%) and SEC31A to be the least mutated marker (60.9%) [11], and our frequencies are in line with a sequencing study demonstrating ACVR2A mutations in 92% and SEC31A mutations in 54% of one hundred MSI CRC cases [27].

In conclusion, our study reinforces the accuracy of the fast and automated Idylla MSI test to detect MSI phenotype in CRC samples. In EC samples, Idylla test is 100% specific, but sensitivity is compromised especially in the case of MSH6 deficient tumors. We conclude that the Idylla test offers a sensitive and specific method for CRC diagnostics, but it should be validated for each tumor type separately in a relatively large material before applying it to diagnostic use.

6. Luchini C, Bibeau F, Ligtenberg MJL, Singh N, Nottegar A, Bosse T, Miller R, Riaz N, Douillard J, Andre F, Scarpa A. ESMO recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a systematic review-based approach. Ann Oncol.2019;30:1232–1243. doi: 10.1093/annonc/mdz116. [PubMed] [CrossRef]

9. Palomäki GE, McClain MR, Melillo S, Hampel HL, Thibodeau SN. EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome. Genet Med.2009;11:42–65. doi: 10.1097/GIM.0b013e31818fa2db. PubMed] [CrossRef]

10. Stinton C, Fraser H, Al-Khudairy L, Court R, Jordan M, Grammatopoulos D, Taylor-Phillips S. Testing for lynch syndrome in people with endometrial cancer using immunohistochemistry and microsatellite instability-based testing strategies – a systematic review of test accuracy. Gynecol Oncol.2021;160:148–160. doi: 10.1016/j.ygyno.2020.10.003. [PubMed] [CrossRef]

21. Zwaenepoel K, Holmgaard Duelund J, De Winne K, Maes V, Weyn C, Lambin S, Dendooven R, Broeckx G, Steiniche T, Pauwels P. Clinical performance of the Idylla MSI test for a rapid assessment of the DNA microsatellite status in human colorectal cancer. J Mol Diagn.2019;22:386–395. doi: 10.1016/j.jmoldx.2019.12.002. [PubMed] [CrossRef]

23. Pécriaux A, Favre L, Calderaro J, Charpy C, Derman J, Pujals A. Detection of microsatellite instability in a panel of solid tumours with the Idylla MSI Test using extracted DNA. J Clin Pathol.2020;74:36–42. doi: 10.1136/jclinpath-2020-206581. [PubMed] [CrossRef]

msi lcd panel testing free sample

Step 4: Familiarize yourself with your monitor’s display controls. They may be located on the monitor itself, on the keyboard, or within the operating system control panel.

In older versions of Windows, you can find the Color Calibration utility in the Display section of the Control Panel, which is listed under Appearance and Personalization.

Step 1: In MacOS, the Display Calibrator Assistant is located in the system preferences under the Displays tab, in the Color section. If you are having trouble finding it, try entering calibrate in Spotlight to scan through your computer’s various folders and files. The results should show an option to open the utility in the System Preferences panel.

The Lagom LCD Monitor Test Pages: Handy for both online and offline use, the Lagom LCD Monitor Test Pages not only allow you to adjust various things such as contrast and response time, but also allow you to download the images as a 120KB zip file, so you can check any monitor in-store that you are thinking about purchasing.

msi lcd panel testing free sample

The MSI Optix MAG274QRF-QD and the LG 27GP850-B are similar 1440p, 27-inch monitors, but there are a few differences. The MSI has a few extra features for office use, like an ergonomic stand and a USB-C input that supports DisplayPort Alt Mode. However, colors look oversaturated, and the color accuracy is much better on the LG. The LG is also slightly better for gaming because it supports DP 1.4 bandwidth, allowing you to reach a higher refresh rate, and the motion handling is a bit better with lower frame rate signals.

The MSI Optix MAG274QRF-QD and the Dell S2721DGF are both great monitors with similar gaming features. They"re both 1440p monitors with a 165Hz refresh rate and outstanding response time. The MSI has a BFI feature, which the Dell doesn"t, but it only works within a narrow frequency range. The MSI also has a USB-C input, which supports DisplayPort Alt Mode. On the other hand, the Dell is a better choice if you want to use it in a well-lit room because it has much better reflection handling. It also has a local dimming feature, but it performs terribly.

The MSI Optix MAG274QRF-QD and the Gigabyte M27Q are two excellent gaming monitors with a 1440p resolution. They each have outstanding response time, low input lag, FreeSync support, and G-SYNC compatibility. The MSI has much better ergonomics, making it easy to place the screen how you like. However, the Gigabyte has much better out-of-the-box accuracy, so colors don"t look over-saturated like they do on the MSI. It also has Picture-by-Picture/Picture-in-Picture modes, allowing you to display images from two sources at once.

The Gigabyte M27Q X is a bit better than the MSI Optix MAG274QRF-QD. The Gigabyte has a higher native refresh rate, resulting in slightly clearer motion overall. The Gigabyte is also more accurate out of the box and has better connectivity, with high bandwidth USB-C and a built-in keyboard, video, and mouse switch.

Overall, the MSI Optix MAG274QRF-QD performs better than the ASUS TUF Gaming VG27AQL1A. The MSI has a much faster response time to deliver a clearer image in fast-moving scenes. It has a much wider color gamut for HDR due to its Quantum Dot layer; however, it doesn"t get as bright as the ASUS to bring out highlights in HDR content. The MSI has more USB ports, including a USB-C that supports DisplayPort and 15W charging.

The ASUS ROG Strix XG27AQ performs very similarly to the MSI Optix MAG274QRF-QD. As far as gaming goes, you can"t really go wrong with either one. The MSI has a slightly faster response time at the max refresh rate while the ASUS has a slightly faster response time at 60Hz and a marginally higher refresh rate, but really, these are minor differences. While the MSI has a wider color gamut thanks to its use of Quantum Dot technology, in practice, colors can look oversaturated. The ASUS has a couple of other things that may make it preferable to some. It has wider viewing angles, so the image stays accurate from the sides, and it has much better out-of-the-box color accuracy, so you may not need to calibrate it to get the most out of your monitor. All in all, though, these are both excellent gaming monitors.

The MSI Optix MAG274QRF-QD is better for most uses than the MSI Optix G273QF, but they have similar gaming performance. They both have an incredible response time, very low input lag, and a 165Hz refresh rate for gaming. The MAG274QRF-QD has much better ergonomics, allowing you to easily adjust the screen. It also supports HDR10, which the G273QF doesn"t, and it has many more features, such as a Console Mode that"s designed for PS5 compatibility.

The Acer Nitro XV272U KVbmiiprzx and the MSI Optix MAG274QRF-QD are very similar gaming-wise. The main differences are that the MSI has a USB-C port with DisplayPort Alt Mode and a better color gamut in HDR.

The MSI Optix MAG274QRF-QD and the ASUS TUF VG27AQ are two excellent gaming monitors; they each have a 1440p resolution and a 165Hz refresh rate. Motion looks smoother on the MSI thanks to its quick response time, especially at 60Hz. However, the ASUS has a BFI feature that works at a wider refresh rate range than the MSI, and it can also be used when VRR is enabled, which the MSI can"t do.

The Samsung Odyssey G7 C32G75T and the MSI Optix MAG274QRF-QD are both great monitors with excellent gaming performance; however, they"re different. The Samsung uses a VA panel and is available in a 27-inch or 32-inch size, while the MSI uses an IPS panel and is only available in a 27-inch. If you want better dark room performance, go with the Samsung because it can produce much deeper blacks. It has a higher refresh rate as well, but the difference is often hard to tell for most casual gamers. The MSI isn"t as good for dark rooms due to its IPS panel"s mediocre contrast, but it provides wider viewing angles, and it has USB-C input.

The Dell Alienware AW2723DF is slightly better for gaming than the MSI Optix MAG274QRF-QD. The main difference is that the Dell has a much higher 280Hz max refresh rate compared to 165Hz on the MSI, allowing you to play games at a higher frame rate. The Dell also has lower input lag and better motion handling with lower frame rate signals. On the other hand, the MSI is better if you game in a bright room because it has much better reflection handling.

The MSI Optix MAG274QRF-QD and the Samsung Odyssey G5 S27AG50 are both excellent gaming monitors. They both have a 165Hz refresh rate, native FreeSync VRR support, a 1440p resolution, and fantastic motion handling. There are a few differences in picture quality, though; the MSI displays a wider color gamut in HDR and SDR, but that"s because it over-saturates the colors, meaning some people may prefer the Samsung. The Samsung also gets brighter and has better reflection handling, so it"s a better choice for well-lit rooms. On the other hand, the MSI has a better selection of inputs, such as a USB-C input and two USB inputs that you can connect your keyboard and mouse to.

The Samsung Odyssey G7 S28AG70 and the MSI Optix MAG274QRF-QD are different types of monitors. The Samsung is a 4k monitor with HDMI 2.1 inputs, so it"s a better choice for console gaming, while the MSI has a 1440p resolution and a slightly higher 165Hz refresh rate. Motion handling is fantastic on both, but the MSI has a slightly faster response time at 60Hz. The MSI has a few more office features like better ergonomics and a USB-C input, but the Samsung delivers clearer text thanks to the higher resolution. The MSI is also a better choice for use in well-lit rooms because it gets brighter.

The MSI Optix MAG274QRF-QD and the Acer Predator XB273U GXbmiipruzx are similar monitors, but the Acer has a bit more to recommend it. They both have exceptional color gamuts, but the MSI lacks an sRGB setting, so colors appear oversaturated. The Acer also has a higher refresh rate, gets brighter in SDR and HDR, and has better accuracy out of the box.

The MSI Optix MAG274QRF-QD is a bit better than the LG 27GP83B-B. The MSI has much better ergonomics, a wider color gamut, and better text clarity. The response time is similar between the two, but the MSI has an optional black frame insertion feature to reduce persistence blur. The MSI also has a bit better connectivity, with a built-in USB hub and a USB-C port.

The MSI Optix MAG274QRF-QD is a better gaming monitor than the LG 27GL850-B. They have similar features with a 1440p resolution, FreeSync support, G-SYNC compatibility, and a very quick response time. The MSI has a Black Frame Insertion feature, which the LG doesn"t, but it has a narrow flicker range and can"t be used with VRR enabled. The MSI also has much better ergonomics, including a swivel adjustment, which the LG doesn"t have.

The MSI Optix MAG274QRF-QD is a bit better for most uses than the LG 27GN850-B, but they have many of the same features. They both have outstanding response time and low input lag for gaming, but the MSI has a slightly higher 165Hz refresh rate. It also has a BFI feature, but it only works within a narrow range. However, the LG has better out-of-the-box accuracy and colors don"t look as over-saturated as the MSI, so you may not need to get it calibrated to enjoy it to the fullest.

The MSI Optix MAG274QRF-QD and the ASUS ROG Swift PG279QM offer very similar performance, each with strengths and weaknesses. The ASUS is a bit better for HDR, as it has a local dimming feature, and it"s a bit brighter in HDR. The MSI has better ergonomics and an optional backlight strobing feature, which can help improve the appearance of motion in some games.

The MSI Optix MAG274QRF-QD and the LG 32GP850-B are similar overall, and the differences between them are minor. The MSI has better ergonomics; if that doesn"t matter to you, the LG has better reflection handling and a larger screen. The LG also has better accuracy out of the box and better gray uniformity, but these could vary between units.

The MSI Optix MAG274QRF-QD is better than the Gigabyte G27Q for most uses. The MSI has a slightly higher refresh rate and faster response time to deliver smoother motion. It also has better ergonomics because it allows for swivel and pivot adjustments, and its USB hub includes a USB-C port that supports DisplayPort and 15W charging. It has a wider color gamut than the Gigabyte but doesn"t get as bright to bring out highlights in HDR content.

The Gigabyte AORUS FI27Q and the MSI Optix MAG274QRF-QD are very similar overall. The MSI performs better because it has a faster response time. The Gigabyte has a Picture-in-Picture mode, but on the other hand, the MSI has a USB-C port that supports DisplayPort Alt Mode.

The LG 27GN880-B and the MSI Optix MAG274QRF-QD are very similar. The most notable difference is that the MSI has a higher refresh rate of 165Hz versus the LG"s 144Hz, which results in a slightly smoother and more responsive gaming experience. The MSI also has a wider SDR and HDR color gamut, and more features, like two USB 3.0 ports a USB-C with DisplayPort Alt Mode. On the flip side, the LG has a better stand that takes up less space and provides more ergonomic adjustments.

The MSI Optix MAG274QRF-QD and the Gigabyte M32Q offer very similar performance overall. The biggest difference between them is in their design. If you prefer a larger screen, go with the Gigabyte, but if ergonomics are more important to you and you don"t plan on VESA mounting the monitor, the MSI might be the better choice.

The MSI Optix MAG274QRF-QD is a much better monitor than the MSI Optix G273. The MAG274QRF-QD delivers a much smoother gaming experience, with a faster response time, resulting in less motion blur behind fast-moving objects. The MAG274QRF-QD also has better compatibility with consoles, as it can display a 1440p or downscaled 4k signal, so your favorite games look sharper and more detailed. Finally, the MAG274QRF-QD supports HDR, whereas the G273 is limited to SDR.

The MSI Optix MAG274QRF-QD and the Gigabyte AORUS FI27Q-X are both excellent gaming monitors. They each have an IPS panel with a 1440p resolution, but the main difference is that the Gigabyte has a 240Hz refresh rate, and the MSI is 165Hz. They have quick response times, native FreeSync support, and low input lag for gaming. The MSI uses a traditional RGB subpixel layout, whereas the Gigabyte uses BGR, which not all programs may use and could result in blurry text. However, we don"t expect this to be an issue for most people.

The MSI Optix MAG274QRF-QD and the Razer Raptor 27 165Hz are great 1440p monitors with a 165Hz resolution. The MSI displays a wider color gamut in SDR and HDR, but that"s because it over-saturates colors. The MSI also has a much quicker response time at 60Hz, delivering smoother motion for console gamers, and has better ergonomics. The Razer has better reflection handling and gets slightly brighter, so it"s a better choice for well-lit rooms. Even though the MSI has Adaptive Sync technology and the Razer has native FreeSync, both FreeSync and G-SYNC VRR work on either monitor.

The MSI Optix MAG274QRF-QD and the MSI Optix MAG161V are very different monitors that target different uses, so they"re not really directly comparable. The MAG161V is a portable monitor with a 16 inch 1080p screen, designed for productivity on the go. The MAG274QRF-QD is a desktop gaming monitor with a 27 inch 1440p screen, and it delivers a much better experience overall, but it"s not portable.

The ASUS ProArt PA148CTV and the MSI Optix MAG274QRF-QD are very different products with different intended uses, so the results aren"t really comparable. The MSI is a desktop fixed gaming monitor with a high refresh rate and advanced gaming features. The ASUS is a simple portable monitor designed for extra productivity on the go.

The ASUS ROG Strix XG17AHPE and the MSI Optix MAG274QRF-QD are very different monitors, each with strengths and weaknesses. The ASUS is designed for portability, so it has a smaller screen, much worse ergonomics, and slightly worse viewing angles. The MSI is more feature-rich, as it supports HDR and has an optional black frame insertion feature.

The MSI Optix MAG274QRF-QD and the ViewSonic Vg1655 are two different monitors. The MSI is a standard monitor with a 1440p resolution and 165Hz refresh rate. It has more gaming features than the ViewSonic, like VRR support, a very quick response time, and low input lag. Since it"s a regular desktop monitor, it also has a stand with much better ergonomics. The ViewSonic is a portable monitor with a 1080p resolution, but it has much better text clarity with its smaller screen.

msi lcd panel testing free sample

The ASUS ProArt PA148CTV and the MSI Optix MAG274QRF-QD are very dif

msi lcd panel testing free sample

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