BAT25, ACVR2, and TGFBR2 mononucleotide STR markers: A triplex panel for microsatellite instability testing in colorectal tumors
Paniz Miar1, Mohammad Amin Tabatabaiefar2, Zeinab Abdollahi1, Mahnaz Noruzi3, Mohammad Kazemi2, Azar Naimi4, Mohammad Hasan Emami5, Shahrokh Izadi6, Mehrdad Zeinalian7
1 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences; Pediatric Inherited Disease Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Biology, Faculty of Sciences, Shahid Chamran University, Ahvaz, Iran
4 Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
5 Department of Internal Medicine, School of Medicine; Poursina Hakim Digestive Diseases Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
6 Department of Biostatistics and Epidemiology, School of Health, Isfahan University of Medical Science, Isfahan, Iran
7 Department of Genetics and Molecular Biology, School of Medicine; Pediatric Inherited Disease Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences; Iranians Cancer Control Charity Institute (MACSA), Isfahan, Iran
|Date of Submission||12-Jul-2021|
|Date of Acceptance||12-Mar-2022|
|Date of Web Publication||27-Sep-2022|
Dr. Mehrdad Zeinalian
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: Microsatellite instability (MSI) in colorectal cancer (CRC) patients is considered as a diagnostic and prognostic marker. MSI is a consequence of mismatch repair deficiency which is evaluated using the different microsatellite markers on the whole genome. In this pilot study, the diagnostic value of a novel triplex panel including three mononucleotide markers has been evaluated in comparison to the standard Promega kit for MSI testing in CRC patients with Amsterdam II criteria.
Materials and Methods: DNA extracted from tumors and normal Formalin-Fixed Paraffin-Embedded (FFPE) tissues of index cases from 37 HNPCC (Hereditary non-polyposis colorectal cancer) families were evaluated for MSI state. Primer design for three markers, including BAT25, ACVR2, and TGFBR2, was performed using 19 nucleotides of the M-13 phage. The instability of each marker was assessed through fragment analysis in comparison with Promega kit markers for all patients. The sensitivity and specificity of each marker have been calculated.
Results: The comparative evaluation of MSI in both tumors and normal adjacent FFPE tissues demonstrated a separate sensitivity as 100%, 83.3%, and 76.9% for BAT25, ACVR2, and TGFBR2, respectively, and 100% sensitivity in the form of a triplex. Moreover, the specificity for each of these three markers in MSI testing was estimated as 100%, separately and in the form of the triplex in comparison with the Promega pentaplex standard Kit.
Conclusions: A high sensitivity and specificity for the novel triplex panel in MSI-testing were estimated among Iranian patients. More studies are recommended to confirm this panel as a diagnostic kit for MSI testing.
Keywords: DNA mismatch repair, Lynch syndrome, microsatellite instability, microsatellite markers
|How to cite this article:|
Miar P, Tabatabaiefar MA, Abdollahi Z, Noruzi M, Kazemi M, Naimi A, Emami MH, Izadi S, Zeinalian M. BAT25, ACVR2, and TGFBR2 mononucleotide STR markers: A triplex panel for microsatellite instability testing in colorectal tumors. Adv Biomed Res 2022;11:79
|How to cite this URL:|
Miar P, Tabatabaiefar MA, Abdollahi Z, Noruzi M, Kazemi M, Naimi A, Emami MH, Izadi S, Zeinalian M. BAT25, ACVR2, and TGFBR2 mononucleotide STR markers: A triplex panel for microsatellite instability testing in colorectal tumors. Adv Biomed Res [serial online] 2022 [cited 2022 Nov 29];11:79. Available from: https://www.advbiores.net/text.asp?2022/11/1/79/356999
| Introduction|| |
Microsatellite instability (MSI) is defined as a hypermutable phenotype with alteration in repetitive DNA sequence length. Microsatellites or short tandem repeat (STR) markers are DNA sequences, including repetitive units with 1 to 6 nucleotides distributed in the eukaryotic genome., MSI, which can be a predictive biomarker, based on National Comprehensive Cancer Network (NCCN) is a consequence of mismatch repair (MMR) deficiency due to germline mutation or epigenetic silencing of at least one of the four MMR genes named MLH1, MSH2, MSH6, and PMS2.,,
Lynch syndrome (LS) is the most common inherited colorectal cancer (CRC) due to a germline mutation in one of the MMR genes. In this autosomal dominant syndrome, there is also a high rate of cancer incidence in the endometrium, stomach, ovary, small bowel, hepatobiliary tract, upper urologic tract, glioblastoma, pancreas, prostate, and breast., MSI feature has been reported in more than 90% of LS tumors and about 15% of sporadic CRCs. MSI tumors have distinct clinicopathological features such as mucinous cells, signet-ring cells, and poorly differentiated cells with relatively good prognoses. There are three clinical criteria for LS diagnosis, including Amsterdam I, Amsterdam II, and Bethesda with 60%, 80%, and 96% sensitivity, respectively, [Table 1].
|Table 1: Amsterdam II criteria for clinical screening of colorectal cancer patients at-risk for Lynch syndrome|
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Different microsatellite markers can be used to detect MSI. In 1997, the National Cancer Institute (NCI) established a five-microsatellite-marker panel for MSI testing containing two dinucleotide and three mononucleotide markers (BAT25, BAT26, D2S123, D5S346, D17S250). After a while, studies showed higher sensitivity and specificity for mononucleotide markers compared with dinucleotide, trinucleotide, tetra-nucleotide, and pentanucleotide markers, significantly. Accordingly, the Promega company established a pentaplex panel including five quasi-monomorphic mononucleotide markers (BAT-25, BAT-26, MON0-27, NR-21, and NR-24) with higher sensitivity and specificity than the NCI panel.,, Three categories have been recommended for the MSI description. MSI-high (MSI-H) is defined when more than 30% of STR markers are unstable, MSI-low (MSI-L) is used when fewer than 30% of STR markers are unstable, and if there are no unstable markers, the tumor is classified as microsatellite-stable (MSS). Studies have demonstrated a better prognosis in MSI-CRCs in comparison to MSS ones. Moreover, the therapeutic response to Fluorouracil-based adjuvant chemotherapy in patients with MSI-CRCs is less than MSS-CRCs, according to some studies.,, Moreover, MSI-H is predominantly associated with long-term immunotherapy-related responses in CRC and non-CRC malignancies treated with immune checkpoint inhibitors.
The BAT25 selection was done according to the different previous studies and Promega standard kit panel. ACVR2 and TGFBR2 mononucleotide markers were also selected from molecular studies on MSI in 2016.,,
Based on the previous studies, the BAT25 mononucleotide marker that locates in the c-kit gene with A(25) repetitive sequence has high sensitivity and specificity for MSI detection in different populations. Furthermore, two other mononucleotide markers, including ACVR2 in Actin receptor type 2 gene, and TGFBR2 in transforming growth factor-beta (TGF-β) receptor type 2 gene with A(8) and A(10) repetitive sequences, have also presented instability in MSI-CRCs.,,
Although the Promega kit is commonly used for MSI testing, the evaluation of other markers in different populations may help increase the accuracy of the MSI detection kit. Moreover, the cost of the Promega kit is high in Iran, and it is inaccessible due to political sanctions. Accordingly, this pilot study was run to evaluate the sensitivity and specificity of a novel triplex panel including three mononucleotide markers for MSI testing in colorectal tumors at-risk for LS among the Iranian population compared to the Promega standard kit. The main question is, what percentage of the diagnostic results of the test with the triplex panel produced in the laboratory of the Department of Genetics, Isfahan University of Medical Sciences, are compatible with the Promega kit?
| Materials and Methods|| |
Patients and specimens
Altogether, 280 CRC patients with a positive family history of cancer were enrolled in this study from Iranians Cancer Control Charity Institute (MACSA), a referral charity-based service provider for cancer patients and their families in central Iran, Isfahan, and Poursina Hakim Gastrointestinal Research Center, Isfahan, Iran. Thirty-seven tumors and their adjacent normal Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens were collected based on Amsterdam II criteria in the patients [Table 1]. The fresh samples or those less than five years old were given priority. Tumor tissues and their adjacent FFPE were separated by a pathologist for each patient. This study was supported by an MSc grant from Isfahan University of Medical Sciences (397055) with IR.MUI.RESEARCH.REC.1397.131 as ethical number (Research ethics certificate has been attached).
Primers and PCR
DNA extraction was done for 37 pairs of FFPE tissue samples from both tumors and their adjacent normal tissues, separately, using the Salehi et al., 2008 protocol. The extracted DNA was used for three mononucleotide markers as a template in the Polymerase Chain Reaction (PCR). Primers were designed using Primer3 (V.0.4.0), and their fluorescent labeling was performed based on Schuelke's method [Table 2].
|Table 2: The information of primers for PCR of ACVR2, TGFBR2, and BAT25 mononucleotide markers|
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The final volume of PCR reagents for each marker was considered 10 μl, containing 5 μl Biofact Master Mix (2×), 0.2 μl forward primer (10 μM), 0.2 μl reverse primer (10 μM), 0.3 μl NED*M13 (10 μM), 3.3 μl dH2O, and 1 μl DNA (50 ng). The Touch-down PCR was run on the extracted DNA and the designed primers according to [Table 3].
Moreover, a multiplex PCR was set up as a three-mononucleotide marker panel according to [Table 4]. The Touch-down PCR condition was performed as mentioned before.
All three mononucleotide markers were labeled with NED; hence to avoid overlapping graphs, we did not pool all PCR products in one tube in the first step. PCR products of ACVR2 and TGFBR2, and BAT25 were analyzed in separate tubes for fragment analysis.
For multiplex PCR, amplified sequences were finally sent for fragment analysis. The amplified fragments were detected by ABI PRISM 3100 Genetic Analyser. Moreover, all extracted DNA from tumors and their adjacent normal FFPE tissue specimens were evaluated by MSI Analysis System kit (Promega), as a gold standard based on the MSI Analysis System protocol, Version 1.2.
The instability of more than one marker is considered as MSI-H, and no instability is defined as MSS. Moreover, if just one marker presents instability, the tumor is categorized as MSI-low. [Figure 1] and [Figure 2].
|Figure 1: An example of Promega kit fragment analysis in an MSI-H patient (above the line: Normal, below the line: Tumoric). MSI-H: Microsatellite instability-high|
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|Figure 2: An example of Promega kit fragment analysis in an MSS patient (above the line: Normal, below the line: Tumoric). MSS: Microsatellite stable|
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The fragment analysis is performed by capillary electrophoresis that can detect different fluorescent colors in a microtube and separate the different fragments in terms of size with precision in pairs. The presence of MSI, which leads to the changes in the length of the duplicate sequence, can be detected by fragment analysis. As mentioned before, fragment analysis was performed by capillary electrophoresis ABI PRISM 3100 to reduce or increase the length of repetitive sequences.
Furthermore, raw files extracted from the device were analyzed by Gene marker software (Version 1.85). The tumor sample of each patient was compared with the normal one, and the changes or non-changes in the number of repetitive sequences of each marker were examined and recorded. The sensitivity and specificity of each marker and triplex panel were calculated.
Furthermore, the Kappa statistics that determine what percentage of the two tests (regardless and after eliminating the possibility of chance agreements) agree are calculated, too. Kappa statistics analysis was performed using STATA software version 14.
| Results|| |
Altogether, 37 CRC cases with Amsterdam II criteria were finally included in this study from 280 CRC patients with a positive family history of cancer. The instability state of three selected markers on tumor DNA was evaluated in comparison with the Promega kit as a gold standard. A marker was considered sensitive when the results of its usage were the same as the gold standard. The MSI testing results obtained from the innovative three-mononucleotide marker panel and MSI Analysis System kit (Promega) have been summarized in [Table 5].
|Table 5: Performance of each three mononucleotide markers separately in comparison to the gold standard (Promega kit)|
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Overall, 10 of 37 (27%) samples were defined as MSI-H, and 27 of 37 (72%) samples were reported as MSS by gold standard Promega kit. Based on sensitivity and specificity calculation, among three innovative markers, BAT25 was also unstable in all 10 MSI-H cases (100% sensitivity). ACVR2 and TGFBR2 markers showed instability with 76.9% and 83.3% sensitivity, respectively. Moreover, in all 27 MSS samples, according to the Promega, the BAT25 marker showed stability (100% specificity). ACVR2 and TGFBR2 were also stable in all 27 MSS cases (100% specificity). Meanwhile, no samples were considered as MSI-L by the MSI Analysis System kit (Promega) [Table 5].
Moreover, these three mononucleotide markers were used as a three-mononucleotide marker panel for MSI detection with multiplex PCR [Figure 3] and [Figure 4]. Considering instability in ≥ one marker as MSI-H, the results were the same as the Promega kit, with no MSI-L sample. Furthermore, the sensitivity and specificity of the triplex panel are calculated with 100% sensitivity and 100% specificity based on [Table 6].
|Figure 3: A multiplex PCR agarose gel for ACVR2, TGFBR2, and BAT25 mononucleotide markers (N: Normal, T: Tumor)|
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|Figure 4: Multiplex PCR Fragment analysis for two patients (1 was MSS and 2 was MSI-H). MSI-H: Microsatellite instability-high, MSS: Microsatellite stable|
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|Table 6: The performance of the innovative triplex panel in comparison to Promega kit, as the gold standard (≥1 instable marker as MSI-H)|
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The Kappa statistics for the triplex panel are calculated (Kappa statistics: 1.00; P value <0.0001). Most experts classify the Kappa statistics above 0.75 as “excellent agreement beyond chance”.
| Discussion|| |
MSI status as a good prognostic and predictive molecular marker, in colorectal tumors could be evaluated using different markers. In 2004, a pentaplex panel was suggested for MSI testing with high accuracy,, which is currently used worldwide., Meanwhile, other different markers with diverse accuracy have been suggested by several studies on different populations., To obtain a more cost-effective panel for the Iranian population, we evaluated three mononucleotide markers separately and optimized a triplex panel for MSI testing.
These three mononucleotide markers were analyzed in DNA extracted from tumors and their adjacent normal FFPE tissues of 37 CRC patients with Amsterdam II criteria. The amplification of markers was performed in both separate PCRs and a multiplex PCR, and the results were compared with the Promega MSI testing kit. Our study indicated 100% sensitivity and specificity for the BAT25 marker. Also, both ACVR2 and TGFBR2 presented 76.9% and 83.3% sensitivity with 100% specificity, respectively.
According to the findings, the innovative triplex panel could detect all MSI-H cases, but when the analysis was according to panel 2, it demonstrated one case as MSI-L that had not been detected with the gold standard. Meanwhile, when we defined tumors for panel 1, the MSI-L case was detected as MSI-H. According to the currently accepted protocols,,, MSI-H status is defined when more than 30% of the markers present instability. Thus, considering one marker as a cut-off for MSI-H in the triplex kit, as the current guidelines, may lead to misclassification of MSI-L tumors to MSI-H, an issue that could be prevented by adding more markers to this panel.
The Promega Kit with five STR markers is used commonly for MSI evaluation, but the high price of the kit and its poor accessibility due to the imposed sanctions have limited its application in Iran. Although reducing the number of markers can reduce the kit price, adding more markers would be more reliable for MSI testing. Meanwhile, these three suggested markers can be included in an innovative MSI kit. As mentioned, the primers design technique in this study is affordable and will reduce the price. In this Primer design technique, no extra fluorescent labels are needed, and M13 sequences can play a critical role in one fluorescent label entry into target sequences for all three markers. All PCR products for these three markers were designed in different sizes and detected by one fluorescent label (NED) easily, and all Fragment analysis graphs in multiplex PCRs were detected without overlapping. This technique will reduce the kit price dramatically and can be used for the MSI evaluation.
In addition, the Promega MSI panel is considered the gold standard for MSI detection. However, the utility of the Promega kit is challenging due to the variable degree of sensitivity and specificity as well as the polymorphic features of its markers. Therefore, current guidelines strongly recommended developing new panels with a relatively small number of highly sensitive mononucleotide repeat markers with monomorphic features across the different populations. Integrating the previously identified MSI marker loci with acceptable sensitivity and specificity is a promising approach to developing informative panels.,, To the best of our knowledge, there is no study to apply BAT25, ACVR2, and TGFBR2 as a triplex panel to evaluate MSI in CRC tumors. Our results of validity measuring were encouraging compared to the gold standard. The accuracy of these three markers is shown both separately and as a triplex panel. Altogether, fast and easy performance (due to the multiplex PCR we developed in our study), acceptable accuracy, and simplified interpretation (due to the relatively small number of stutter) of this triplex panel would introduce it as an informative candidate panel for MSI testing.
| Conclusion|| |
The current study demonstrated high accuracy for the three-mononucleotide marker (BAT25, ACVR2, and TGFBR2) as a triplex panel in MSI testing. Moreover, this study reconfirms the importance of molecular screening of tumor DNA through MSI testing in CRC patients at-risk for LS. Given the results, this triplex panel can be considered in more evaluations with more samples for investigating the feasibility of using it as an alternative diagnostic kit for MSI testing.
We appreciate the patients and their families for permitting us to use their samples. We also appreciate the health workers in the Iranian Cancer Control Charity Institute (MACSA), Isfahan, Iran, and Poursina Hakim Digestive Diseases Research Center, Isfahan, Iran.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]