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Head-to-head comparative study: evaluating three panels for MSI-PCR testing in patients with colorectal and gastric cancer
  1. Xinhui Fu1,2,
  2. Jinglin Huang1,
  3. Xinjuan Fan1,
  4. Chao Wang1,
  5. Weihao Deng1,
  6. Xiaoli Tan1,
  7. Zhiting Chen1,
  8. Yacheng Cai1,
  9. Lin Hanjie1,
  10. Liang Xu1,
  11. Jiaxin Zou2,3,
  12. Huanmiao Zhan1,
  13. Shuhui Huang1,
  14. Yongzhen Fang2,4,
  15. Yan Huang1
  1. 1 Department of Pathology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
  2. 2 Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
  3. 3 Department of General Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
  4. 4 Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
  1. Correspondence to Dr Yan Huang; huangy27{at}mail.sysu.edu.cn

Abstract

Aims Due to the lack of large clinical cohorts in the Chinese populations with colorectal cancer (CRC) and gastric cancer (GC), there is no consensus among the preferred panel for microsatellite instability (MSI)-PCR testing. This study aims to evaluate a more appropriate panel.

Methods We tested the MSI status of 2572 patients with CRC and GC using the NCI panel and 2 mononucleotide panels (5 and 6 mononucleotide panels). Immunohistochemistry (IHC) was employed to perform mismatch repair protein testing in 1976 samples.

Results We collected 2572 patients with CRC and GC. The National Cancer Institute (NCI) panel failed to detect 13 cases. Of the 2559 cases that received results from all three panels, 2544 showed consistent results. In the remaining 15 cases, 9 showed discrepancies between MSI-H and MSI-L, and 6 showed discrepancies between MSI-L and microsatellite stability (MSS). The misdiagnosis rate of MSI-L was significantly lower in two mononucleotide panels than in the NCI panel (12.5% vs 87.5%, p=0.010) in CRC. In patients with GC, only the NCI panel detected three MSI-L cases, while the results of the two mononucleotide panels were one MSI-H and two MSS. Based on their IHC results, the MSI-L misdiagnosis rate of the NCI panel was 33.3%. Furthermore, compared with two mononucleotide panels, the NCI panel had a much lower rate of all loci instability in CRC (90.8% and 90.3% vs 25.2%) and GC (89.5% and 89.5% vs 12.0%).

Conclusion In Chinese patients with CRC and GC, the five and six mononucleotide panels have advantages for detecting MSI over the NCI panel.

  • COLORECTAL CANCER
  • GASTRIC CANCER
  • Pathology, Molecular
  • MOLECULAR BIOLOGY

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jcp-2023-209089

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Three microsatellite instability (MSI)-PCR detection panels are widely used in clinical laboratories: the NCI panel and the five-mononucleotide and six-mononucleotide panels. Previous studies compared two of these three panels in small cohorts of patient with colorectal cancer (CRC).

    WHAT THIS STUDY ADDS

    • We compared these three MSI-PCR panels in a large cohort of patients with CRC, and also studied this issue in patients with gastric cancer, which has not been reported before.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Our results showed that the mononucleotide panel is more suitable for Chinese patients with CRC and gastric cancer than the NCI panel. Our study may be helpful for optimal MSI-PCR panel selection in clinical laboratories.

    Introduction

    Colorectal cancer (CRC) and gastric cancer (GC) are prevalent malignancies with diverse genetic phenotypes. Determining microsatellite instability (MSI) carries substantial clinical implications1–3 in cancer management, prognostication and the evaluation of hereditary cancer susceptibility.4–9 In 2021, the National Comprehensive Cancer Network (NCCN) recommended MSI testing for all patients with CRC and GC.10 11 The mismatch repair (MMR) system, responsible for monitoring and correcting mutations during DNA replication, can directly lead to MSI when defects in the MMR pathway proteins occur.12–14 Immunohistochemistry (IHC) is the preferred screening method to evaluate the expression of MMR protein,15–17 and MSI-PCR is the gold standard for detecting MSI status and MMR function.18 Various microsatellite panels are available for MSI-PCR, including the widely used NCI panel (BAT25, BAT26, D2S123, D17S250, and D5S346),18 the five-mononucleotide panel (BAT25, BAT26, NR21, NR24 and MONO27),4 19 and the six-mononucleotide panel (BAT25, BAT26, NR21, NR24, NR27 and MONO27).20 In addition to the NCI panel, the five-mononucleotide panel was recommended for MSI detection in the 2021 NCCN guidelines.10 However, Suraweera et al showed that the detection sensitivity and specificity of loci are not consistent in different populations, which indicates that the optimal detection panel may be different in different ethnic groups.21

    In China, CRC has become the third most prevalent cancer and the incidence of GC also ranks third, emphasising the importance of determining the optimal MSI detection panel for diagnosis and treatment.22 23 Several studies have reported MSI-H detection rates ranging from 6.3% to 10.0% using the six-mononucleotide panel,24–26 9.4% to 22.2% by the five-mononucleotide panel,24 27 28 and 9.1%–25.3% using the NCI panel.27 29 30 However, the data are not easily comparable due to the independent nature of these studies and the lack of direct comparisons between all three panels. Recently, two head-to-head studies have suggested that the mononucleotide loci panel is more suitable than the NCI panel for the Chinese population.31 32 However, these studies only had small sample sizes and only compared the NCI panel with one mononucleotide panel. In addition, there are still no relevant studies on the head-to-head comparison of these panels among populations with GC in China.

    Therefore, further large-scale head-to-head studies are necessary to validate these findings. Our study enrolled a large cohort of Chinese patients with CRC and GC. We compared the accuracy, interpretability of MSI results, and DNA sample quality requirements among the NCI, five-mononucleotide, and six-mononucleotide panels. Our study aims to provide valuable insights for clinical detection.

    Materials and methods

    Patients

    This study collected 2572 patients from the Department of Pathology, the Sixth Affiliated Hospital of Sun Yat-sen University. These patients were diagnosed between August 2021 and February 2023, of which 2178 were pathologically confirmed as CRC and 394 were confirmed as GC. These samples were all formalin-fixed paraffin-embedded (FFPE) biopsy or surgical specimens, including 976 biopsy specimens and 1596 surgical specimens.

    Immunohistochemical detection of MMR protein expression

    Paraffin-embedded tumour tissue sections of 4 µm were dried at 65°C for 15 min and then placed in an automatic IHC machine (BenchMark XT, Roche) for MMR protein staining (MLH1: Cat No. MAB-0789, MXB; MSH2: Cat No. IR376, LBP; MSH6: Cat No. ZA-0541, ZSGB-BIO; PMS2: Cat No. ZA-0542, ZSGB-BIO). Qualified pathologists interpreted IHC results according to the College of American Pathologists (Colon and Rectum Biomarker Reporting Template) manual, any positive reaction of MMR proteins in tumour cell nuclei would be considered as intact expression. The expression and loss of tumour cells can be explained only when the internal control cells (such as interstitial nuclei or inflammatory cells) are positive. Otherwise, the experiment needs to be repeated. Loss of expression of any of the four MMR proteins was defined as defective mismatch repair (dMMR) and expression of all four proteins were defined as proficient mismatch repair (pMMR).

    MSI status detection with the NCI, five-mononucleotide and six-mononucleotide panel

    Genomic DNA was extracted from FFPE tumour tissue and normal tissue or peripheral blood using a DNA extraction kit (FFPE: Cat No. IVD 3126, Magen, China, blood: Cat No. D3111-02, Magen, China) according to the manufacturer’s standard protocol. Our laboratory established the five-mononucleotide and six-mononucleotide panels detection system based on the nucleotide loci and primer sequences. The primers and PCR amplification conditions for detection are shown in online supplemental table 1. The NCI panel detections were done with a commercialisation kit (Tongshu BioTech, Shanghai, China). The extracted DNA template was added to the system of three panels for PCR amplification. The amplified products were identified by capillary electrophoresis using ABI 3500DX Genetic Analyzer (Applied Biosystems, Foster City, California, USA) and then analysed with GeneMapper V.4.1 software (Applied Biosystems, Foster City, California, USA). Mononucleotide and dinucleotide loci were defined as unstable if the tumour tissue peak deviated from the normal control by more than two and four base pairs, respectively. According to the number of unstable loci, tumours were diagnosed as MSI-H (≥2), MSI-L (1), and MSS (0).

    Supplemental material

    Results

    Comparison of overall characteristics between three MSI-PCR panels

    A total of 2572 patients diagnosed with CRC and GC were included in this study. The detection results of the three panels in these 2572 cases are shown in figure 1, and the clinicopathological characteristics of the patients are presented in table 1. The NCI panel had a detection failure rate of 0.5% (13/2572), while the two mononucleotide panels provided results in all cases. Therefore, only 2559 cases had the detection results of the 3 panels, comprising 2167 CRCs and 392 GCs.

    Figure 1
    Figure 1

    The flow chart of three MSI panels in testing patients with CRC and GC. CRC, colorectal cancer; GC, gastric cancer; MSI, microsatellite instability; 5-M, five-mononucleotide panel; 6-M, six-mononucleotide panel.

    View this table:
    Table 1

    Clinical characteristics of patients with gastrointestinal tumours

    Of these cases, 2544 showed consistent results across the 3 panels, while 15 displayed inconsistent results between the NCI panel and the 2 mononucleotide panels. Among the 2544 samples with consistent results in the 3 panels, the CRC and GC MSI-H rates were 11.0% (238/2155) and 9.8% (38/389), respectively. Among the 15 inconsistent specimens, 9 showed inconsistencies between MSI-H and MSI-L (8 cases of CRC and 1 case of GC), and 6 showed inconsistencies between MSI-L and MSS (figure 1). The detailed detection results of these 15 cases and their corresponding IHC results are listed in table 2.

    View this table:
    Table 2

    IHC and MSI-PCR results of 15 patients with inconsistent results

    Among the 2559 cases, 1970 cases had IHC results (pMMR n=1728; dMMR n=242). In the 1728 pMMR patients, the results of the 3 panels’ results were 16 MSI-H, 1703 MSS and 9 MSI-L/MSS. In the 242 dMMR patients, more MSI-H patients were detected by 2 mononucleotide panels than the NCI panel (233 vs 226). Using IHC data as ground truth, the specificity of the 3 panels were all 99.07%. The sensitivities of the 2 mononucleotide panels were slightly higher than that of the NCI panel (96.28%, 96.28% vs 93.38%).

    Three panels detection results in patients with CRC

    Among 2167 patients with CRC with results from the 3 panels, the MSI-H rates of the NCI panel and 2 mononucleotide panels were 11.0% (239/2167) and 11.3% (245/2167), respectively. The rate of MSI-L detected by the 2 mononucleotide panels at 0.4% (8/2167) was lower than that of the NCI panel at 0.8% (18/2167). The NCI panel and the 2 mononucleotide panels detected 19 MSI-L specimens. The IHC and MSI-PCR results of these specimens are shown in table 3. Among the eight patients with CRC who displayed inconsistent MSI-L and MSI-H results between the NCI panel and two mononucleotide panels, seven cases were misdiagnosed as MSI-L by the NCI panel, and one case was misdiagnosed as MSI-L by the two mononucleotide panels based on IHC. Furthermore, the MSI-L misdiagnosis rate of the two mononucleotide panels (12.5%, 1/8) was significantly lower than that of the NCI panel (87.5%, 7/8) (p=0.010).

    View this table:
    Table 3

    IHC and MSI-PCR results of 19 patients with CRC with MSI-L detected in at least 1 of 3 panels

    The 238 MSI-H type CRC samples show the instability rates of each locus of the three panels in figure 2A. BAT25 exhibited the highest instability rate among the mononucleotide loci, with 99.6% (237/238) of samples showing instability. D2S123 was the most unstable dinucleotide locus (59.7%, 142/238). The average frequency of mononucleotide loci instability was 97.9%, and the average frequency of dinucleotide loci instability was 54.9%. Furthermore, the instability rates of each mononucleotide locus exceeded 95% (figure 2A). In MSI-H CRC, the frequency of simultaneous instability of all five loci detected by the NCI panel was only 25.2% (60/238) (figure 3A). This rate was much lower than the frequency of simultaneous instability at all 5 loci of 90.8% (216/238) (figure 3B) and at 6 loci of 90.3% (215/238) (figure 3C) detected with the five-mononucleotide and six-mononucleotide panels, respectively.

    Figure 2
    Figure 2

    The frequency of instability at various loci in MSI-H colorectal cancers (A) and MSI-H gastric cancers (B). MSI, microsatellite instability.

    Figure 3
    Figure 3

    Distribution of cases by number of multiple unstable loci among the three panels in MSI-H cases. MSI, microsatellite instability.

    Three panel detection results in patients with GC

    Among 392 patients with GC with results from the three panels, the MSI-H rates of the NCI panel and two single nucleotide panels were 9.7% (38/392) and 9.9% (39/392), respectively. The MSI-L frequencies were 0.0% (0/392) and 0.8% (3/392). Among the three MSI-L samples detected by the NCI panel, one was found to have dMMR based on IHC and MSI-H detected by the two mononucleotide panels. The agreement rate with IHC was 66.7% (2/3) based on the IHC results.

    In the 38 samples of MSI-H type GC, the instability rates of each locus of the three panels are presented in figure 2B. Among the mononucleotide loci, BAT25, NR27 and MONO27 exhibited the highest instability rate, with 100.0% (38/38) of samples showing instability. D2S123 had the highest instability rate (65.8%, 25/38) in the dinucleotide loci. The mean instability frequency of mononucleotide loci was 98.3%, and that of dinucleotide loci was 53.5% (figure 2B). In MSI-H GC, the simultaneous instability rate of the five loci of the NCI panel was even lower, accounting for only 13.2% (figure 3D), while the simultaneous instability rate of all loci of the two mononucleotide panels was similar in CRC and GC (figure 3E,F).

    Challenges in interpreting capillary electrophoresis signals of dinucleotide loci

    Figure 4A shows an example of microsatellite stable results of the D5S346 dinucleotide locus. However, due to the different amplification efficiency of dinucleotide, the peak types of tumour tissue and normal tissue are quite different, which leads to controversy in interpretation among different technicians. Figure 4B shows an example of microsatellite stable results of the D17S250 dinucleotide locus. Due to the heterozygosity of the dinucleotide, the peak types of tumour tissue and normal tissue are also quite different, making it easily misjudged as unstable. Figure 4C shows MSI at the D2S250 dinucleotide locus, which can also be prone to misjudgment due to heterozygosity. In comparison, the amplification efficiency of the single nucleotide locus was high, and the peak map was relatively fixed, which eliminated the need to consider changes in peak patterns caused by the amplification efficiency of tumour tissue and normal tissue. The single nucleotide loci of stable samples generally exhibit a five-finger peak, while the peak type of unstable samples is noticeably changed.

    Figure 4
    Figure 4

    Representative samples of controversially interpreted dinucleotide loci.

    Discussion

    This study compared the performance of the NCI, five-mononucleotide and six-mononucleotide panels in detecting MSI status in the largest cohort of Chinese patients with CRC and GC to date. In our study, the results of the six-mononucleotide panel and the five-mononucleotide panel were identical. If adding the NR27 loci would increase the additional detection cost, using the five-mononucleotide panel would be sufficient. The incidences of MSI-H in the NCI panel and the two mononucleotide panels in patients with CRC were 11.0% and 11.3%, respectively, similar to other studies.24 In patients with GC, the prevalence of MSI-H was 9.7% and 9.9%, respectively. There was no statistically significant difference in the detection rate of MSI-H among the three panels, but the two mononucleotide panels have a slightly higher detection rate than the NCI panel. This suggests that more patients with MSI-H can be identified by mononucleotide panels in patients with CRC and GC. In previous reports on Chinese populations, the detection rate of MSI-H in different panels were controversial.28 32 The first report about comparison of NCI panel and mononucleotide panel in Chinese CRC patients was published in 2018.28 In a non-random cohort of 245 cases of CRC, the NCI panel detected 27 MSI-H cases, while the five-mononucleotide panel only detected 23 MSI-H cases. So the authors indicated that the NCI panel performed better than the five-mononucleotide panel. The sensitivities of the five mononucleotide loci were: 78.6% (BAT-25), 75.0% (BAT-26), 28.5% (MONO-27), while NR-21 and NR-24 loci were not showed.28 These data were much lower than previously reports.19 21 These contradictory conclusions may due to the small sample size and sampling bias. The second report about comparison of the NCI and mononucleotide panel in Chinese patients with CRC was published in 2023.32 In 468 CRC cases, the authors concluded that the 6-mononucleotide panel may be more suitable than the NCI panel for Chinese, which supported our results.

    There are few reports on the detection rate and misdiagnosis rate to MSI-L among the different panels. In this study, we found that the incidence of MSI-L in the NCI panel was higher than that of the mononucleotide panel (0.8% vs 0.4%) in patients with CRC. There is a low rate of misdiagnosis of MSI-H as MSI-L in all three panels, as well as a low rate of diagnosis of MSS as MSI-L in the NCI panel only (table 2). Based on IHC results, the misdiagnosis rate of MSI-L by the NCI panel was significantly higher than that of the two mononucleotide panels (87.5% vs 12.5%, p=0.010). Thus, we recommend using a different panel to confirm MSI-L results, especially for the NCI panel’s result.

    In patients with GC, only the NCI panel detected three MSI-L cases, while the results of the two mononucleotide panels were one MSI-H and two MSS. Based on their IHC results, the MSI-L misdiagnosis rate of the NCI panel was 33.3%. Thus, if MSI-L was detected by the NCI panel in patients with GC, retesting with a mononucleotide panel was strongly recommended.

    In MSI-H patients with CRC, we found that the frequency of simultaneous instability in all loci was much higher in the two mononucleotide panels (90.8% and 90.3%) than in the NCI panel (25.2%), which enabled an easier final diagnosis by the two mononucleotide panels. A similar situation was found in MSI-H patients with GC. In all patients, the average rate of mononucleotide loci instability was above 95%, while the average rate of dinucleotide loci instability was below 55%. The sensitivity of dinucleotide loci in detecting MSI was much lower than that of mononucleotide loci, which was consistent with previous studies,4 19 32 33 and mononucleotide loci were qualitatively easier to interpret.

    The interpretation process of the signal peaks of capillary electrophoresis still depends on manual recognition. However, due to the considerable variations in the amplification efficiency of dinucleotide loci in different specimens, even the signal peak of microsatellite stability loci also presented significant variations. Since the variations caused by the amplification efficiency and MSI are not easy to distinguish, this may lead to different interpretations by different technicians. Moreover, variations in alleles at these dinucleotide loci can make interpretation challenging. Most of the mononucleotide loci (NR-21, BAT-25, BAT-26 and MONO-27) were homozygous in this cohort, and NR-21 is known to show less than 5% heterozygosity in the Chinese population.33 Nevertheless, heterozygotes are very common at the dinucleotide loci.

    Among the 2572 cases in this study, there were 13 instances of detection failure with the NCI panel, possibly due to sample quality issues. However, the mononucleotide panel successfully detected these samples, indicating its compatibility with low-quality samples.

    The two mononucleotide panels have more advantages than the NCI panel in terms of the detection success rate and interpretation process. The results of five-mononucleotide and six-mononucleotide detection were identical, and the Mono27 locus of the six-mononucleotide panel did not increase the detection rate of MSI-H.

    In patients with CRC, MSI-H was rarely misdiagnosed as MSI-L in all three panels, but the misdiagnosis rate of the NCI panel was slightly higher than that of the two mononucleotide panels. In patients with GC, when MSI-L was detected, it was only detected by the NCI panel with the two mononucleotide panels categorising these cases as either MSI-H or MSS, correlating with IHC results. In Chinese patients with CRC and GC, the five and six mononucleotide panels have advantages for detecting MSI than the NCI panel. These findings provide valuable information for testing laboratories in selecting the optimal MSI-PCR testing panel for patients with CRC and GC, particularly in the Chinese population.

    Data availability statement

    All data relevant to the study are included in the article or uploaded as supplementary information.

    Ethics statements

    Patient consent for publication

    Ethics approval

    The studies involving human participants were reviewed and approved by the ethics committee of the Sixth Affiliated Hospital of Sun Yat-sen University (2020ZSLYEC-148). The patients/participants provided their written informed consent to participate in this study.

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    Footnotes

    • Handling editor Deepa Patil.

    • XF and JH contributed equally.

    • Contributors XFu and JH: conceptualisation, methodology, funding acquisition, writing original draft. XFan and CW: methodology, investigation, data curation, writing original draft. WD and XT: investigation, data curation, formal analysis. JZ, ZC and YC: investigation, data curation, software, validation. LH and LX: data curation. HZ, SH and YF: investigation, data curation. YH: responsible for the overall content, writing review and editing. All authors contributed to the article and approved the submitted version.

    • Funding This work was supported by the National Key Research and Development Program of China (2017YFC1308800 to Ping Lan), National Natural Science Foundation of China (81971999 to Xin-hui Fu), Science and Technology Achievements Transformation Project of Sun Yat-sen University (88000-18843232 to Xin-hui Fu), Young Teacher Training Program of Sun Yat-sen University (14YKPY31 to Xin-hui Fu), “985” Project of Sun Yat-sen University (4202037 to Jian-ping Wang), the program of Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004) and National Key Clinical Discipline.

    • Disclaimer The funder did not influence the study design, data collection, analysis, the decision to publish, or manuscript preparation.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.