由于微衛(wèi)星不穩(wěn)定（MSI）在泛癌種中具有明確且重要的臨床檢測意義，現(xiàn)已被國內(nèi)外眾多臨床指南共識強(qiáng)烈推薦常規(guī)檢測。臨床對于MSI的檢測需求越來越明確，是否檢測MSI現(xiàn)已不再是問題，如何選擇檢測位點(diǎn)給患者帶來最準(zhǔn)確的檢測結(jié)果才更加需要注意。

人類基因組中已知的微衛(wèi)星位點(diǎn)數(shù)目已有1900萬個[1]，而如何在眾多位點(diǎn)中選擇最具有代表性的微衛(wèi)星位點(diǎn)，國內(nèi)外指南共識其實早有定論。縱觀國內(nèi)外指南，明確推薦用于MSI檢測的Panel都是由五個位點(diǎn)組成，即美國癌癥研究中心（NCI）提出的由兩個單核苷酸位點(diǎn)和三個雙核苷酸位點(diǎn)組成的2B3D NCI Panel（BAT-26、BAT-25、D5S346、D2S123、D17S250）和由5個單核苷酸位點(diǎn)組成的Promega Panel。并且，5個位點(diǎn)的判定標(biāo)準(zhǔn)也是一致的：5個位點(diǎn)中，2個及以上的位點(diǎn)不穩(wěn)定為MSI-H；5個位點(diǎn)中1個位點(diǎn)不穩(wěn)定為MSI-L，5個位點(diǎn)均穩(wěn)定為MSS[2]。目前沒有任何指南推薦6個及6個以上的單核苷酸位點(diǎn)。

圖 1 NCCN指南明確推薦5個位點(diǎn)組成的兩套Panel以及判定標(biāo)準(zhǔn)

指南共識所推薦的兩套位點(diǎn)中，2B3D NCI Panel從被提出以來一直被指南共識所推薦，其推薦地位從未被動搖。而5個單核苷酸位點(diǎn)Panel還是經(jīng)過了較為復(fù)雜的演進(jìn)過程，最初是法國人類多態(tài)性研究中心基于高加索人群數(shù)據(jù)提出的Pentaplex Panel（BAT25、BAT26、NR-21、NR-22、NR-24）[3]，該P(yáng)anel被Revised Bethesda指南推薦[4]。后來，美國Promega公司在Pentaplex Panel上做了進(jìn)一步改進(jìn)，將NR-22替換為Mono-27推出了Promega Panel（BAT-25、BAT-26、NR-21、NR-24、MONO-27）�；�于此，NCCN指南推薦2B3D NCI Panel和以Promega Panel為主的5個單核苷酸位點(diǎn)Panel用于MSI檢測，而沒有任何指南推薦6個單核苷酸位點(diǎn)。

表 1 不同檢測Panel指南推薦與臨床研究應(yīng)用情況

同時，國內(nèi)外各種PD-1/L1免疫檢查點(diǎn)抑制劑的大型臨床研究入組MSI-H患者的篩選位點(diǎn)都遵循了指南推薦，選擇的2B3D NCI Panel或Promega Panel由5個位點(diǎn)組成的檢測Panel，比如帕博利珠單抗的臨床研究中以上兩種Panel都使用過；納武利尤單抗、阿維魯單抗、德瓦魯單抗的臨床研究則更多使用的是2B3D NCI Panel。而目前公開的數(shù)據(jù)和研究中，沒有任何臨床研究是用的是5個位點(diǎn)以上的MSI檢測Panel。因此，6個位點(diǎn)的檢測Panel并沒有免疫治療伴隨診斷的數(shù)據(jù)支持。

圖 2 大型臨床研究采用5個位點(diǎn)的2B3D NCI Panel或單核苷酸位點(diǎn)

近日，市面上有些聲音將某個由6個單核苷酸位點(diǎn)組成的Panel與指南共識推薦和臨床研究中使用的Pentaplex Panel或Promega Panel混同，企圖混淆視聽�？梢栽敿�(xì)比較一下Pentaplex Panel、Promega Panel 和6個單核苷酸的Panel。

表 2 單核苷酸Panel位點(diǎn)信息

可以看出，6個單核苷酸位點(diǎn)與Pentaplex相比，少了NR-22位點(diǎn)，且多了NR-27和MONO-27位點(diǎn)，同時也比Promega Panel多一個NR-27位點(diǎn)。而基于國內(nèi)人群不同檢測Panel的MSI-H檢出率的META分析結(jié)果顯示，6個單核苷酸位點(diǎn)Panel的MSI-H整體檢出率遠(yuǎn)低于5個單核苷酸的Promega Panel，所有組合中，檢出率最高的還是2B3D NCI Panel[9]。

表 3 中國人群不同MSI檢測Panel檢出率匯總

那么同樣是單核苷酸Panel，為什么6個單核苷酸比Promega的檢出率低呢？我們發(fā)現(xiàn)，6個單核苷酸位點(diǎn)相比Promega位點(diǎn)多出了NR-27位點(diǎn)，這個位點(diǎn)是從哪里來的呢？我們在這一篇發(fā)表于Disease Markers（影響因子=3.434）雜志的文獻(xiàn)中，發(fā)現(xiàn)了NR-27的選擇原因。

圖 3 由于NR22位點(diǎn)無法兼容，NR27位點(diǎn)引入Pentaplex Panel

在20年前，我們進(jìn)行MSI檢測時，大多是進(jìn)行的單重PCR，操作是較為復(fù)雜的，于是研究者們希望能夠開發(fā)多重PCR在一個單管中完成多個微衛(wèi)星位點(diǎn)的檢測進(jìn)而簡化流程。但是，在將Pentaplex Panel與多重PCR技術(shù)結(jié)合時，研究團(tuán)隊發(fā)現(xiàn)NR-22位點(diǎn)無法與其余四個位點(diǎn)在同一個單管中兼容，因此將NR-27位點(diǎn)引入，替代了Pentaplex的NR-22位點(diǎn)[24]。但是原文中，并沒有闡述為什么會選擇NR-27，也并沒有對NR-27在人群中的敏感性。

這個NR-27位點(diǎn)在中國人群中并沒有公開的研究數(shù)據(jù)或文獻(xiàn)報道來證實其檢測性能。而基于國外人群數(shù)據(jù)顯示，NR-27位點(diǎn)的檢測敏感性僅6%左右[25]，6個位點(diǎn)組合加入NR-27位點(diǎn)很有可能會降低檢測Panel的整體敏感性。并且6個單核苷酸位點(diǎn)的判讀標(biāo)準(zhǔn)是不明確的，因為指南中根本沒有推薦過任何大于5個位點(diǎn)的檢測Panel。這些因素有可能就是6個單核苷酸位點(diǎn)MSI-H檢出率相較5個位點(diǎn)的組合更低，更容易造成漏檢的原因。

圖 4 數(shù)據(jù)提示NR-27位點(diǎn)靈敏度不高

綜上，無論是經(jīng)典2B3D還是單核苷酸位點(diǎn)組合，指南推薦和有數(shù)據(jù)驗證的主流位點(diǎn)都是5個位點(diǎn)組成的Panel，6個位點(diǎn)的檢測panel沒有任何指南推薦，且無任何伴隨診斷的用藥的數(shù)據(jù)基礎(chǔ)。

1.Bai, W., et al., Screening of MSI detection loci and their heterogeneity in East Asian colorectal cancer patients. Cancer Med, 2019. 8(5): p. 2157-2166.

2.Network, N.C.C., NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Gastric Cancer Version 1.2021 2021.

3.Suraweera, N., et al., Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology, 2002. 123(6): p. 1804-11.

4.Umar, A., et al., Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst, 2004. 96(4): p. 261-268.

5.Marabelle, A., et al., Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study. J Clin Oncol, 2020. 38(1): p. 1-10.

6.Overman, M.J., et al., Durable Clinical Benefit With Nivolumab Plus Ipilimumab in DNA Mismatch Repair-Deficient/Microsatellite Instability-High Metastatic Colorectal Cancer. J Clin Oncol, 2018. 36(8): p. 773-779.

7.Kim, J.H., et al., A Phase II Study of Avelumab Monotherapy in Patients with Mismatch Repair-Deficient/Microsatellite Instability-High or POLE-Mutated Metastatic or Unresectable Colorectal Cancer. Cancer Res Treat, 2020. 52(4): p. 1135-1144.

8.Clinicaltrials-NCT03435107

9.Zhang, C., et al., Incidence and detection of high microsatellite instability in colorectal cancer in a Chinese population: a meta-analysis. 2020, 2020.

10.LI X, LIU QH. Study on Microsatellite Instability in Colorectal Cancer Patients with Familial Predisposition. J China Med Univ 2006;37:410-408.

11.Jin HY, Lai RS, Ding YJ, Xie L, Yang BL, Liu F, Ding SQ, Ge YS. Detection of microsatellite instability in colorectal cancer by fluorescence multiplex polymerase chain reaction and its clinical value. Zhonghua Wei Chang Wai Ke Za Zhi. 2007 May;10(3):217-20.

12.Yang BL, Gu YF. Microsatellite instability in sporadic colorectal cancer and its relationship with clinicopathological features. World Chinese Journal of Digestology 2007;15:1160-1164.

13.Jin P, Meng XM, Sheng JQ, et al. Clinicopathological Features of Non-familial Colorectal Cancer with High-frequency Microsatellite Instability. Chin Med Sci J 2010;25:228-232.

14.Meng WJ, Wang L, Yu YY, et al. Significance of microsatellite instability in sporadic stage II and III rectal cancer. Chongqing Med J 2010;39:2420-2426.

15.Peng JL, Tang T, Ye ZL, et al. The relationship of microsatellite instability state with loss of mismatch repair proteins and clinical pathological characteristics in sporadic colorectal cancers. Chin J Cancer Biother 2015;22:479-483.

16.Zhou LY, Wan MZ, Liu YP, et al. Assessment of Microsatallite Instability in Colorectal Carcinoma: A Comparison Between Immunohistochemistry and PCR Method. Cancer Res Prev Treat 2015;42:1231-1234.

17.Yan WY, Hu J, Xie L, et al. Prediction of biological behavior and prognosis of colorectal cancer patients by tumor MSI/MMR in the Chinese population. Onco Targets Ther 2016;9:7415-7424.

18.Zheng JM, Huang BX, Nie X, et al. The clinicopathological features and prognosis of tumor MSI in East Asian colorectal cancer patients using NCI panel. Future Oncol 2018;14:1355-1364.

19.Jiang N. Relationship between microsatellite instability and clinicopathological features and prognosis in sporadic colorectal cancer. Acta Universitatis Medicinalis Anhui 2019;54:139-142.

20.Huang YQ, Yuan Y, Ge WT, et al. Comparative features of colorectal and gastric cancers with microsatellite instability in Chinese patients. J Zhejiang Univ-SC B 2010;11:647-653.

21.Li WQ, Li HN, Liu RQ, et al. Comprehensive Analysis of the Relationship Between RAS and RAF Mutations and MSI Status of Colorectal Cancer in Northeastern China. Cell Physiol Biochem 2018;50:1496-1509.

22.Wang Z, Tang XL, Wu XQ, et al. Mismatch Repair status between primary colorectal tumor and metastatic tumor, a retrospective consistent study. Biosci Rep 2019;39:BSR20190730.

23.Song YL, Wang LL, Ran WW, et al. Effect of Tumor Location on Clinicopathological and Molecular Markers in Colorectal Cancer in Eastern China Patients: An Analysis of 2,356 Cases. Front Genet 2020;11:96.

24.Buhard, O., et al., Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis. Dis Markers, 2004. 20(4-5): p. 251-7.

25.Nouri Nojadeh, J., et al., Evaluation of microsatellite instability in tumor and tumor marginal samples of sporadic colorectal cancer using mononucleotide markers. EXCLI J, 2018. 17: p. 945-951.