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| 导读 | 无论是经典2B3D还是单核苷酸位点组合，指南推荐和有数据验证的主流位点都是5个位点组成的Panel，6个位点的检测panel没有任何指南推荐，且无任何伴随诊断的用药的数据基础。 |
由于微卫星不稳定（MSI）在泛癌种中具有明确且重要的临床检测意义，现已被国内外众多临床指南共识强烈推荐常规检测。临床对于MSI的检测需求越来越明确，是否检测MSI现已不再是问题，如何选择检测位点给患者带来最准确的检测结果才更加需要注意。
人类基因组中已知的微卫星位点数目已有1900万个[1]，而如何在众多位点中选择最具有代表性的微卫星位点，国内外指南共识其实早有定论。纵观国内外指南，明确推荐用于MSI检测的Panel都是由五个位点组成，即美国癌症研究中心（NCI）提出的由两个单核苷酸位点和三个双核苷酸位点组成的2B3DNCIPanel（BAT-26、BAT-25、D5S346、D2S123、D17S250）和由5个单核苷酸位点组成的PromegaPanel。并且，5个位点的判定标准也是一致的：5个位点中，2个及以上的位点不稳定为MSI-H；5个位点中1个位点不稳定为MSI-L，5个位点均稳定为MSS[2]。目前没有任何指南推荐6个及6个以上的单核苷酸位点。
这个NR-27位点在中国人群中并没有公开的研究数据或文献报道来证实其检测性能。而基于国外人群数据显示，NR-27位点的检测敏感性仅6%左右[25]，6个位点组合加入NR-27位点很有可能会降低检测Panel的整体敏感性。并且6个单核苷酸位点的判读标准是不明确的，因为指南中根本没有推荐过任何大于5个位点的检测Panel。这些因素有可能就是6个单核苷酸位点MSI-H检出率相较5个位点的组合更低，更容易造成漏检的原因。
参考文献
1.Bai,W.,etal.,ScreeningofMSIdetectionlociandtheirheterogeneityinEastAsiancolorectalcancerpatients.CancerMed,2019.8(5):p.2157-2166.
2.Network,N.C.C.,NCCNClinicalPracticeGuidelinesinOncology(NCCNGuidelines®)GastricCancerVersion1.20212021.
3.Suraweera,N.,etal.,EvaluationoftumormicrosatelliteinstabilityusingfivequasimonomorphicmononucleotiderepeatsandpentaplexPCR.Gastroenterology,2002.123(6):p.1804-11.
4.Umar,A.,etal.,RevisedBethesdaGuidelinesforhereditarynonpolyposiscolorectalcancer(Lynchsyndrome)andmicrosatelliteinstability.JNatlCancerInst,2004.96(4):p.261-268.
5.Marabelle,A.,etal.,EfficacyofPembrolizumabinPatientsWithNoncolorectalHighMicrosatelliteInstability/MismatchRepair-DeficientCancer:ResultsFromthePhaseIIKEYNOTE-158Study.JClinOncol,2020.38(1):p.1-10.
6.Overman,M.J.,etal.,DurableClinicalBenefitWithNivolumabPlusIpilimumabinDNAMismatchRepair-Deficient/MicrosatelliteInstability-HighMetastaticColorectalCancer.JClinOncol,2018.36(8):p.773-779.
7.Kim,J.H.,etal.,APhaseIIStudyofAvelumabMonotherapyinPatientswithMismatchRepair-Deficient/MicrosatelliteInstability-HighorPOLE-MutatedMetastaticorUnresectableColorectalCancer.CancerResTreat,2020.52(4):p.1135-1144.
8.https://clinicaltrials.gov/ct2/show/NCT03435107?term=NCT03435107&draw=2&rank=1
9.Zhang,C.,etal.,IncidenceanddetectionofhighmicrosatelliteinstabilityincolorectalcancerinaChinesepopulation:ameta-analysis.2020,2020.
10.LIX,LIUQH.StudyonMicrosatelliteInstabilityinColorectalCancerPatientswithFamilialPredisposition.JChinaMedUniv2006;37:410-408.
11.JinHY,LaiRS,DingYJ,XieL,YangBL,LiuF,DingSQ,GeYS.Detectionofmicrosatelliteinstabilityincolorectalcancerbyfluorescencemultiplexpolymerasechainreactionanditsclinicalvalue.ZhonghuaWeiChangWaiKeZaZhi.2007May;10(3):217-20.
12.YangBL,GuYF.Microsatelliteinstabilityinsporadiccolorectalcanceranditsrelationshipwithclinicopathologicalfeatures.WorldChineseJournalofDigestology2007;15:1160-1164.
13.JinP,MengXM,ShengJQ,etal.ClinicopathologicalFeaturesofNon-familialColorectalCancerwithHigh-frequencyMicrosatelliteInstability.ChinMedSciJ2010;25:228-232.
14.MengWJ,WangL,YuYY,etal.SignificanceofmicrosatelliteinstabilityinsporadicstageIIandIIIrectalcancer.ChongqingMedJ2010;39:2420-2426.
15.PengJL,TangT,YeZL,etal.Therelationshipofmicrosatelliteinstabilitystatewithlossofmismatchrepairproteinsandclinicalpathologicalcharacteristicsinsporadiccolorectalcancers.ChinJCancerBiother2015;22:479-483.
16.ZhouLY,WanMZ,LiuYP,etal.AssessmentofMicrosatalliteInstabilityinColorectalCarcinoma:AComparisonBetweenImmunohistochemistryandPCRMethod.CancerResPrevTreat2015;42:1231-1234.
17.YanWY,HuJ,XieL,etal.PredictionofbiologicalbehaviorandprognosisofcolorectalcancerpatientsbytumorMSI/MMRintheChinesepopulation.OncoTargetsTher2016;9:7415-7424.
18.ZhengJM,HuangBX,NieX,etal.TheclinicopathologicalfeaturesandprognosisoftumorMSIinEastAsiancolorectalcancerpatientsusingNCIpanel.FutureOncol2018;14:1355-1364.
19.JiangN.Relationshipbetweenmicrosatelliteinstabilityandclinicopathologicalfeaturesandprognosisinsporadiccolorectalcancer.ActaUniversitatisMedicinalisAnhui2019;54:139-142.
20.HuangYQ,YuanY,GeWT,etal.ComparativefeaturesofcolorectalandgastriccancerswithmicrosatelliteinstabilityinChinesepatients.JZhejiangUniv-SCB2010;11:647-653.
21.LiWQ,LiHN,LiuRQ,etal.ComprehensiveAnalysisoftheRelationshipBetweenRASandRAFMutationsandMSIStatusofColorectalCancerinNortheasternChina.CellPhysiolBiochem2018;50:1496-1509.
22.WangZ,TangXL,WuXQ,etal.MismatchRepairstatusbetweenprimarycolorectaltumorandmetastatictumor,aretrospectiveconsistentstudy.BiosciRep2019;39:BSR20190730.
23.SongYL,WangLL,RanWW,etal.EffectofTumorLocationonClinicopathologicalandMolecularMarkersinColorectalCancerinEasternChinaPatients:AnAnalysisof2,356Cases.FrontGenet2020;11:96.
24.Buhard,O.,etal.,Quasimonomorphicmononucleotiderepeatsforhigh-levelmicrosatelliteinstabilityanalysis.DisMarkers,2004.20(4-5):p.251-7.
25.NouriNojadeh,J.,etal.,Evaluationofmicrosatelliteinstabilityintumorandtumormarginalsamplesofsporadiccolorectalcancerusingmononucleotidemarkers.EXCLIJ,2018.17:p.945-951.
人类基因组中已知的微卫星位点数目已有1900万个[1]，而如何在众多位点中选择最具有代表性的微卫星位点，国内外指南共识其实早有定论。纵观国内外指南，明确推荐用于MSI检测的Panel都是由五个位点组成，即美国癌症研究中心（NCI）提出的由两个单核苷酸位点和三个双核苷酸位点组成的2B3DNCIPanel（BAT-26、BAT-25、D5S346、D2S123、D17S250）和由5个单核苷酸位点组成的PromegaPanel。并且，5个位点的判定标准也是一致的：5个位点中，2个及以上的位点不稳定为MSI-H；5个位点中1个位点不稳定为MSI-L，5个位点均稳定为MSS[2]。目前没有任何指南推荐6个及6个以上的单核苷酸位点。
图1NCCN指南明确推荐5个位点组成的两套Panel以及判定标准
指南共识所推荐的两套位点中，2B3DNCIPanel从被提出以来一直被指南共识所推荐，其推荐地位从未被动摇。而5个单核苷酸位点Panel还是经过了较为复杂的演进过程，最初是法国人类多态性研究中心基于高加索人群数据提出的PentaplexPanel（BAT25、BAT26、NR-21、NR-22、NR-24）[3]，该Panel被RevisedBethesda指南推荐[4]。后来，美国Promega公司在PentaplexPanel上做了进一步改进，将NR-22替换为Mono-27推出了PromegaPanel（BAT-25、BAT-26、NR-21、NR-24、MONO-27）。基于此，NCCN指南推荐2B3DNCIPanel和以PromegaPanel为主的5个单核苷酸位点Panel用于MSI检测，而没有任何指南推荐6个单核苷酸位点。
表1不同检测Panel指南推荐与临床研究应用情况
图2大型临床研究采用5个位点的2B3DNCIPanel或单核苷酸位点
近日，市面上有些声音将某个由6个单核苷酸位点组成的Panel与指南共识推荐和临床研究中使用的PentaplexPanel或PromegaPanel混同，企图混淆视听。可以详细比较一下PentaplexPanel、PromegaPanel和6个单核苷酸的Panel。
表2单核苷酸Panel位点信息
可以看出，6个单核苷酸位点与Pentaplex相比，少了NR-22位点，且多了NR-27和MONO-27位点，同时也比PromegaPanel多一个NR-27位点。而基于国内人群不同检测Panel的MSI-H检出率的META分析结果显示，6个单核苷酸位点Panel的MSI-H整体检出率远低于5个单核苷酸的PromegaPanel，所有组合中，检出率最高的还是2B3DNCIPanel[9]。
表3中国人群不同MSI检测Panel检出率汇总
那么同样是单核苷酸Panel，为什么6个单核苷酸比Promega的检出率低呢？我们发现，6个单核苷酸位点相比Promega位点多出了NR-27位点，这个位点是从哪里来的呢？我们在这一篇发表于DiseaseMarkers（影响因子=3.434）杂志的文献中，发现了NR-27的选择原因。
图3由于NR22位点无法兼容，NR27位点引入PentaplexPanel
在20年前，我们进行MSI检测时，大多是进行的单重PCR，操作是较为复杂的，于是研究者们希望能够开发多重PCR在一个单管中完成多个微卫星位点的检测进而简化流程。但是，在将PentaplexPanel与多重PCR技术结合时，研究团队发现NR-22位点无法与其余四个位点在同一个单管中兼容，因此将NR-27位点引入，替代了Pentaplex的NR-22位点[24]。但是原文中，并没有阐述为什么会选择NR-27，也并没有对NR-27在人群中的敏感性。这个NR-27位点在中国人群中并没有公开的研究数据或文献报道来证实其检测性能。而基于国外人群数据显示，NR-27位点的检测敏感性仅6%左右[25]，6个位点组合加入NR-27位点很有可能会降低检测Panel的整体敏感性。并且6个单核苷酸位点的判读标准是不明确的，因为指南中根本没有推荐过任何大于5个位点的检测Panel。这些因素有可能就是6个单核苷酸位点MSI-H检出率相较5个位点的组合更低，更容易造成漏检的原因。
图4数据提示NR-27位点灵敏度不高
综上，无论是经典2B3D还是单核苷酸位点组合，指南推荐和有数据验证的主流位点都是5个位点组成的Panel，6个位点的检测panel没有任何指南推荐，且无任何伴随诊断的用药的数据基础。
参考文献
1.Bai,W.,etal.,ScreeningofMSIdetectionlociandtheirheterogeneityinEastAsiancolorectalcancerpatients.CancerMed,2019.8(5):p.2157-2166.
2.Network,N.C.C.,NCCNClinicalPracticeGuidelinesinOncology(NCCNGuidelines®)GastricCancerVersion1.20212021.
3.Suraweera,N.,etal.,EvaluationoftumormicrosatelliteinstabilityusingfivequasimonomorphicmononucleotiderepeatsandpentaplexPCR.Gastroenterology,2002.123(6):p.1804-11.
4.Umar,A.,etal.,RevisedBethesdaGuidelinesforhereditarynonpolyposiscolorectalcancer(Lynchsyndrome)andmicrosatelliteinstability.JNatlCancerInst,2004.96(4):p.261-268.
5.Marabelle,A.,etal.,EfficacyofPembrolizumabinPatientsWithNoncolorectalHighMicrosatelliteInstability/MismatchRepair-DeficientCancer:ResultsFromthePhaseIIKEYNOTE-158Study.JClinOncol,2020.38(1):p.1-10.
6.Overman,M.J.,etal.,DurableClinicalBenefitWithNivolumabPlusIpilimumabinDNAMismatchRepair-Deficient/MicrosatelliteInstability-HighMetastaticColorectalCancer.JClinOncol,2018.36(8):p.773-779.
7.Kim,J.H.,etal.,APhaseIIStudyofAvelumabMonotherapyinPatientswithMismatchRepair-Deficient/MicrosatelliteInstability-HighorPOLE-MutatedMetastaticorUnresectableColorectalCancer.CancerResTreat,2020.52(4):p.1135-1144.
8.https://clinicaltrials.gov/ct2/show/NCT03435107?term=NCT03435107&draw=2&rank=1
9.Zhang,C.,etal.,IncidenceanddetectionofhighmicrosatelliteinstabilityincolorectalcancerinaChinesepopulation:ameta-analysis.2020,2020.
10.LIX,LIUQH.StudyonMicrosatelliteInstabilityinColorectalCancerPatientswithFamilialPredisposition.JChinaMedUniv2006;37:410-408.
11.JinHY,LaiRS,DingYJ,XieL,YangBL,LiuF,DingSQ,GeYS.Detectionofmicrosatelliteinstabilityincolorectalcancerbyfluorescencemultiplexpolymerasechainreactionanditsclinicalvalue.ZhonghuaWeiChangWaiKeZaZhi.2007May;10(3):217-20.
12.YangBL,GuYF.Microsatelliteinstabilityinsporadiccolorectalcanceranditsrelationshipwithclinicopathologicalfeatures.WorldChineseJournalofDigestology2007;15:1160-1164.
13.JinP,MengXM,ShengJQ,etal.ClinicopathologicalFeaturesofNon-familialColorectalCancerwithHigh-frequencyMicrosatelliteInstability.ChinMedSciJ2010;25:228-232.
14.MengWJ,WangL,YuYY,etal.SignificanceofmicrosatelliteinstabilityinsporadicstageIIandIIIrectalcancer.ChongqingMedJ2010;39:2420-2426.
15.PengJL,TangT,YeZL,etal.Therelationshipofmicrosatelliteinstabilitystatewithlossofmismatchrepairproteinsandclinicalpathologicalcharacteristicsinsporadiccolorectalcancers.ChinJCancerBiother2015;22:479-483.
16.ZhouLY,WanMZ,LiuYP,etal.AssessmentofMicrosatalliteInstabilityinColorectalCarcinoma:AComparisonBetweenImmunohistochemistryandPCRMethod.CancerResPrevTreat2015;42:1231-1234.
17.YanWY,HuJ,XieL,etal.PredictionofbiologicalbehaviorandprognosisofcolorectalcancerpatientsbytumorMSI/MMRintheChinesepopulation.OncoTargetsTher2016;9:7415-7424.
18.ZhengJM,HuangBX,NieX,etal.TheclinicopathologicalfeaturesandprognosisoftumorMSIinEastAsiancolorectalcancerpatientsusingNCIpanel.FutureOncol2018;14:1355-1364.
19.JiangN.Relationshipbetweenmicrosatelliteinstabilityandclinicopathologicalfeaturesandprognosisinsporadiccolorectalcancer.ActaUniversitatisMedicinalisAnhui2019;54:139-142.
20.HuangYQ,YuanY,GeWT,etal.ComparativefeaturesofcolorectalandgastriccancerswithmicrosatelliteinstabilityinChinesepatients.JZhejiangUniv-SCB2010;11:647-653.
21.LiWQ,LiHN,LiuRQ,etal.ComprehensiveAnalysisoftheRelationshipBetweenRASandRAFMutationsandMSIStatusofColorectalCancerinNortheasternChina.CellPhysiolBiochem2018;50:1496-1509.
22.WangZ,TangXL,WuXQ,etal.MismatchRepairstatusbetweenprimarycolorectaltumorandmetastatictumor,aretrospectiveconsistentstudy.BiosciRep2019;39:BSR20190730.
23.SongYL,WangLL,RanWW,etal.EffectofTumorLocationonClinicopathologicalandMolecularMarkersinColorectalCancerinEasternChinaPatients:AnAnalysisof2,356Cases.FrontGenet2020;11:96.
24.Buhard,O.,etal.,Quasimonomorphicmononucleotiderepeatsforhigh-levelmicrosatelliteinstabilityanalysis.DisMarkers,2004.20(4-5):p.251-7.
25.NouriNojadeh,J.,etal.,Evaluationofmicrosatelliteinstabilityintumorandtumormarginalsamplesofsporadiccolorectalcancerusingmononucleotidemarkers.EXCLIJ,2018.17:p.945-951.
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