UOG Journal Club: January 2016 妇产科超声杂志期刊俱乐部：2016年1月 Clinical implementation of routine screening for fetal trisomies in the UK NHS: cell-free DNA test contingent on results from first-trimester combined test 在英国国民健康体系临床实施常规筛查预测胎儿三体: 根据妊娠早期的联合测试结果决定是否进行DNA测试 MM Gil, R Revello, LC Poon, R Akolekar, KH Nicolaides Volume 47, Issue 1, Date: January 2016 (pages 42–52) Journal Club slides prepared by Dr Shireen Meher (UOG Editor for Trainees) 1

Introduction 前言 游离DNA测试的临床实施 MM Gil et al., UOG 2016 2 Screening for trisomy 21 in UK NHS hospitals is currently based on the first-trimester combined test, which has a potential prenatal detection rate of about 90% for trisomy 21 and 95% for trisomies 18 or 13, at a false-positive rate (FPR) of 5%. 21三体筛查在英国公立医院目前是基于早期联合测试,它在产前对21三体的检出率达90%,对18或13三体的检出率达95%，假阳性率为5%。 Cell-free DNA (cfDNA) analysis of maternal blood for detection of trisomies 21, 18 and 13 is superior to other methods of screening, with detection rates of 99%, 96% and 91% respectively, at a FPR of 0.35%. 母体外周血游离DNA(cfDNA)分析对21，18，13三体的检出率优于其他方法的筛选，检出率分别为99%, 96%和91%，假阳性率为0.35%。 cfDNA analysis may be used as a first-line method of screening or contingent on the results of the first-trimester combined test. cfDNA分析可作为一线的筛查方法或视早期联合筛查的结果而定。 As cfDNA is currently an expensive test, one strategy to maximize screening performance at reduced cost is to offer cfDNA testing as contingent screening. cfDNA目前作为一个昂贵的检验,需要一种策略最大化筛查性能并降低成本作为一种有条件的筛查。 2

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 研究目的 To report the feasibility of implementing cfDNA screening contingent on the results of the first-trimester combined test. 阐述有条件的实施cfDNA筛查基于早期妊娠联合筛查的可行性。 To examine factors affecting patient decisions concerning their options for screening and decisions on the management of affected pregnancies. 测试影响病人选择筛查和决定治疗的因素。 To report the prenatal diagnosis of fetal trisomies and outcome of affected pregnancies following contingent screening. 报告胎儿三体综合征的产前诊断和进行有条件筛查后的妊娠结局。 3

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Methods方法 Study design: prospective study between October 2013 and February 2015 研究设计：2013年10月至2015年2月之间的前瞻性研究 Setting: two hospitals in England 背景:两家英格兰的医院 Inclusion criteria: 入选标准 women with singleton pregnancy between 11 and 13 weeks’ gestation 单胎妊娠11至13周的女性 attending hospital for routine care 到医院进行常规产检 Exclusions: 除外： pregnancies ending in termination, miscarriage or stillbirth with no known karyotype 终止妊娠的、流产或死产没有已知核型的 pregnancies lost to follow-up 失访的 Intervention: routine clinical implementation of contingent screening 干预:常规临床实施有条件筛查 All women were offered combined screening, and on the basis of their result, divided into 所有女性都提供联合筛查,在她们结果的基础上,分为 ： High-risk group (risk ≥1 in 100): offered invasive testing, or cfDNA testing or no further testing 高危人群（风险值≥１/１００）：提供侵入性检查,或cfDNA测试或没有进一步检查 Intermediate-risk group (risk between 1 in 101 and 1 in 2500): offered cfDNA testing or no further testing 中度风险组（风险值在１/１０１至１/２５００之间 ）：提供cfDNA测试或没有进一步检查 Low-risk group (risk <1 in 2500): reassured, no further testing offered 低风险组（风险值小于１/２５００）：消除疑虑，不进行更多的检查 4

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 方法 Outcomes:结局 Trisomic status of the pregnancies as determined by prenatal or postnatal karyotyping or by examination of the neonates, and outcome of these pregnancies following screening. 妊娠三体综合征状态由产前或产后染色体核型分析或检查新生儿决定, 这些妊娠结局建立在筛查基础上。 Factors affecting parental decision making in choosing options for testing and management of pregnancies. 影响父母选择测试和妊娠管理的因素 Statistical analysis:统计分析： To compare outcome groups, the Mann–Whitney U-test was used for continuous variables and χ2-test/Fisher’s exact test for categorical variables. 比较结局各组,Mann-WhitneyＵ-检验用于连续变量，对分类变量用χ2-检验 /Fisher’s确切概率法。 Logistic regression using SPSS for significant predictors of opting for CVS in the high-risk group and cfDNA testing in the intermediate-risk group. 选择在高危人群组的绒毛穿刺和中度风险组用cfDNA测试者的重要的预测因子使用SPSS逻辑回归。 Ethics approval obtained获得伦理批准

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results结果 12 134 women offered combined screening, 11 921 accepted (98.2%) 给12 134名妇女提供联合筛查,11 921名接受(98.2%) 229 (1.9%) excluded from analysis due to pregnancy loss with no known karyotype (n=169) or lost to follow-up (n=60) 229人(1.9%)分析报告排除掉，由于妊娠丢失并没有已知的核型(n = 169)或失访(n = 60) Mean maternal age in study population: 31 years研究人群的平均孕龄:31岁 Based on results of the combined screening, the 11 692 pregnancies were classified as 基于联合筛查的结果, 11 692名孕妇被归类为 ： High risk: 460 women (3.9%)　高风险:460名妇女(3.9%) Intermediate risk: 3552 women (30.4%)中间风险:3552名妇女(30.4%) Low risk: 7680 women (65.7%)低风险:7680名妇女(65.7%) Diagnosis of trisomies in the study population of 11 692 pregnancies was 研究11692名孕妇三体综合征如下： Trisomy 21: 47 cases　　　21三体综合征:47例 Trisomy 18: 24 cases　　　18三体综合征:24例 Trisomy 13: 4 cases　　　 13三体综合征:4例 No trisomies: 11 617 pregnancies　　　没有三体综合征的:11617孕妇 6

Results: potential performance of combined screening Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: potential performance of combined screening 结果:联合筛选的潜能 The combined test result was ≥ 1 in 100 (high risk) in 联合测试结果是≥1/ 100(高风险) 87% (41/47) of fetuses with trisomy 21　　87%(41/47)的胎儿为21三体综合征 92% (22/24) of fetuses with trisomy 18　　92%(22/24)的胎儿为18三体综合征 100% (4/4) of fetuses with trisomy 13 　　100%(4/4)的胎儿 为13三体综合征 3.4% (393/11 617) of non-trisomic pregnancies 3.4%(393/11 617)的妊娠不是三体综合征 Five cases of trisomy 21 and two of trisomy 18 were in the intermediate-risk group 5例21三体和2例18三体在中度风险组中 One case of trisomy 21 was in the low-risk group １例21三体在低风险组 The combined test followed by invasive testing for all high-risk women could potentially have detected 87% of trisomy 21 and 93% of trisomies 18 or 13, at a FPR of 3.4%. 对所有高风险的妇女联合筛查后进行侵入性检测可能会检测到87%的21三体和93%的18三体或13三体，阳性预测值为3.4%。 However, in the high-risk group, only 38% of women chose invasive testing. 然而,高危人群中,只有38%的女性选择了侵入性测试。

Results: potential performance of cfDNA testing Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: potential performance of cfDNA testing 结果:cfDNA测试潜能 The cfDNA test was carried out in 3698 pregnancies from high- and intermediate-risk groups. cfDNA测试是在高风险和中度风险组的3698个孕妇中实行。 The test provided a result for 97.3% after first sampling and 98.2% after second sampling 97.3%的第一采样得出测试结果，98.2%在第二次取样得出测试结果 65 pregnancies had no results from cfDNA testing 65例没有cfDNA测试的结果 5/65 had results from invasive testing (couples chose to have both CVS and cfDNA testing) 5/65行侵入性检查得出结果(夫妇选择绒毛穿刺和cfDNA测试) 60/65 had no further tests – all cases had normal outcomes 60/65没有进一步的检查——所有都是正常的结果 The median time interval for results was 8 (range, 4–21) days, with 98.7% being available within 14 days. 结果的平均时间间隔是8天(范围，4-21),98.7%是14天内获得 The cfDNA test was screen positive for cfDNA测试筛查以下情况是肯定的 98% (43/44) of tested fetuses with trisomy 21 (1 false negative) 胎儿测试98%(43/44) 为21三体 (1例假阴性) 88% (21/24) of tested fetuses with trisomy 18 (no test result in 3 cases) 胎儿测试88%(21/24)为18三体(3例没有测试结果) 50% (2/4) of tested fetuses with trisomy 13 (2 false negatives) 胎儿测试50%(2/4)为13三体(2假阴性) 0.25% (9/3564) of non-trisomic pregnancies (FPR) 无三体综合征妊娠风险为0.25%(9/3564)(假阳性率)

Results: performance of contingent screening Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: performance of contingent screening 结果:条件性筛查的性能 Contingent screening led to the prenatal detection of • 条件性筛查导致产前检测 92% (43/47) of fetuses with trisomy 21 92%(43/47)的胎儿为21三体综合征 100% (28/28) of fetuses with trisomies 18 or 13 100%(28/28)的胎儿为18或13三体 Contingent screening did not detect four cases of trisomy 21 条件性筛查未发现4例21三体综合征 1 case in low-risk group – no further tests offered 1例在低风险组中,没有提供进一步的测试 2 cases in high/intermediate-risk groups but parents declined further testing 2例在高/中度风险组但父母拒绝进一步测试 1 false-negative result 1例假阴性结果

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Summary of screening performance总结筛查性能 Karyotype 染色体核型 No. of fetuses 胎儿数 Screen positive by combined screening 联合筛查阳性 Screen positive by cfDNA screening cfDNA筛查阳性 Screen positive by contingent screening 条件性筛查阳性 Trisomy 21 21三体 47 87% (41/47) 98% (43/44) 92% (43/47) Trisomy 18 18三体 24 92% (22/24) 88% (21/24) 100% (24/24) Trisomy 13 13三体 4 100% (4/4) 50% (2/4) No trisomy 无三体综合征 11 617 3.4% (393/11 617) 0.25% (9/3564) 10

Results: parental decision regarding further investigations Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: parental decision regarding further investigations 结果:夫妻双方决定进一步调查 In the high-risk group, 38% opted for CVS, 60% for cfDNA and 2% for no further investigation 在高危人群中,38%选择了绒毛穿刺,60%选择了cfDNA检查，2%没有进一步检查。 In the intermediate-risk group 92% opted for cfDNA and 9% for no further investigation 中度风险组中92%选择了cfDNA检查，9%没有进一步检查

Results: parental decisions in high-risk group Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: parental decisions in high-risk group 结果:高危组的父母决定 With implementation of contingent screening in the high-risk group (n=460) 高危人群中行条件性筛查 (n = 460) 38% opted for CVS 38%选择了绒毛穿刺 60% opted for cfDNA testing 60%选择了cfDNA检查 2% did not want further tests -2%不希望进一步检查 Without contingent screening in the previous year, among the high-risk group (n=407) 在高危人群组中，前一年没有行条件性筛查 (n = 407) 66% had opted for invasive testing 66%的人选择了侵入性检查 34% had no further investigations 34%没有进一步的检查 Contingent screening was associated with a 43% reduction in invasive testing 条件性筛查与降低43%的侵入性检测相关

Results: parental decision in high-risk group Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: parental decision in high-risk group 结果:高危组的父母决定 Significant independent predictors for opting for CVS 选择绒毛穿刺的重要独立预测因子 Increasing risk for trisomies 增加三体综合征风险 Increasing fetal nuchal translucency measurement 增加胎儿NT测量 Significant independent predictors for opting against CVS 不选择绒毛穿刺的重要独立预测因子 Afro-Caribbean racial origin 非洲-加勒比海种族 Attending hospital in which results from combined screening were not given on the same visit as the scan 去医院超声扫查的同时没有给予联合筛查结果

Results: parental decisions in high-risk group Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: parental decisions in high-risk group 结果:高危组的父母决定 41 cases of trisomy 21 in high-risk group 41例高危人群为21三体综合症 27 chose CVS 27例选择绒毛穿刺 13 chose cfDNA test 13例选择cfDNA检查 1 chose no further investigations 1例选择不做进一步检查 13 cases with a positive cfDNA result 13例有确定的cfDNA结果 9 parents had confirmatory CVS 9例父母选择做绒毛穿刺确定 4 parents did not want further testing 4例父母不想做进一步检查 Rate of pregnancy termination in trisomy 21 21三体综合征的终止妊娠率 93% (25/27) in those choosing invasive testing 选择侵入性检查里有93%(25/27) 36% (5/14) in those choosing cfDNA testing or no tests 选择cfDNA检查或没有检查的有 36%(5/14)

Results: parental decision in intermediate-risk group Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: parental decision in intermediate-risk group 结果:中度风险组父母的决定 Significant independent predictors for opting for cfDNA testing 选择cfDNA检查的重要独立预测因子 Increasing risk for trisomies 三体综合征的风险增加 Increasing maternal age 孕妇年龄的增加 Significant independent predictors for opting against cfDNA testing 选择不做cfDNA检查的重要的独立预测因子 Afro-Caribbean racial origin 非洲-加勒比黑人种族起源 Smoking 吸烟 Being parous 经产妇 Attending hospital in which results from combined screening were not given at the same visit as the scan 去医院超声扫查的同时没有给予联合筛查结果 5 cases of trisomy 21 in intermediate-risk group 5例中度风险组的21三体综合征 4 parents chose cfDNA testing 4例父母选择行cfDNA检查 1 chose no further investigations 1例选择不做进一步的检查 3 cases of trisomy 21 with a positive cfDNA result 3例21三体综合征的cfDNA结果为阳性 2 had termination of pregnancy 2例终止妊娠 1 continued pregnancy 1例继续妊娠

Results: pregnancy outcome in trisomies Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Results: pregnancy outcome in trisomies 结果:三体综合征的妊娠结局 Among 43 prenatally detected cases of trisomy 21 在43个产前检测为21三体综合征的病例中 74% (32/43) opted for pregnancy termination 74%(32/43)选择了终止妊娠 26% (11/43) chose to continue with pregnancy 26%(11/43)选择继续妊娠 32% (15/47) of trisomy 21 fetuses were live born 32%(15/47)的21三体综合征胎儿出生 Among 28 prenatally detected cases of trisomy 18 or 13 在28例产前检查到的18或13三体中 82% (23/28) opted for termination of pregnancy 82%(23/28)选择了终止妊娠 18% (5/28) chose to continue with pregnancy (resulting in 3 miscarriages or fetal deaths and 2 neonatal deaths) 18%(5/28)选择继续妊娠 (3例最终流产或胎儿死亡，2例新生儿死亡)

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Discussion讨论 This study demonstrates the feasibility of introducing contingent cfDNA testing for major trisomies in routine clinical practice. 此研究表明,在常规临床实践中，对主要的三体综合征引入条件性cfDNA检查是可行的 。 In the high-risk group, 38% opted for invasive testing, 60% for cfDNA testing and 2% for no further tests. In the intermediate-risk group, 91.5% opted for cfDNA testing and 8.5% for no further tests. 在高危人群,38%选择了侵入性检查,60%选择cfDNA检查，2%没有做进一步的检查。在中度风险组中,91.5%的人选择了cfDNA检查，8.5%没有做进一步的检查。 Introduction of cfDNA testing reduced uptake of invasive testing by an estimated 43%. 引入cfDNA检查估计可以降低43%的侵入性检查。 In the high-risk group, uptake of cfDNA testing was partly at the expense of invasive testing but mainly as a new option in women who would have previously chosen to have no further investigations. 在高危人群组, 做cfDNA检查部分是以侵入性测试为代价的,但主要是在那些之前选择不做进一步的检查的妇女中作为一个新选择。 Choice between CVS and cfDNA was influenced by a number of factors including 在绒毛穿刺和cfDNA检查之间做选择受许多因素的影响 risk of trisomy 三体综合征的风险 racial origin: Afro-Caribbean more averse to invasive testing 种族起源:非洲-加勒比人更反对侵入性检查 parental attitudes about termination of pregnancy: termination was chosen by 93% of trisomy cases in CVS group compared to 36% of those opting for cfDNA or no testing. 关于终止妊娠的父母的态度:在绒毛穿刺组93%的三体综合征病例选择终止妊娠，而行cfDNA检查或没有检查组中36%的人选择终止妊娠

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Discussion讨论 Contingent screening could have potentially detected most trisomies with a lower invasive rate. 条件性筛查可发现潜在的大多数三体综合征，并且侵入性检查率很低。 However the theoretical performance of contingent screening does not equate to rate of prenatal diagnosis and termination because 然而条件性筛选的理论性能并不等同于产前诊断和终止妊娠率,因为 many high-risk women chose invasive testing 许多高风险妇女选择入侵性检测 some declined confirmatory testing 一些拒绝做确认测试 many chose to continue the pregnancy with an affected fetus 许多人选择继续妊娠有问题的胎儿 Based on these findings, health economics analyses may not be entirely valid as they assume that 基于这些发现,卫生经济学分析可能不是完全有效的假设，他们假定： cfDNA will replace the more expensive invasive testing, and cost savings could be utilized to offer cfDNA testing to the intermediate-risk group cfDNA将取代更昂贵的侵入性测试,提供中间风险组做cfDNA检查可以节约成本 improved prenatal detection of trisomies would result in lower rates of affected live births, therefore cost savings from postnatal care 提高产前检测三体综合征将降低有问题的胎儿出生率,因此节约产后护理成本

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Limitations of the study 研究的局限性 The study was not designed for comparison of screening performance between the combined and cfDNA tests as the number of trisomic pregnancies was too small for valid conclusion. 这项研究没有设计联合筛查和cfDNA检查之间的性能比较，三体综合征妊娠的数量太少已致不能做有效的结论。 Rates of uptake of specific screening options may not be generalizable to all populations from different racial and socioeconomic backgrounds in different countries and healthcare systems. 特殊筛查的选择率，可能无法适用于来自不同种族和不同国家的社会经济背景和医疗保健系统的所有人群。

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 讨论:与其他研究进行比较 Study 研究 Type of study 研究类型 No. of pregnancies 妊娠数 Affected cases 异常病例 Traditional screening 传统筛查 cfDNA test cfDNA检查 Nicolaides 2012 比较联合筛查与存储血浆cfDNA检查样本——cfDNA结果非临床使用 1949例单胎，11–13 周 10例 三倍体 100%检测 假阳性率4.5% 100%检测 假阳性率0.1% Bianchi 2014 相比第一/第二阶段筛查与cfDNA检查-cfDNA结果不用于临床 1914例 17周 (范围, 8–39周) 8例 三倍体 100%检测 假阳性率4.2% 100%检测 假阳性率0.5% Norton 2015 15 841 例 10–14 周 38例 21-三体 79%检测 假阳性率5.4% 100%检测 假阳性率0.06% Song 2012 相比孕中期血清学三联筛查，cfDNA应用研究 1741例 16 例(范围, 11–21周) 11例 三倍体 55%检测 假阳性率14.1% Quezada 2015 cfDNA测试与联合筛查相比,cfDNA应用研究 2785例 cfDNA: 10–11 周 联合筛查:11–13 周 32例 21-三体 10例 18-三体 5例 13-三体 100%检测 假阳性率4.4% 21三体为100%,18三体为90%, 13三体为40%, 假阳性率0.3%

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 Conclusion结论 In healthcare systems offering routine screening for trisomy 21 by the first-trimester combined test, incorporating the option of cfDNA testing for some patients is feasible. 在医疗保健系统中，常规在早期妊娠进行21三体综合症的联合筛查,对有的病人选择cfDNA测试是可行的。 Such contingent screening could potentially lead to the prenatal detection of a higher proportion of affected pregnancies and a lower invasive-testing rate than in screening by the combined test alone. 相比单纯使用联合筛查，这样的条件性筛查可能会检出更高比例的不良妊娠和降低侵入性检查率。 In clinical practice, prenatal detection of trisomies and pregnancy outcome depend not only on performance of screening tests but also on parental choice. 在临床实践中,产前检测三倍体和妊娠结局不仅取决于筛查的性能，也取决于父母的选择。 Clinical implementation of cfDNA testing, contingent on results of the combined test, may have only a modest impact in reducing the rate of invasive testing and a small effect on the rate of live births with trisomy 21. 临床进行cfDNA测试,取决于联合测试的结果, 可能在降低侵入性检测率上影响甚微，对21三体活产率的影响很小。

Clinical implementation of cfDNA testing MM Gil et al., UOG 2016 未来的展望 Further studies are needed on economic analyses of contingent screening, taking into account impact of parental choice on uptake of invasive testing and termination rates. 关于条件性筛查，还需要进一步的经济学分析研究,结合考虑父母对选择侵入性检查和终止妊娠的接受度。 讨论关键点 testing for trisomies be introduced into routine clinical practice as contingent screening Should cfDNA or first-line screening? 三倍体的检查作为条件性筛查被引入常规临床实践，cfDNA或一线筛查项目? 22