Noninvasive Prenatal Testing for Fetal Aneuploidies Using Cell-Free Fetal DNA

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1 Subject: Noninvasive Prenatal Testing for Fetal Page: 1 of 20 Last Review Status/Date: March 2015 Noninvasive Prenatal Testing for Fetal Aneuploidies Using Cell-Free Fetal DNA Description National guidelines recommend that all pregnant women be offered screening for fetal chromosomal abnormalities, the majority of which are aneuploidies (an abnormal number of chromosomes). The trisomy syndromes are aneuploidies involving 3 copies of one chromosome. Trisomies 21, 18, and 13 are the most common forms of fetal aneuploidy that survive to birth. There are numerous limitations to standard screening for these disorders using maternal serum and fetal ultrasound. Commercial noninvasive, sequencing-based testing of maternal serum for fetal trisomy 21, 18, and 13 has recently become available and has the potential to substantially alter the current approach to screening. Background Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. Most fetal chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes. The trisomy syndromes are aneuploidies involving 3 copies of 1 chromosome. The most important risk factor for trisomy syndromes is maternal age, with an approximate risk of 1 in 1500 in young women that increases to nearly 1 in 10 by age 48. Trisomy 21 (T21, Down syndrome) is the most common cause of human birth defects and provides the impetus for current maternal serum screening programs. Other trisomy syndromes include Trisomy 18 (T18; Edwards syndrome), and trisomy 13 (T13; Patau syndrome) are the next most common forms of fetal aneuploidy that survive to birth. The prevalence of these other aneuploidies is much lower than the prevalence of trisomy 21 and identifying them is not currently the main intent of prenatal screening programs. Also, the clinical implications of identifying trisomy 18 and 13 are unclear, as most fetuses with trisomy 18 and 13 do not survive to term. Sex chromosome aneuploidies (e.g. 45,X [Turner Syndrome]; 47,XXY, 47,XYY) occur in approximately 1 in 400 live births. These aneuploidies are typically diagnosed postnatally, sometimes not until adulthood, such as during an evaluation of diminished fertility. Alternatively, sex chromosome aneuploidies may be diagnosed incidentally during invasive karyotype testing of pregnant women at high-risk for Down syndrome. The net clinical value of prenatal diagnosis of sex chromosome aneuploidies is unclear. Potential benefits of early identification such as the opportunity for early

2 Subject: Noninvasive Prenatal Testing for Fetal Page: 2 of 20 management of the manifestations of the condition must be balanced against potential harms that can include stigmatization and distortion of a family s view of the child. Current national guidelines recommend that all pregnant women be offered screening for fetal aneuploidy (referring specifically to trisomy 21, 18, and 13) before 20 weeks of gestation, regardless of age. (1) Combinations of maternal serum markers and fetal ultrasound done at various stages of pregnancy are used, but there is not a standardized approach. The detection rate for various combinations of non-invasive testing ranges from 60-96% when the false positive rate is set at 5%. When tests indicate a high risk of a trisomy syndrome, direct karyotyping of fetal tissue obtained by amniocentesis or chorionic villous sampling (CVS) is required to confirm that trisomy 21 or another trisomy is present. Both amniocentesis and CVS are invasive procedures and have an associated risk of miscarriage. A new screening strategy that reduces unnecessary amniocentesis and CVS procedures and increases detection of trisomy 21, 18, and 13 has the potential to improve outcomes. Commercial, non-invasive, sequencing-based testing of maternal serum for fetal trisomy syndromes has recently become available and has the potential to substantially alter the current approach to screening. The test technology involves detection of fetal cell-free DNA fragments present in the plasma of pregnant women. As early as 8 to 10 weeks of gestation, these fetal DNA fragments comprise 6% to 10% or more of the total cell-free DNA in a maternal plasma sample. The tests are unable to provide a result if fetal fraction is too low, that is, below about 4%. Fetal fraction can be affected by maternal and fetal characteristics. For example, fetal fraction was found to be lower at higher maternal weights and higher with increasing fetal crown-rump length. (2) Sequencing-based tests use 1 of 2 general approaches to analyzing cell-free DNA. The first category of tests uses quantitative or counting methods. The most widely used technique to date uses massively parallel shotgun sequencing (MPS; also known as next generation or next-gen sequencing). DNA fragments are amplified by polymerase chain reaction (PCR); during the sequencing process, the amplified fragments are spatially segregated and sequenced simultaneously in a massively parallel fashion. Sequenced fragments can be mapped to the reference human genome in order to obtain numbers of fragment counts per chromosome. The sequencing-derived percent of fragments from the chromosome of interest reflects the chromosomal representation of the maternal and fetal DNA fragments in the original maternal plasma sample. Another technique is direct DNA analysis which analyzes specific cell-free DNA fragments across samples and requires approximately a tenth the number of cell-free DNA fragments as MPS. The digital analysis of selected regions (DANSR ) is an assay that uses direct DNA analysis. The second general approach is single-nucleotide polymorphism (SNP)-based methods. These use targeted amplification and analysis of approximately 20,000 SNPs on selected chromosomes (e.g., 21, 18 and 13) in a single reaction. A statistical algorithm is used to determine the number of each type of chromosome. In order to be clinically useful, the technology must be sensitive enough to detect a slight shift in DNA fragment counts among the small fetal fragment representation of a genome with a trisomic chromosome against a large euploid maternal background. Whether sequencing-based assays require

3 Subject: Noninvasive Prenatal Testing for Fetal Page: 3 of 20 confirmation by invasive procedures and karyotyping depends on assay performance. However, discrepancies between sequencing and invasive test results that may occur for biological reasons could make confirmation by invasive testing necessary at least in some cases, regardless of sequencing test performance characteristics. Regulatory Status None of the commercially available sequencing assays for detection of trisomy 21, 18 and 13 or other chromosomal abnormalities has been submitted to or reviewed by the U.S. Food and Drug Administration (FDA). Clinical laboratories may develop and validate tests in-house (laboratorydeveloped tests or LDTs; previously called home-brew ) and market them as a laboratory service; LDTs must meet the general regulatory standards of the Clinical Laboratory Improvement Act (CLIA). Laboratories offering LDTs must be licensed by CLIA for high-complexity testing. Information on commercially available tests is as follows: In October 2011, Sequenom (San Diego, CA) introduced its MaterniT21 test to test for trisomy 21, 18 and 13. The test is offered through the company s CLIA laboratory, the Sequenom Center for Molecular Medicine. (Uses MPS; reports results as positive or negative). As of October 2014, Sequenom is offering the MaterniT21 PLUS test which tests for trisomies 21, 18 and 13 and also reports fetal sex aneuploidies, trisomies 16 and 22, and selected microdeletions as additional findings. In March 2012, Verinata Health (Redwood, CA) launched its Verifi prenatal test for trisomy 21, 18, and 13. (Uses MPS and calculates a normalized chromosomal value [NPS]; reports results as 1 of 3 categories: No Aneuploidy Detected, Aneuploidy Detected, or Aneuploidy Suspected. In May 2012, Ariosa Diagnostics(San Jose, CA) (formerly Aria) launched its Harmony test for trisomy 21 and 18, which is available from Integrated Genetics, a division of LabCorp. (Uses directed DNA analysis, results reported as risk score) In March 2013, Natera (San Carlos, CA) introduced its Panorama prenatal test for detecting trisomy 21, 18 and 13 as well as for detecting select sex chromosome abnormalities. The test is available at ARUP Laboratories. (Uses SNP technology; results reported as risk score). Related Policies First-Trimester Detection of Down Syndrome Using Fetal Ultrasound Markers Combined with Maternal Serum Assessment Policy *This policy statement applies to clinical review performed for pre-service (Prior Approval, Precertification, Advanced Benefit Determination, etc.) and/or post-service claims. Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 may be considered medically necessary in women with high-risk singleton pregnancies (see Policy Guidelines) undergoing screening for trisomy 21. (Karyotyping would be necessary to exclude the possibility of a false positive

4 Subject: Noninvasive Prenatal Testing for Fetal Page: 4 of 20 nucleic acid sequencing based test. Before testing, women should be counseled about the risk of a false positive test. See Policy Guidelines.) Concurrent nucleic acid sequencing-based testing of maternal plasma for trisomy 13 and/or 18 may be considered medically necessary in women who are eligible for and are undergoing nucleic acid sequencing-based testing of maternal plasma for trisomy 21. Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 is considered not medically necessary in women with average-risk singleton pregnancies. Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 is considered investigational in women with twin or multiple pregnancies. Nucleic acid sequencing-based testing of maternal plasma for trisomy 13 and/or 18, other than in the situations specified above, is considered investigational. Nucleic acid sequencing-based testing of maternal plasma for fetal sex chromosome aneuploidies is considered investigational. Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 in women who do not meet the above criteria is considered investigational. Policy Guidelines High-risk singleton pregnancies, as defined by the American College of Obstetricians and Gynecologists (ACOG) Committee Opinion, Number 454, December 2012 include women who meet at least one of the following criteria: Maternal age 35 years or older at delivery; Fetal ultrasonographic findings indicating increased risk of aneuploidy; History of previous pregnancy with a trisomy; Standard serum screening test positive for aneuploidy; or Parental balanced robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21. Studies published to date report rare but occasional false positives. In these studies, the actual false positive test results were not always borderline; some were clearly above the assay cutoff value, and no processing or biological explanations for the false positive results were reported. In the decision model conducted for the 2012 TEC Assessment, using an overall estimate for predictive value calculations, even in a high risk population, the predictive value of a positive result was only 83%. Thus, in the absence of substantial data to confidently characterize the false positive rate, a karyotyping test would be necessary to confirm a positive result.

5 Subject: Noninvasive Prenatal Testing for Fetal Page: 5 of 20 In some cases, tissue samples from CVS or amniocentesis may be insufficient for karyotyping; confirmation by specific fluorescent in situ hybridization (FISH) assay is acceptable for these samples. Rationale Literature Review The policy is informed by 2 TEC Assessments. A 2013 TEC Assessment focused on detection of trisomy 21(3) and a 2014 TEC Assessment addressed detection of fetal aneuploidies other than trisomy 21, specifically trisomies 13 and 18, and fetal sex chromosome aneuploidies (4). Additionally, the policy limits its scope to the evaluation of tests that are available in the United States. Assessment of a diagnostic technology such as maternal plasma DNA sequencing tests typically focuses on 3 parameters: (1) analytic validity; (2) clinical validity (i.e., sensitivity and specificity) in appropriate populations of patients; and (3) demonstration that the diagnostic information can be used to improve patient health outcomes (clinical utility). The evidence on these 3 questions is described next. What is the analytic validity of the available maternal plasma DNA sequencing-based tests? No studies were identified that provided direct evidence on analytic validity. Each of the commercially available tests uses massively parallel sequencing (MPS; also called next generation sequencing), a relatively new technology but not an entirely new concept for the clinical laboratory. Currently, there are no recognized standards for conducting clinical sequencing by MPS. On June 23, 2011, the U.S. Food and Drug Administration (FDA) held an exploratory, public meeting on the topic of MPS, in preparation for an eventual goal of developing a transparent evidence-based regulatory pathway for evaluating medical devices/products based on NGS [next generation sequencing] that would assure safety and effectiveness of devices marketed for clinical diagnostics. (5) The discussion pointed out the differences among manufacturers sequencing platforms and the diversity of applications, making it difficult to generate specific regulatory phases and metrics. It was suggested that the process may need to be judged by the accuracy and fidelity of the final result. A consistent discussion trend was that validation be application-specific. Thus, technical performance may need to be more closed linked to intended use and population and may not be generalizable across all sequencing applications. Each of the companies currently offering a maternal plasma DNA sequencing test for fetal trisomy 21 has developed a specific procedure for its private, CLIA-licensed laboratory where all testing takes place. Section Summary: Although all currently available commercially available tests use MPS, actual performance and interpretive procedures vary considerably. Clinical sequencing in general is not standardized or regulated by the FDA or other regulatory agencies, and neither the routine quality control procedures used for each of these tests, nor the analytic performance metrics have been published.

6 Subject: Noninvasive Prenatal Testing for Fetal Page: 6 of 20 What is the clinical validity of the available maternal plasma DNA sequencing-based tests for fetal aneuploidies compared to the gold standard of karyotype analysis? High-risk pregnancies A 2014 meta-analysis by Gil et al searched for studies published through December 2013 on the diagnostic performance of sequencing-based tests in identifying T21, T18 and T13. (6 ) A total of 18 studies on T21 were identified; the studies included a combined total of 809 T21 and 12,272 non-t21 pregnancies. Sixteen of the 18 studies were conducted in high-risk singleton pregnancies, so these data are considered in the section on high-risk pregnancies. The pooled weighted detection rate for T21 was 99.0% (95% confidence interval [CI], 98.2 to 99.6) and the pooled weighted false positive rate was 0.08% (95% CI, 0.03 to 0.14). Fifteen studies reported on T18; a total of 301 T18 and 11,646 non-t18 singleton pregnancies were included. The pooled weighted detection rate for T18 was 96.8% (95% CI: 94.5 to 98.4) and the pooled false positive rate was 0.15% (95% CI: 0.08 to 0.25). Finally, 11 studies reported on the performance of the tests in identifying T13. The studies included a total of 85 T13 and 8,339 non-t13 singleton pregnancies. The pooled weighted detection rate and false positive rate were 92.1% (95% CI: 85.9 to 96.7%) and 0.2% (95% CI: 0.04 to 0.46). This systematic review was not limited to tests available in the United States. The 2014 TEC Assessment included a meta-analysis of sequencing-based studies on published through April 15, 2014 that reported on T18, T13 and/or sex chromosome anomalies. (4) Analyses were conducted on the overall population, and, for T18 and T13, separately for the studies on high-risk and low-risk pregnancies. Findings in the high-risk pregnancy population are presented in Table 1. Table 1: Findings from 2014 TEC Assessment Meta-Analysis Overall and in High-Risk Pregnancies (T13 and T18) Aneuploidy No Studies Pooled Sensitivity Pooled Specificity T13 Overall 18 84% (95% CI: 71 to 92%) (115 cases, 8 false-negatives) 99% (95% CI: 99 to 100%) (22 false-positives) High risk 15 84% (95% CI: 71 to 92%) (110 cases, 8 false-negatives) 99% (95% CI: 99 to 99%) (21 false-positives) T18 Overall 15 Sens 22 Spec 95% (95% CI, 90 to 97%) (392 cases, 21 false-positives) 100% (95% CI, 99 to 100%) (22 false-positives) High risk 17 95% (95% CI, 90 to 97%) (344 cases, 10 false-negatives) 100% (95% CI, 99 to 100%) (17 false-positives) For sex chromosome anomalies, the largest number of studies (n=14, total of 137 cases) addressed detection of monosomy X. Pooled sensitivity for detecting monosomy X was 86% (95% CI: 79 to 92%) and pooled specificity was 100% (95% CI: 99 to 100%). In addition, 5 studies with a total of 37 cases were identified on the performance of sequencing-based tests in identifying other sex chromosome anomalies. Pooled sensitivity was 91% (95% CI: 77 to 97%) and pooled specificity was 100% (99 to

7 Subject: Noninvasive Prenatal Testing for Fetal Page: 7 of %). The meta-analysis of studies on sex chromosome aneuploidies did not differentiate between high- and low-risk populations. Key studies evaluating sequencing-based tests for detecting T21 (and, when available T18 and T13) in high-risk singleton pregnancies are summarized in Table 1 in the Appendix. (7-16) Sensitivity and specificity of the tests, as shown in Table 1, were uniformly high. Sensitivity ranged from 99.1% to 100%, and specificity from 99.7% to 100%. Studies are available from all 4 companies currently marketing tests in the United States. Most were prospective, and most were industry-funded. Studies generally included women at a wide range of gestational ages (e.g., 8-36 weeks or weeks) spanning first and second trimesters. The approach to analysis varied. Some studies analyzed samples from all enrolled women and others analyzed samples from all women with pregnancies known to have a trisomy syndrome and selected controls (i.e., nested case-control analysis within a cohort). The studies evaluated the results of maternal fetal DNA testing in comparison with the criterion standards of karyotyping or, in individual cases when a sample did not allow karyotyping, fluorescence in situ hybridization for specific trisomies. Section Summary Data from the available published studies consistently reported a very high sensitivity and specificity of maternal plasma DNA sequencing-based tests for detecting trisomy 21 in high-risk women with singleton pregnancies. There are fewer data on the diagnostic performance of sequencing-based tests for detecting T13, T18 and sex chromosome aneuploidies. The available data suggest that diagnostic performance for detecting these other fetal aneuploidies is not as high as it is for detection of T21. Average-risk pregnancies Fewer studies have been published on maternal plasma DNA sequencing-based tests for detection of T21 in average-risk women. The available studies are described next: One study, published in 2014 by Bianchi et al, evaluated the Illumina test (Verifi) in a general population sample. (17) The study enrolled 2052 women with singleton pregnancies at least 8 weeks gestation. Another eligibility criterion was a completed or planned standard prenatal serum screening during the first and/or second trimester. The blood sample for sequencing-based testing was not required to be taken at the same time as standard screening, so women beyond the second trimester remained eligible for study participation. A total of 40% of the sample were in their first trimester, 32% in the second trimester and 28% in the third trimester. The reference standard was newborn physical examination in 97% of cases and karyotype analysis in the remaining 3% of cases. Screening was incomplete for 39 patients, and 10 others did not have an adequate blood sample. A total of 1914 patients remained, although numbers varied somewhat in the different analyses. The primary study outcome was the false positive rate of sequencing-based testing compared with standard prenatal screening; this analysis excluded all cases of true aneuploidy. For the detection of T21, there were 6 of 1909 (0.3%) false positives with sequencing-based testing and 69 of 1909 (3.6%) false positive with standard testing. The difference between groups was statistically significant, favoring sequencing-based testing. The relative sensitivity of the tests was a secondary outcome. There were 5

8 Subject: Noninvasive Prenatal Testing for Fetal Page: 8 of 20 cases of T21; both techniques correctly identified all of these cases. A limitation of the study was the small number of T21 cases included in the analysis. Moreover, most patients were in the second or third trimester of pregnancy when blood was drawn and had a higher fetal fraction of DNA than samples drawn earlier in pregnancy at the time that the test would most likely be used in practice. Two studies have evaluated sequencing-based tests available in the U.S. for detecting trisomy 21 in average-risk singleton pregnancies. The studies were conducted by the same research group in the U.K. and both used the Ariosa (Harmony) test, which provides risk-scores rather than a positive versus negative result. The first study, by Nicolaides and colleagues did a preliminary analysis of the accuracy of cell-free DNA testing in a general population sample. (18) The authors evaluated archived samples from 2,049 women attending their routine first pregnancy visit at weeks gestation. Karyotyping results were available for only a small percentage of women in the study; for the rest of the enrollees, ploidy was imputed by phenotype at birth obtained from medical records. This study was judged to have a high risk of bias due to a high number of exclusions from analysis. Twenty-eight pregnancies ending in stillbirth or miscarriage were excluded for lack of karyotype; while unavoidable, these exclusions likely affect the case detection rate. Cases were verified primarily by phenotype at birth from medical records. Resultswere available for 1949 of 2049 cases (95%). In the remaining 5%, either the fetal fraction was too low or the assay failed. Overall, using the risk cutoff for the Harmony test, the trisomy detection rate was 100% (i.e., 10 of 10 cases identified) and there was a false-positive rate of 0.1%. The risk score was over 99% in all of the 8 cases of trisomy and both cases of trisomy 18. In the 1939 known or presumed euploid cases, risk scores for trisomy 21 and trisomy 18 were <,01% in 1939 (99.9%). Next, the investigators conducted a 2-part prospective study that evaluated a testing strategy consisting of analysis of serum markers (i.e., pregnancy-associated plasma protein-a [PAPP-A] and free betahuman chorionic gonadotropin) and cell-free DNA at 10 weeks and ultrasound markers (i.e., nuchal translucency and presence or absence of fetal nasal bone) at 12 weeks. In the first part of the study, Gill and colleagues prospectively studied 1005 pregnant women. (19) They evaluated a testing strategy that included analysis of serum markers (i.e., pregnancy-associated plasma protein-a [PAPP-A] and free beta-human chorionic gonadotropin [b-hcg]) and cell-free DNA at 10 weeks and ultrasound markers (i.e., nuchal translucency and presence or absence of fetal nasal bone) at 12 weeks. Parents were counseled primarily on the finding of the Harmony test if it indicated either a high- or low-risk of trisomy. If no results were available on the Harmony tests, parents were counseled based on combined first-trimester serum marker and ultrasound findings. Risk scores from cell-free DNA testing were available for 984 cases (98%), 27 of these required a second round of sampling. Risk scores were >99% for trisomy 21 in 11 cases and for trisomy 18 in 5 cases. In 1 case, the risk score for trisomy 13 was 34%. Sixteen of the 17 women with a high-risk score for aneuploidy underwent CVS and the suspected abnormality was confirmed in 15 of the 16 cases. There was 1 case with a high risk-score for trisomy 21 and a negative CVS; at the time the article was written, the woman was still pregnant so the presence or absence of trisomy 21 could not be confirmed. In part 2 of the study, published in 2014 by Quezada et al., results of the combined test were used to estimate risk of each trisomy for all patients. (20) A total of 2,905 women were included in this second analysis (it is not clear whether there is overlap between patients included here and in the 2013 study by Gil et al). According to the reference standard (i.e. fetal karyotyping or clinical examination of

9 Subject: Noninvasive Prenatal Testing for Fetal Page: 9 of 20 neonates, there were 34 cases of T21, 10 of T18 and 5 of T13. Cell-free DNA identified 32 of 34 (94%) cases of T21 and all cases of T18 and T13 as high-risk. Combined testing with maternal serum markers and fetal ultrasound markers identified all cases of T21, T18 and T13. Out of 2,787 non-trisomic cases, cell-free DNA correctly identified 2,730 (97.95%) as low-risk and combined testing identified 2,663 (95.55%) as low-risk. With cell-free DNA, 8 non-trisomic cases were considered high-risk and there was no result for 49. Combined testing incorrectly identified 124 non-trisomic cases as high-risk. The 2014 TEC Assessment, discussed previously, included a meta-analysis of studies on the diagnostic performance of sequencing-based tests for detecting T13 and T18 in average-risk women. (4) Findings in the high-risk pregnancy population are presented in Table 2. Table 2: Findings from 2014 TEC Assessment Meta-Analysis in Average-Risk Pregnancies (T13 and T18) Aneuploidy No Studies Pooled Sensitivity Pooled Specificity T13 Average risk 2 100% (95% CI: 0 to 100%) (2 cases, 0 false-negatives) T18 Average risk 5 100% (95% CI, 99 to 100%) (68 cases, 0 false-negatives) 99% (95% CI: 99 to 100%) (2 false-positives) 100% (95% CI, 100 to 100%) (5 false-positives) The number of studies and number of cases of T18 and T13 in the average-risk population were low. Findings of the meta-analysis on sensitivity of the assays for detecting T13 was very imprecise, as evidenced by the wide confidence interval (0 to 100%). Section Summary: There are fewer data on the diagnostic accuracy of cell-free DNA testing of women with average-risk singleton pregnancies. A 2014 prospective study reported that performance characteristics in average-risk women were similar to those in high-risk women and that there was a lower false positive rate with cell-free DNA testing compared with standard screening. The studies conducted in average-risk women identified a small number of trisomies (e.g., 5 cases of T21 in the 2014 study) and did not confirm negative or positive findings in all cases. Thus, the evidence on accuracy of sequencing-based tests is less definitive for women with average-risk pregnancies as it is for women with high-risk pregnancies. Twin and multiple pregnancies Detection of trisomy 21 in twin pregnancies was systematically evaluated in only one study, published in 2012 by Canick and colleagues; the study used the Sequenom test. (21) All 7 cases of twin pregnancies with Down syndrome were correctly classified. Five of these were discordant, where one twin had T21 aneuploidy and the other did not; 2 were concordant where both twins had T21 aneuploidy. There is a lack of data on detection of T18, T13 and sex chromosome aneuploidies in twin and multiple pregnancies. Section Summary; For women with multiple pregnancies, there is insufficient evidence to draw conclusions about the diagnostic accuracy of these tests for detecting fetal aneuploidies.

10 Subject: Noninvasive Prenatal Testing for Fetal Page: 10 of 20 What is the clinical utility of the available maternal plasma DNA sequencing-based tests for aneuploidy? No comparative studies were evaluated that compared health outcomes in patients managed using the maternal plasma DNA tests compared to standard screening tests. The 2013 and 2014 TEC Assessments each constructed decision models to predict health outcomes of sequencing-based testing compared with standard testing. The model in the 2013 TEC assessment focused on T21. In this model, the primary health outcomes of interest included the number of cases of aneuploidy correctly identified, the number of cases missed, the number of invasive procedures potentially avoided (i.e., with a more sensitive test), and the number of miscarriages potentially avoided as a result of fewer invasive procedures. The results were calculated for a high-risk population of women age 35 years or older (estimated antenatal prevalence of T21: 0.95%), and an average risk population including women of all ages electing an initial screen (estimated antenatal prevalence of T21: 0.25%). For women testing positive on initial screen and offered an invasive, confirmatory procedure, it was assumed that 60% would accept amniocentesis or CVS. Sensitivities and specificities for both standard and sequencing-based screening tests were varied to represent the range of possible values; estimates were taken from published studies whenever possible. According to the model results, sequencing-based testing improved outcomes for both high-risk and average risk women. As an example, assuming there are 4.25 million births in the United States per year and two-thirds of the population of average risk pregnant women (2.8 million) accepted screening, the following outcomes would occur for the 3 screening strategies under consideration: Standard screening. Of the 2.8 million screened with the stepwise sequential screen, 87,780 would have an invasive procedure (assuming 60% uptake after a positive screening test and a recommendation for confirmation), 448 would have a miscarriage, and 3,976 of 4,200 (94.7%) trisomy 21/Down syndrome cases would be detected. Sequencing as an alternative to standard screening. If sequencing-based testing were used instead of standard screening, the number of invasive procedures would be reduced to 7,504 and the number of miscarriages reduced to 28, while the cases of Down syndrome detected would increase to 4,144 of 4,200 (97.6%of total), using conservative estimates. Sequencing following standard screening. Another testing strategy would be to add sequencingbased testing only after a positive standard screen. In this scenario, invasive procedures would be further decreased to 4,116, miscarriages would remain at 28, but fewer Down syndrome cases would be detected (3,948 of 4,200, 94.0% of total).. Thus, while this strategy has the lowest rate of miscarriages and invasive procedures, it detects fewer cases than sequencingbased testing alone. The model in the 2014 TEC Assessment included T13 and T18 (but not sex chromosome aneuploidies, due to the difficulty of defining relevant health outcomes). The model was similar but not identical to the one previously used to evaluate T21. As in earlier model, outcomes of interest included the number of cases of aneuploidy correctly detected and the number of cases missed, and findings were calculated

11 Subject: Noninvasive Prenatal Testing for Fetal Page: 11 of 20 separately for a high-risk population of women aged 35 or older and a low-risk population. The model assumed that 75% of high-risk and 50% of low-risk women who tested positive on the initial screen would proceed to an invasive test. (The T21 model assumed a 60% uptake rate of invasive confirmatory testing). A distinctive feature of the 2014 modelling study was that it assumed that screening for T21 was done concurrently to screening for T13 and T18, and that women who choose invasive testing do so because of a desire to detect T21. Consequently, miscarriages associated with invasive testing were not considered an adverse effect of T13 or T18 screening. The model compared 2 approaches to screening: 1) a positive sequencing-based screen followed by diagnostic invasive testing; and 2) a positive standard noninvasive screen followed by diagnostic invasive testing. As in the T21 modelling study, sensitivities and specificities for both standard and sequencing-based screening tests were varied to represent the range of possible values; estimates were taken from published studies whenever possible. Assuming that a hypothetical population of 100,000 pregnant women was screened, the model had the following findings: High-risk women: Assuming 75% uptake after a positive screen, the maximum cases detectable in the hypothetical population of 100,000 pregnancies is 127 T18 cases and 45 T13 cases. Standard noninvasive screening would identify 123 of the 127 T18 cases and sequencing-based screening would identify 121 of 127 cases. In addition, standard noninvasive screening would identify 37 of 45 T13 cases and sequencing-based screening would identify 39 of 45 T13 cases. Low-risk women: Assuming 50% uptake after a positive screen, the maximum cases detectable in the hypothetical population of 100,000 pregnancies is 20 T18 cases and 6 T13 cases. Each initial screening test would identify 19 of the 20 T18 cases and 5 of the 6 T13 cases. Results of the modelling suggest that sequencing-based tests detect a similar number of T13 and T18 cases and miss fewer cases compared to standard noninvasive screening. Even in a hypothetical population of 100,000 women, however, the potential number of detectable cases is low, especially for T13 and for low-risk women. In addition to the TEC Assessments, several other published articles presented decision models in published articles and these are described next. Garfield and Armstrong published a study modeling use of the Verinata test. (22) In the model, women were eligible for screening following a positive first-trimester or second-trimester screening test or following a second-trimester ultrasound. The model assumed that 71% of women at average risk and 80% of women at high risk would choose the test. In a theoretical population of 100,000 pregnancies, the detection rate of T21 increased from 148 with standard testing to 170 with Verifi testing and detection of T18 increased from 44 to 45. In addition, the number of miscarriages associated with invasive testing (assumed to be 0.5% for amniocentesis and 1% with CVS) was reduced from 60 to 20. In 2012, Palomaki et al modeled use of the Sequenom sequencing-based test offered to women after a positive screening test, with invasive testing offered only in the case of a positive sequencing-based test.(7) The model included cases positive for T21 or T18 (but not T13 due to its lower prevalence). As in the TEC Assessment, they assumed 4.25 million births in the U.S. per year, with two-thirds of these receiving standard screening. The model assumed a 99% detection rate, 0.5% false positive rate, and

12 Subject: Noninvasive Prenatal Testing for Fetal Page: 12 of % failure rate for sequencing-based testing. Compared to the highest performing standard screening test, the addition of sequencing-based screening would increase the Down syndrome detection rate from 4,450 to 4,702 and decrease the number of miscarriages associated with invasive testing from 350 to 34. In 2013, Ohno and Caughey published a decision model comparing use of sequencing-based tests in high-risk women with confirmatory testing (i.e. as a screening test) and without confirmatory testing (i.e., as a diagnostic test). (23) Results of the model concluded that using sequencing-based tests with a confirmatory test results in fewer losses of normal pregnancies compared to sequencing-based tests used without a confirmatory test. The model made their estimates using the total population of 520,000 high-risk women presenting for first-trimester care each year in the U.S. Sequencing-based tests used with confirmatory testing resulted in 1441 elective terminations (all with Down syndrome). Without confirmatory testing, sequencing-based tests resulted in 3873 elective terminations, 1449 with Down syndrome and 2424 without Down syndrome. There were 29 procedure-related pregnancies losses when confirmatory tests were used. The decision model did not address T18 or T13. It is important to note that all of the above models include confirmatory invasive testing for positive screening tests. Sequencing-based testing without confirmatory testing carries the risk of misidentifying normal pregnancies as positive for trisomy. Due to the small but finite false positive rate, together with the low baseline prevalence of trisomy in all populations, a substantial percent of positive results on sequencing tests could be false positive results. Section Summary: There is no published direct evidence that managing patients using sequencingbased testing improves health outcomes compared to standard screening. Modeling studies using published estimates of diagnostic accuracy and other parameters predict that sequencing-based testing as an alternative to standard screening will lead to an increase in the number of Down syndrome cases detected and a large decrease in the number of invasive tests and associated miscarriages. The number of T18 and T13 cases detected is similar or higher with sequencing-based testing, although this is more difficult to estimate because of the lower prevalence of these aneuploidies, especially with T13. The impact of screening for sex chromosome aneuploidies has not been modelled in published studies. Ongoing Clinical Trials Study of the Efficacy of New Non-invasive Prenatal Tests for Screening for Fetal Trisomies Using Maternal Blood (PEGASUS) (NCT ) (24): This study is comparing non-invasive assays and strategies in a real-life setting in order to develop an evidence-based and cost-effective screening program in Canada. The following interventions will be compared: integrated prenatal screening for Down syndrome; serum QUAD assay for aneuploidy screening; targeted noninvasive assay using cell-free DNA in maternal blood; untargeted noninvasive assay using cell-free DNA in maternal blood; Harmony Test (Ariosa Diagnostics). The primary outcome measure is the number of cases of trisomy 21, 18 or 13, confirmed by fetal karyotype or at or after delivery. Both a high risk and low risk arm will be included. The study aims to recruit 5600 women and the expected date of completion is January 2017.

13 Subject: Noninvasive Prenatal Testing for Fetal Page: 13 of 20 Prenatal Non-invasive Aneuploidy Test Utilizing SNPs [single nucleotide polymorphism] Trial (PreNATUS) (NCT ) (25): This is a prospective, blinded study evaluating the diagnostic accuracy of the Natera test for diagnosing aneuploidies (chromosomes 13, 18, 21) and sex aneuploidy (X and Y). It includes women with singleton pregnancies at high or moderate risk for trisomy who were planning on undergoing invasive testing. Gestational age of the fetus is between 8 weeks 0 days and 23 weeks 6 days. The estimated enrollment is 1000 participants. As of October 2014, the study continues to recruit participants. Non-invasive Chromosomal Examination of Trisomy study (NEXT) (NCT ) (26): This is a prospective blinded case-control study comparing the Aria test for T21 with standard first-trimester prenatal screening (maternal serum testing and nuchal translucency). Cases will consist of patients with T21 pregnancies confirmed by genetic testing, and controls will consist of patients without T21 pregnancies, as confirmed by genetic testing or live birth. The study is sponsored by Aria Diagnostics. The estimated enrollment is 25,000 subjects. As of October 2014, the study continues to recruit participants. Clinical Evaluation of the SEQureDx T21 Test in Low-Risk Pregnancies (NCT )27: This is a prospective study and includes pregnant women between 10 to 22 weeks gestation who are at low risk for T21 aneuploidy (i.e., no positive prenatal screening tests, and no personal or family history of Down syndrome). Blood samples will be collected at a scheduled prenatal care visit and analyzed with the SEQureDx T21 test; pregnancies will be followed until the birth outcome is recorded. The study is sponsored by Sequenom; estimated enrollment is As of October 2014, the study is ongoing, but not recruiting participants Practice Guidelines and Position Statements National Society of Genetic Counselors (NSGC): (28) In 2013, the NSGC published a position statement regarding noninvasive prenatal testing of cell-free DNA in maternal plasma. The NSGC supports non-invasive cell-free DNA testing as option in women who want testing for aneuploidy. The document states that the test has been validated primarily in pregnancies considered to be at increased risk of aneuploidy and the organization does not support routine first-tier screening in lowrisk populations. In addition, the document states that test results should not be considered diagnostic and abnormal findings should be confirmed through conventional diagnostic procedures such as CVS and amniocentesis. American College of Medical Genetics and Genomics: (29) In 2013, the ACMG published a statement on non-invasive prenatal screening for fetal aneuploidy that addresses challenges in incorporating non-invasive testing into clinical practice. Limitations identified by the organization include that chromosomal abnormalities such as unbalanced translocations, deletions and duplications, singlegene mutations and neural tube defects cannot be detected by the new tests. Moreover, it currently takes longer to obtain test results than with maternal serum analytes. The ACMG also stated that pretest and posttest counseling should be performed by trained individuals.

14 Subject: Noninvasive Prenatal Testing for Fetal Page: 14 of 20 International Society for Prenatal Diagnosis (ISPD): (30) n 2013, the ISPD published a position statement regarding prenatal diagnosis of chromosomal abnormalities. The statement included the following discussion of maternal cell-free DNA screening:..although rapid progress has been made in the development and validation of this technology, demonstration that in actual clinical practice, the testing is sufficiently accurate, has low failure rates, and can be provided in a timely fashion, has not been provided. Therefore, at the present time, the following caveats need to be considered Reliable noninvasive maternal cfdna (cell-free) aneuploidy screening methods have only been reported for trisomies 21 and 18. There are insufficient data available to judge whether any specific cfdna screening method is most effective. The tests should not be considered to be fully diagnostic and therefore are not a replacement for amniocentesis and CVS Analytic validity trials have been mostly focused on patients who are at high risk on the basis of maternal age or other screening tests. Efficacy in low-risk populations has not yet been fully demonstrated American College of Obstetricians and Gynecologista (ACOG) and Society for Maternal-Fetal Medicine: (31) In November 2012, ACOG released a committee opinion on noninvasive testing for fetal aneuploidy. The Committee Opinion was issued jointly with the Society for Maternal-Fetal Medicine Publications Committee. ACOG recommended that maternal plasma DNA testing be offered to patients at increased risk of fetal aneuploidy. They did not recommend that the test be offered to women who are not at high risk or women with multiple gestations. ACOG further recommended that women be counseled prior to testing about the limitations of the test and recommended confirmation of positive findings with CVS or amniocentesis. The document noted that the content reflected emerging clinical and scientific advances and is subject to change as additional information becomes available. The Committee Opinion did not include an explicit review of the literature. U.S. Preventive Services Task Force Recommendations The U.S. Preventive Services Task Force (USPSTF) does not currently address screening for Down Syndrome. This topic had been addressed in the 1990s, but the topic is no longer listed on the USPSTF website. Summary Published studies on all commercially available tests, and meta-analyses of these studies, have consistently demonstrated very high sensitivity and specificity for detecting Down syndrome (trisomy 21 [T21]) in singleton pregnancies. Most of the studies included only women at high risk of T21. For average risk women, available studies suggest test performance similar to that reported in high-risk women, but there is less evidence in this population.

15 Subject: Noninvasive Prenatal Testing for Fetal Page: 15 of 20 Direct evidence of clinical utility is not available. A 2012 TEC Assessment modeled comparative outcomes based on the published data on test performance, published estimates of standard screening performance, patient uptake of confirmatory testing, and miscarriage rates associated with invasive procedures. For each comparison and in each risk population, sequencing-based testing improved outcomes, i.e., increased the rate of Down syndrome detection and reduced the number of invasive procedures and procedure-related miscarriages. In the modeling, the negative predictive value of testing approached 100% across the range of aneuploidy risk, while the positive predictive value varied widely according to baseline risk. The variable positive predictive value highlights the possibility of a false positive finding and thus testing using karyotyping is necessary to confirm a positive result. Based on the available evidence, including modeling in the TEC Assessment, as well as recommendations from national organizations, nucleic acid sequencing-based testing for trisomy 21 may be considered medically necessary in women with high-risk singleton pregnancies who meet criteria and investigational in other women. There is less published evidence on the diagnostic performance of sequencing-based tests for detecting T18, T13 and sex chromosome anomalies, and most of the available studies were conducted in high-risk pregnancies. Meta-analyses of available data suggest high sensitivities and specificities, but the small number of cases, especially for T13, makes definitive conclusions difficult. The findings of a decision analysis study included in the 2014 TEC Assessment suggest similar rates of T13 and T18 detection to standard noninvasive screening; the analysis assumed that T13 and T18 screening would be done in conjunction with T21 screening. Due to the low survival rate, the clinical benefit of identifying trisomy 18 and 13 are unclear. The clinical utility of early sex chromosome aneuploidy detection is also unclear. Thus, sequencing-based tests for T18 and T13 are considered medically necessary in women who are eligible for and are undergoing sequencing-based testing of maternal plasma for trisomy 21 and investigational otherwise, and sequencing-based testing for fetal sex chromosome aneuploidies is considered investigational. Medicare National Coverage No national coverage determination. References 1. American College of Obstetricians and Gynecologists (ACOG). Practice Bulletin No. 77: screening for fetal chromosomal abnormalities. Obstet Gynecol. Jan 2007;109(1): PMID Ashoor G, Syngelaki A, Poon LC, et al. Fetal fraction in maternal plasma cell-free DNA at weeks' gestation: relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol. Jan 2013;41(1): PMID Blue Cross Blue Shield Association Technology Evaluation Center (TEC). Sequencing-based tests to determine fetal down syndrome (trisomy 21) from maternal plasma DNA. TEC Assessments 2013; Volume 27, Tab 10. PMID

16 Subject: Noninvasive Prenatal Testing for Fetal Page: 16 of Blue Cross Blue Shield Association Technology Evaluation Center (TEC). Noninvasive maternal plasma sequencing-based screening for fetal aneuploides other than trisomy 21. TEC Assessments 2014; Volume 28, Tab TBD. PMID 5. U.S. Food and Drug Administration (FDA). Ultra High Throughput Sequencing for Clinical Diagnostic Applications - Approaches to Assess Analytical Validity, June 23, 2011 (Archived Content). Accessed April Gil MM, Akolekar R, Quezada MS, et al. Analysis of Cell-Free DNA in Maternal Blood in Screening for Aneuploidies: Meta-Analysis. Fetal Diagn Ther. Feb 8, PMID Palomaki GE, Deciu C, Kloza EM, et al. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genet Med. Mar 2012; 14(3): PMID Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med. Nov 2011; 13(11): PMID Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol. Mar 2011; 204(3):205 e PMID Bianchi DW, Platt LD, Goldberg JD, et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. May 2012; 119(5): PMID Sehnert AJ, Rhees B, Comstock D, et al. Optimal detection of fetal chromosomal abnormalities by massively parallel DNA sequencing of cell-free fetal DNA from maternal blood. Clin Chem. Jul 2011; 57(7): PMID Norton ME, Brar H, Weiss J, et al. Non-Invasive Chromosomal Evaluation (NICE) Study: Results of a Multicenter, Prospective, Cohort Study for Detection of Fetal Trisomy 21 and Trisomy 18. Am J Obstet Gynecol PMID 13. Ashoor G, Syngelaki A, Wagner M, et al. Chromosome-selective sequencing of maternal plasma cell-free DNA for first-trimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol. Apr 2012; 206(4):322 e PMID Sparks AB, Struble CA, Wang ET, et al. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol. Apr 2012; 206(4):319 e PMID Nicolaides KH, Syngelaki A, Gil M, et al. Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y. Prenat Diagn. Jun 2013; 33(6): PMID Porreco RP, Garite TJ, Maurel K, et al. Noninvasive prenatal screening for fetal trisomies 21, 18, 13 and the common sex chromosome aneuploidies from maternal blood using massively parallel genomic sequencing of DNA. Am J Obstet Gynecol. Mar 19, PMID Bianchi DW, Parker RL, Wentworth J, et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. Feb 27, 2014; 370(9): PMID