Medical Policy: 02.04.38
Original Effective Date: February 2016
Reviewed: February 2018
Revised: February 2018
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This Medical Policy document describes the status of medical technology at the time the document was developed. Since that time, new technology may have emerged or new medical literature may have been published. This Medical Policy will be reviewed regularly and be updated as scientific and medical literature becomes available.
Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. Most fetal chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes, which are the structures that contain genetic information. The trisomy syndromes are aneuploidies involving 3 copies of 1 chromosome. The most important risk factor for trisomy syndromes is maternal age. Trisomy 21 (T21) Down syndrome is the most common chromosomal aneuploidy and is the driving force for current maternal serum screening programs. Other trisomy syndromes include T18 (Edwards syndrom) and T13 (Patau syndrom), which are the next most common forms of fetal aneuploidy, although the percentage of cases surviving to birth is low and survival beyond birth is limited. The prevalence of these other aneuploidies is much lower than the prevalence of T21, and identifying them is not currently the main intent of prenatal screening programs. Also, the clinical implications of identifying T18 and T13 are unclear, because survival beyond birth is limited for both conditions.
There are numerous limitations to standard screening for these disorders using maternal serum and fetal ultrasound. Noninvasive prenatal screening (NIPS) analyzing cell-free DNA in maternal serum is a potential complement or alternative to conventional serum screening. Noninvasive prenatal screening (NIPS) using cell-free DNA has also been proposed to screen for microdeletions and sex chromosome aneuploidies.
Current national guidelines have recommend that all pregnant women be offered screening for fetal aneuploidy, referring specifically to T21, T18 and T13 before 20 weeks of gestation, regardless of age. Standard aneuploidy screening involves combinations of maternal serum markers and fetal ultrasound done at various stages of pregnancy. The detection rate for various combinations of noninvasive testing ranges from 60% to 96% when the false-positive rate is set at 5%. When tests indicate a high risk of trisomy syndrome, direct karyotyping of fetal tissue obtained by amniocentesis or chronic villous sampling (CVS) is required to confirm that T21 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 T21, T18 and T13 could improve outcomes. Confirmation of positive noninvasive screening tests with amniocentesis or CVS is recommended; with more accurate tests, fewer geno typical women could receive positive screening results.
Commercial, noninvasive, sequencing based testing of maternal serum for fetal trisomy syndromes is now available. 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 test are unable to provide a result if fetal fraction is too low, that is, below 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.
Sequencing-based tests use one of two general approaches to analyzing cell-free fetal DNA. The first category of tests uses quantitative or counting methods. The most widely used technique to date uses massively parallel sequencing (MPS; also known as next-generation or “next gen” sequencing). DNA fragments are amplified by polymerase chain reaction; 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 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) â€’ based methods. These use targeted amplification and analysis of approximately 20,000 SNPs on selected chromosomes (e.g., 21, 18, 13) in a single reaction. A statistical algorithm is used to determine the number of each type of chromosome.
The purpose of noninvasive prenatal screening (NIPS) using cell-free fetal DNA is to screen for fetal chromosomal abnormalities. It can be used a complement or as an alternative to conventional serum screening. The timing for testing is generally in the first trimester of pregnancy and can be early in the second trimester. Positive cell-free fetal DNA tests need to be confirmed using invasive testing.
No studies were identified that provided direct evidence on analytic validity. Each of the commercially available tests use massively parallel sequencing (MPS; also called next-generation sequencing [NGS]), 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 MPS, in preparation for an eventual goal of developing “a transparent evidence-based regulatory pathway for evaluating medical devices/products based on next generation sequencing, NGS, that would assure safety and effectiveness of devices marketed for clinical diagnostics.” The discussion pointed out the difference among manufacturers’ sequencing platforms and the diversity of applications, making it difficult to generate specific regulatory phases and metric. 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 has developed a specific procedure for its private, Clinical Laboratory Improvement Act-licensed laboratory where all testing takes place.
Although all currently available commercial tests use MPS (massively parallel sequencing - also called next generation sequencing), actual performance and interpretive procedures vary considerably. Clinical sequencing is not standardized or regulated by Food and Drug Administration 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.
Several systemic reviews and meta-analyses of studies on the diagnostic accuracy of sequencing based tests for the detection of fetal aneuploidies have been published. In 2016, Taylor-Phillips et. al. published a comprehensive systematic review and meta-analysis on the accuracy of noninvasive prenatal testing using cell-free DNA for detection of Down syndrome (T21), Edwards (T18) and Patau (T13) syndromes. To be included, studies had to confirm trisomy status using an invasive test, fetal pathologic examination, or postnatal phenotype assessment. Most studies were limited to samples of high risk women and singleton pregnancies. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% confidence interval (CI), 98.9 to 99.6%) for Down syndrome, 97.4% (95% CI, 95.8% to 98.4%) for Edwards, and 97.4% (955 CI, 86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. The authors concluded NIPT using cell-free fetal DNA was very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases.
A 2016 study by Norton et. al. reported on the performance of sequential DNA screening in a large cohort of women who participated in the California Prenatal Screening Program and compared findings with an estimations of cell-free fetal DNA findings. A total of 452,901 women underwent sequential screening, 2575 (0.57%) had a fetal chromosomal abnormality; 2101 were detected for a detection rate (DR) of 81.6%, and 19,929 euploid fetuses had positive sequential screening for a false positive rate (FPR) of 4.5%. If no results, cases were presumed normal, cell-free DNA (cfDNA) screening would have detected 1820 chromosome abnormalities (70.7%) with an FPR of 0.7%. If no results, cases were considered screen positive, 1985 (77.1%) cases would be detected at a total screen positive rate of 3.7%. in either case, the detection rate of sequential screening for all aneuploidies in the cohort was greater than cfDNA (P<0.001). A limitation of this study was that results of cell-free fetal DNA tests were estimated using statistical modeling and were not observed. The authors concluded for primary population screening, cfDNA providers lower DR (detection rate) than sequential screening if considering detection of all chromosomal abnormalities. Assuming that no results of cfDNA are high risk improves cfDNA detection but with a greater FPR (false positive rate). cfDNA should not be adopted as primary screening without further evaluation of the implications for detection of all chromosomal abnormalities and how to best evaluate no results cases.
In 2017, a systemic review and meta-analysis was published by Iwarsson et. al., the aim of this study was to review the performance of noninvasive prenatal testing (NIPT) for detection of trisomy 21, 18 and 13 (T21, T18, and T13) in a general pregnant population as well as on high risk pregnancies. In a general pregnant population, there is moderate evidence that the pooled sensitivity is 0.993 (95% CI, 0.955-0.999) and specificity was 0.999 (95% CI, 0.998-0.999) for the analysis of T21. Pooled sensitivity and specificity for T13 and T18 was not calculated in this population due to the low number of studies. In a high risk pregnant population, there is moderate evidence that the pooled sensitivities for T21 and T18 are 0.998 (95% CI, 0.981-0.999) and 0.977 (95% CI, 0.958-0.987) respectively, and low evidence that the pooled sensitivity for T13 is 0.975 (95% CI, 0.819-0.997). The pooled sensitivity for all three trisomies is 0.999 (95% CI, 0.998-0.999). The authors concluded, this is the first meta-analysis using GRADE that shows that NIPT performs well as a screen for trisomy 21 in general pregnant population. Although the false positive rate is low compared with first trimester combined screening, women should still be advised to confirm a positive result by invasive testing if termination of pregnancy is under consideration.
Several studies have evaluated noninvasive prenatal screening for fetal aneuploidies (T21, and when available T18 and T13) in high risk singleton pregnancies. The sensitivity and specificity of the tests were uniformly high. Sensitivity ranged from 99.1% to 100% and the specificity from 99.7% to 100%. Several companies market this testing, studies available are mostly prospective and are industry funded. Studies generally included women at a wide range of gestational ages (e.g. 8-36 weeks or 11-20 weeks) spanning first and second trimesters. The approach to analysis varied. Some studies analyzed samples from enrolled women, and other analyzed samples from all women with pregnancies known to have a trisomy syndrome and selected controls. The studies compared the results of cell-free DNA testing with the criterion standards of karyotyping for specific trisomies.
Data from the available peer reviewed medical literature have consistently reported a very high sensitivity and specificity of maternal plasma DNA sequencing based tests for detecting T21 in high risk women with singleton pregnancies. The available prospective studies in general population samples providing data separately for low risk women have found high sensitivity and specificity rates, similar to rates seen in high risk women. Based on study results, although PPV was lower in the subsample of low risk women than the general population, PPV of cell-free fetal DNA testing was much higher than standard screening.
There are fewer studies and data on the diagnostic performance of sequencing based tests for detecting T13 and T18 aneuploidies. The available data have suggested that diagnostic performance for detecting these other fetal aneuploidies is not has high as it is for detection of T21 and there is a higher rate of false positive tests.
The 2013 and 2014 BlueCross BlueShield Association 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, number of cases missed, number of invasive procedures potentially avoided (ie, 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:
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 that 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 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 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:
Results of the modeling suggest that sequencing-based tests detect a similar number of T13 and T18 cases and miss fewer cases than 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 2012, Palomaki et. al. modeled the 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. The model included cases positive for T21 or T18 (but not T13 due to its lower prevalence). As in the 2013 TEC Assessment, Palomaki assumed 4.25 million births in the United States per year, with two-thirds of those receiving standard screening. The models assumed a 99% detection rate, 0.5% false positive rate, and 0.9% failure rate for sequencing based testing. Compared with the highest performing standard screening test, the addition of sequencing based screening would increase the Down syndrome detection rate from 4450 to 4702 and decrease the number of miscarriages associated with invasive testing from 350 to 34.
In 2013, Ohno and Caughey published a decision model comparing the use of sequencing based tests in high risk women with confirmatory testing (i.e. as screening test) and without confirmatory testing (i.e. as diagnostic test). Results of the model concluded that using sequencing based tests with confirmatory test results in fewer losses of normal pregnancies compared with sequencing based tests used without a confirmatory test. The model assumed estimates using the total population of 520,000 high risk women presenting for first trimester care each year in the United States. Sequencing based tests used with confirmatory testing resuled in 1441 elective terminations (all with Down syndrome). Without confirmatory testing, sequencing based testing resulted in 3873 elective terminations, 1449 with Down syndrome and 2424 without Down syndrome. There were 29 procedure-related pregnancy losses when confirmatory tests were used. The decision model did not address T18 or T13.
It is important to note 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 percentage of positive results on sequencing test could be false positive results.
Modeling studies using published estimates of diagnostic accuracy and other parameters predict that sequencing-based testing as an alternative to standard screening would increase the number of T21 (i.e. Down syndrome) cases detected and, when included in the model, a large decrease in the number of invasive tests and associated miscarriages. A 2016 modeling study conducted in a general population sample, which compared data on standard screening with estimated cell-free fetal DNA results, found a significantly higher detection rate of Down syndrome cases with sequencing based tests than with standard screening.
A 2016 modeling study conducted in a general population sample, which compared data on standard screening and estimated cell-free fetal DNA results, found similar rates of T18 detection. However, models for T18 and T13 are more difficult to estimate because of lower prevalence of these aneuploidies and the limited number of cases detected in screening studies.
For individuals who have a singleton pregnancy who receive noninvasive prenatal screening (NIPS) for T21 using cell-free fetal DNA, the evidence includes observational studies and systematic reviews. Published studies on commercially available tests and meta-analyses (systematic reviews) of these studies have consistently demonstrated a very high sensitivity and specificity for detecting Down syndrome (T21) in singleton pregnancies. Most studies included only women at high risk of T21, but several studies, including one with a large sample size, have reported similar levels of diagnostic accuracy in average risk women. Compared with standard serum screening, both the sensitivity and specificity of cell-free fetal DNA screening are considerably higher. As a result, screening with cell-free fetal DNA will result in fewer missed cases of Down syndrome, fewer invasive procedures, and fewer cases of pregnancy loss following invasive procedures. The evidence is sufficient to determine this testing results in a meaningful improvement in net health outcomes for both high risk and average risk singleton pregnancies.
Based on review of the peer reviewed medical literature, for individuals who have a singleton pregnancy who receive noninvasive prenatal screening (NIPS) for aneuploidies using cell-free fetal DNA for T18 (Edwards syndrome) and T13 (Patau syndrome) there are few studies and data on the diagnostic performance detecting T13 and/or T18 aneuploidies, due to small number of cases. The available data based on meta-analysis have suggested that diagnostic performance for detecting these aneuploidies is not has high as it is for detection of T21 and there is a higher rate of false positives. The evidence is insufficient to determine the effects of this testing on net health outcomes.
In 2017, a meta-analysis by Gil et. al., completed an analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies (fetal trisomies 21, 18, 13) and sex chromosome aneuploidies (SCA) in both single and twin pregnancies. The inclusion criteria was peer reviewed study reporting on clinical validation for implementation of maternal cfDNA testing in screening for aneuploidies in which data on pregnancy outcome were provided for more than 85% of the study population. In total 35 relevant studies were identified and these were used for the meta-analysis on the performance of cfDNA testing in screening for aneuploidies. These studies reported cfDNA results in relation to fetal karyotype from invasive testing for clinical outcome. In the combined total of 1963 cases of trisomy 21 and 223 932 non-trisomy 21 singleton pregnancies, the weighted pooled DR and FPR were 99.7% (95% CI, 99.1-99.9%) and 0.04% (95% CI, 0.02-0.07%), respectively. In a total of 563 cases of trisomy 18 and 222 013 non-trisomy 18 singleton pregnancies, the weighted pooled DR and FPR were 97.9% (95% CI, 94.9-99.1%) and 0.04% (95% CI, 0.03-0.07%), respectively. In a total of 119 cases of trisomy 13 and 212 883 non-trisomy 13 singleton pregnancies, the weighted pooled DR and FPR were 99.0% (95% CI, 65.8-100%) and 0.04% (95% CI, 0.02-0.07%), respectively. In a total of 36 cases of monosomy X and 7676 unaffected singleton pregnancies, the weighted pooled DR and FPR were 95.8% (95% CI, 70.3-99.5%) and 0.14% (95% CI, 0.05-0.38%), respectively. In a combined total of 17 cases of SCA other than monosomy X and 5400 unaffected singleton pregnancies, the weighted pooled DR and FPR were 100% (95% CI, 83.6-100%) and 0.004% (95% CI, 0.0-0.08%), respectively. For twin pregnancies, in a total of 24 cases of trisomy 21 and 1111 non-trisomy 21 cases, the DR was 100% (95% CI, 95.2-100%) and FPR was 0.0% (95% CI, 0.0-0.003%), respectively. The authors concluded screening by analysis of cfDNA in maternal blood in singleton pregnancies could detect >99% of fetuses with trisomy 21, 98% of trisomy 18 and 99% of trisomy 13 at a combined FPR of 0.13%. The number of reported cases of SCA is too small for accurate assessment of performance of screening. In twin pregnancies, performance of screening for trisomy 21 is encouraging but the number of cases reported is small.
In 2017, Du et. al. assessed the performance of massively parallel sequencing (MPS) testing of cell-free fetal DNA (cfDNA) from maternal plasma for trisomies 21, 18, and 13 in twin pregnancies. MPS technology has been widely used to screen for trisomies 21, 18 and 13 in singleton pregnancies. Ninety-two women with twin pregnancies were recruited. The results were identified through karyotypes of amniocentesis or clinical examination and follow-up of the neonates. Cell-free fetal DNA testing correctly identified two T21 pregnancies, and there was 1 false positive T13 test. No cases of T18 were identified.
Foster et. al. in 2017, evaluated two sets of maternal blood samples from twin pregnancies using noninvasive prenatal testing (NIPT) for fetal aneuploidy. Clinical study A, 115 stored samples from pregnancies with known outcome and Clinical Study B 487 prospectively collected samples for which outcomes were requested from providers. NIPT was used to screen for the presence of fetal aneuploidy on chromosomes 13, 18, 21, X and Y in all cases, and results were compared with outcomes when known. In Clinical Study A, all 115 samples were classified correctly by NIPT: three cases of trisomy 21 (one fetus affected), one of monochorionic trisomy 18 (both fetuses affected) and 111 euploid (normal number of chromosomes). In Clinical Study B, a NIPT result was reported for 479 (98.4%) of the 487 samples. Aneuploidy was detected or suspected in nine (1.9%) cases: seven cases of trisomy 21 detected, one case of trisomy 21 suspected, and one case with trisomy 21 detected and trisomy 18 suspected. Information on aneuploidy outcome was available for 171 (35.75) cases in Clinical Study B. Of the nine cases with aneuploidy detected or suspected, six were confirmed to be a true positive in at least one twin based on karyotype or birth outcome and two were suspected to be concordant based on ultrasound findings; the one known discordant result was for aneuploidy suspected case. No false negatives were reported. Limitations of this study include the number of affected pregnancies was small and the majority were trisomy 21. This precluded determination of detection rates of trisomies 13 and 18. Another limitation was incomplete clinical outcomes with aneuploidy outcome information available for only 35.7% of cases in Clinical Study B. Authors concluded, although there is considerable evidence for robust NIPT performance in singleton pregnancies, there is still relatively little published data about its performance in twins. In this study, the detection rate for trisomy 21 in twin pregnancies appears to be in line with that in singletons. The limited number of affected cases for other trisomies precluded conclusive determination of those detection rates. In summary, the findings reported support the view that cfDNA NIPT performs well in twin pregnancies, with overall very low false-positive frequencies.
Based on review of the peer reviewed medical literature there is considerable evidence for robust noninvasive prenatal testing (NIPT) using cell-free DNA performance in singleton pregnancies, however, there is still relatively little published data about its performance in twins and multiple gestation pregnancy. Meta-analyses (systematic reviews) found that the total number of cases of T21 aneuploidy identified were small and there were even fewer cases of T18 and T13. The quantity of evidence is insufficient for drawing conclusions about clinical validity.
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Direct evidence is not available for the evaluation of noninvasive prenatal testing to detect fetal aneuploidies in women pregnant with twins or multiples. Additionally, it is not possible to construct a chain of evidence for clinical utility due to the lack of sufficient evidence on clinical validity.
For individuals who have twin or multiple pregnancies who receive noninvasive prenatal screening (NIPS) for aneuploidies using cell-free fetal DNA, the evidence includes observational studies and systematic reviews. The total number of cases of aneuploidy identified in these studies is small and is insufficient to draw conclusions about clinical validity. There is a lack of direct evidence of clinical utility, and a chain of evidence cannot be constructed due to insufficient evidence on clinical validity. The evidence is insufficient to determine the effects of the technology on net health outcomes.
Some of the commercially available cell-free DNA prenatal tests also test for other abnormalities including sex chromosome aneuploidies and selected microdeletions.
Microdeletions (also known as submicroscopic deletions) are defined as chromosomal deletions that are too small to be detected by microscopy or conventional cytogenetic methods. They can be as small as 1 and 3 megabases (mb) long. Microdeletions, along with microduplications, are collectively known as copy number variations (CNVs). CNVs can lead to disease when the change in copy number of a dose-sensitive gene or genes disrupts the ability of the gene(s) to function and effects the amount of protein produced. A number of genomic disorders associated with microdeletions have been identified. The disorders have distinctive and, in many cases, serious clinical features, such as cardiac anomalies, immune deficiency, palatal defects and developmental delay. A contributing factor is that the breakpoints of the microdeletions may vary, and there may be a correlation between the number of haplo-insufficient genes and phenotypic severity. Examples of microdeletion syndromes include: DiGeorge syndrome or velocardiofacial syndrome (most common), Prader-Willi syndrome, Angelman syndrome, Neurofibromatosis type I, Neurofibromatosis type II, Williams syndrome, Miller-Dieker syndrome, Smith-Magenis syndrome, Rubinstein-Taybi syndrome and Wolf-Hirschhorn syndrome.
A proportion of microdeletions are inherited and some are de novo (a genetic alteration that is present for the first time in one family member as a result of a variant (mutation) in a germ cell (egg or sperm) of one of the parents; new variant/mutation). Accurate estimates of the prevalence of microdeletion syndromes during pregnancy or at birth are not available. The risk of fetus with a microdeletion syndrome is independent of maternal age. There is little population based data and most studies published to date base estimates on phenotypic presentation. The 22q11.2 (DiGeorge) deletion is the most common microdeletion associated with a clinical syndrome.
Routine prenatal screening for microdeletion syndromes is not recommended by national organizations. Current practice is to offer invasive prenatal diagnostic testing in select cases to women when a prenatal ultrasound indicates anomalies (e.g. heart defects, cleft palate) that could be associated with a particular microdeletion syndrome. Samples are analyzed using fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA) or karyotyping. In addition, families at risk, those known to have the deletion or with a previous affected child generally receive genetic counseling. Most affected individuals are identified postnatally based on clinical presentation and may be confirmed with genetic testing.
Maternal plasma DNA sequencing based tests for fetal microdeletions have been proposed for use in a similar setting as noninvasive screening for fetal aneuploidies. However, there is currently no widely accepted clinical use of screening for microdeletions and microduplications in early pregnancy. Other potential uses are for diagnosis of suspected genetic disorders.
In 2015, Wapner et. al. published a study evaluating the ability of the Natera single-nucleotide variant based cell-free DNA test to identify microdeletions. The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP) based noninvasive prenatal test for 5 microdeletion syndromes: 22q11.2, 1p36, cri-du- chat, Prader-Willi and Angelman deletions. A cohort of 469 samples were evaluated, 358 plasma samples from pregnant women and 111 artificial plasma mixtures (PlasmArts). The PlasmArts samples mimicked the fetal fraction found in cell-free DNA from pregnant plasma and were enriched with microdeletions (in half of the samples). Twenty- three (6.4%) of the pregnancy sample and 3 of the PlasmArts samples failed quality control; all pregnancy samples were from unaffected pregnancies. The detection rates were 97.8% for 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1) and cri-du-chat syndromes (24/24). False positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442) or 1p36 deletion syndromes (0/422). This study had several limitations to include a significant limitation of lack of sufficient maternal plasma samples from affected pregnancies at appropriate gestational ages.
Data on the analytic validity of single-nucleotide variant based cell-free DNA testing have been reported on a constructed sample. The validity of testing in such samples are not well understood. Moreover, all patients did not receive criterion standard test for microdeletions, so it is not possible to accurately identify all false negatives or false positives. Data on analytic validity in a clinical population (rather than artificially constructed samples) are needed. Additionally, more data are needed on the ability of sequencing based tests to identify microdeletions of different sizes (e.g. 10 megabases vs 3 megabases) and the ability to identify microdeletions of fetal origin by the fetal fraction of DNA present in the maternal plasma sample.
Studies from 2 companies offering microdeletion testing have evaluated data from clinical samples submitted for screening. In 2016, Gross et. al. published a study evaluating the performance of the Natera cell-free DNA test to identify 22q11.2 deletion syndrome. The study retrospectively analyzed 21,949 samples submitted for screening. After 1172 cases were excluded (919 failed quality control, 46 were twins/triploidy, 207 were out of specification), 20,776 cases were evaluated for the microdeletion. A total of 97 (0.46%) of the 20,776 cases were considered at high risk for the 22q11.2 deletion. One of these was confirmed to be a 22q11.2 microdeletion in the mother, not in the fetus, and another was suspected to be a maternal deletion. Diagnostic testing results were available for 61 (64.2%) cases, which confirmed 11 (18.0%) true positives and identified 50 (82.0%) false positives, resulting in a positive predictive value (PPV) of 18.0%. Information regarding invasive testing was available for 84 (88.4%) high risk cases: 57.1% (48/84) had invasive testing and 42.9% (36/84) did not. Ultrasound anomalies were present in 81.8% of true positive and 18.0% of false-positive cases. Limitations of the analysis included lack of follow up data on both high risk and low risk cases. Although attempts were made to follow-up all high risk cases, confirmatory diagnostic information was unavailable in 34 cases (36%). This included cases for which patients chose not to have confirmatory testing results or were reluctant to share confirmatory testing results, as well as cases for which patients was lost to follow-up. Providers were encouraged to report false-negative cases, but no such cases were reported. However, because follow-up on low-risk cases was not carried out, calculation of negative predictive value was not carried out, calculation of the negative predictive value was not possible. Authors concluded the decision to add 22q11.2 deletion screening as an adjunct to existing NPIT needs to balance the medical benefits of early diagnosis of 22q11.2 deletions against efficacy of the test, the prevalence in the referral group (which would be expected to be higher when NIPT referrals include patients with positive combined test results and abnormal ultrasound findings), additional clinical service consideration and cost. The data on clinical experience presented in this study may be helpful in this assessment.
In 2015, a study by Helgeson et. al. used the Sequenom MPS-based test and the investigators analyzed 175,393 blood samples from high risk pregnant women. Between October 2013 and July 2014, 123,096 samples were tested for 4 microdeletions: 1p36, 5p-, 15q-, and 22q11.2. From August 2014 to October 2014, 52,297 samples were tested for those 4 microdeletions plus an additional 3: 4p-, 8q- and 11q-. The preferred reference standard was diagnostic testing (chromosomal microarray analysis, fluorescence in situ hybridization or karyotype analysis). Cases were considered “confirmed” if the deletion was detected in the pregnant woman and/or fetus, and considered “false-positive” if diagnostic testing was negative for the deletion in either the fetus or pregnant woman (maternal plasma samples contain DNA fragments from both the pregnant woman and the fetus; microdeletions detected could be either or both of them). In the absence of diagnostic testing, cases were considered “suspected” if diagnostic testing was not peformed and phenotypic data were consistent with the clinical presentation common to the deletion. Fifty-five (0.03%) of the samples had one of the testing microdeletions. Nearly half (48%) of the positive tests were in pregnancies referred for testing due to ultrasound findings. Two patients were lost to follow-up, and diagnostic testing and/or clinical phenotype information was available for the remaining 53 patients. Microdeletions were confirmed (in the pregnant woman and/or fetus) 41 (77.4%) of 53 cases, and an additional 9 cases did not have confirmatory testing but had clinical features consistent with one of the microdeletions. There were 3 false positive cases, 1 case of 1p36 deletion and 2 cases of 5p deletion. The PPV ranged from 60% to 100% for cases with diagnostic and/or clinical follow-up information. The false-positive rate was 0.0017% for confirmed cases; if cases lost to follow-up were all false positives, the rate would be 0.0029%. In the 25 of 55 microdeletions identified by NIPS, a maternal component was identified. Twenty of these cases were associated with a 22q11.2 deletion, four with a 15q deletion, and one with an 8q deletion. In at least 5 cases, deletions were confirmed in the pregnant woman but not in the fetus. Clinical outcomes were unavailable for most pregnancies in which a deletion was not detected. There false negatives were reported, all for 22q11.2 based on phenotypic presentation, but data on false negatives were incomplete. Not all patients had confirmatory testing, so it is not possible to identify all false negatives or false positives accurately.
Several studies on clinical validity of microdeletion testing have been published, based on large numbers of samples submitted to the testing companies. These studies have limitations (e.g. substantial missing data on confirmatory testing, lack of complete data on false negatives). As demonstrated in one of the studies, many of the cases of microdeletion syndromes are currently initially detected by characteristic anomalies seen on prenatal ultrasound.
The clinical utility of testing for any particular microdeletion or any panel of microdeletions is uncertain. There is no direct data on whether sequencing-based testing for microdeletions improve outcomes compared with standard care. There is a potential that prenatal identification of individuals with microdeletion syndromes could improve health outcomes due to the ability to allow for informed reproductive decision making and/or initiate earlier treatment; however, data demonstrating improvement are unavailable. Given the variability of expressivity of microdeletion syndromes and the lack of experience with routine genetic screening for microdeletions, clinical decision making based on genetic test results is not well defined. It is not clear what follow-up testing or treatments might be indicated for screen-detected individuals.
Most treatment decisions would be made after birth, and it is unclear whether testing in utero would lead to earlier detection and treatment of clinical disease after birth. Moreover, clinical decision making when a maternal microdeletion is detected in pregnant women without previous knowledge of a genetic variant is unclear.
The clinical utility of noninvasive prnatal screening (NIPS) for microdeletions is not well-established. Although there is a potential for clinical utility in screening for some syndromes associated with microdeletions early in pregnancy, the clinical management changes that would be associated with early diagnosis of these syndromes are not well-established and the potential outcome improvements associated with early diagnosis (i.e. before the diagnosis would be suspected on the basis of physical exam findings or findings on routine imaging) is not well-established. The incidence of microdeletions syndromes is low, and not all individuals with microdeletion will have clinical symptoms.
For individuals with who receive noninvasive prenatal screening (NIPS) for microdeletions using cell-free fetal DNA, the evidence includes several observational and retrospective studies. The available studies on clinical validity have limitations (e.g. missing data on confirmatory testing, false negatives), and the added benefit of NIPS compared with current approaches is unclear. The clinical utility of NIPS for microdeletions has not been evaluated in published studies. A joint practice bulletin by the American College of Obstetricians and Gynecologists (ACOG) and the Society of Maternal-Fetal Medicine (SMFM) states cell-free DNA screening tests for microdeletions have not been validated clinically and are not recommended at this time. The evidence is insufficient to determine the effects of the technology on net health outcomes.
Sex chromosome aneuploidies belong to a group of genetic conditions that are caused or affected by the loss or damage of sex chromosomes (genosomes). This may refer to: 47, XXX; 48, XXXX; 49 XXXXY syndrome; 49, XXXXX; Klinefelter’s syndrome, XXY; Turner syndrome, X; XXX gonadal dysgenesis; XX male syndrome; XXYY syndrome; XYY syndrome, and occur in approximately 1 in 400 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. Potential benefits of early identification (e.g. the opportunity for early 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.
The evidence for noninvasive prenatal screening (NIPS) using cell free DNA to detect sex chromosome aneuploidies in individuals pregnant with singletons includes several observational studies, mainly in high-risk pregnancies and systematic reviews. Meta-analyses of available data suggests high sensitivities and specificities, but the small number of cases, makes definitive conclusions difficult. The evidence is insufficient to determine the effects of this testing on net health outcomes.
Counseling regarding the limitations of cell-free DNA should include a discussion about how the screening methods provide information regarding only trisomies 13, 18, and 21. If a sex chromosome analysis has been requested or is part of the standard panel, then this information should be conveyed as well.
Patients should be counseled that cell-free DNA screening does not replace the precision obtained with diagnostic tests, such as chronic villus sampling or amniocentesis and, therefore, is limited in its ability to identify all chromosome abnormalities. Not only can there be false-positive test results, but a positive cell-free DNA test result for aneuploidy does not determine if the trisomy is due to a translocation, which affects the risk of recurrence. If a fetal structural anomaly is identified on ultrasound examination, diagnostic testing should be offered rather than cell-free DNA screening.
The cell-free DNA screening test should not be considered in isolation from other clinical findings and test results. Given the potential for inaccurate results and to understand the type of trisomy for recurrence-risk counseling, a diagnostic test should be recommended for a patient who has a positive cell-free DNA test result. Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone. False-positive results do occur and diagnostic testing with amniocentesis or chronic villus sampling (CVS) should be recommended before any pregnancy termination decision. Causes of false-positive test results have been reported, which include but are not limited to placental mosaicism, vanishing twins, and maternal malignancies.
Before offering cell-free DNA screening, counseling is recommended. The family history should be reviewed to determine if the patient should be offered other forms of screening or prenatal diagnosis for a particular disorder. In order to ensure accuracy and testing of the appropriate patient population, a baseline ultrasound examination also should be considered to confirm viability, the number of fetuses, and gestational dating, if not performed previously. Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy. A negative test result still carries a residual risk of one of the common trisomies and does not ensure that the fetus does not have another chromosome abnormality or genetic diagnosis. Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects. Patients who are undergoing cell-free DNA should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment. Parallel or simultaneous testing with multiple screening methodologies for aneuploidy is not cost effective and should not be performed. However, use of cell-free DNA screening as a follow-up test for patients with positive traditional screening test is reasonable for patients who want to avoid a diagnostic test.
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests (LDTs) must meet the general regulatory standards of the Clinical Laboratory Improvement Act (CLIA). Laboratories that offer LDTs must be licensed by CLIA for high complexity testing. To date, the U.S. Food and Drug Administration has chosen not to require any regulatory review of noninvasive prenatal screening tests using cell-free fetal DNA. Commercially available tests include but are not limited to the following:
Also, when a clear result is seen, fetal sex aneuploidies and select microdeletions will be reported as additional findings.
In 2012, The American College of Obstetricians and Gynecologists issued a committee opinion, number 545 for noninvasive prenatal testing for fetal aneuploidy that included the following indications for considering the use of cell-free fetal DNA:
September 2015, ACOG and the Society of Maternal Fetal Medicine related an updated committee opinion (number 640) on cell-free DNA screening for fetal aneuploidy (this document replaces committee opinion number 545). The list of recommendations in the 2015 committee opinion includes the following:
In 2015, the Society for Maternal-Fetal Medicine (SMFM) published a special report clarifying its recommendations regarding cell-free DNA aneuploidy screening, as follows:
“The purpose of this statement is to clarify that SMFM does not recommend that cell free DNA (cfDNA) aneuploidy screening be offered to all pregnant women, nor does it suggest a requirement for insurance coverage for cfDNA screening in women in low risk of aneuploidy. However, SMFM believes, due to the ethics of patient autonomy, that the option should be available to women who request additional testing beyond what is currently recommended by professional societies. SMFM recognizes the value of cfDNA screening for women at higher risk for aneuploidy but considers that cfDNA screening is not the appropriate choice for first-line screening for low risk obstetric population at the present time. For this population, conventional screening methods remain the preferred approach.”
In 2016, the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM) released a joint practice bulletin (No. 163) on screening for fetal aneuploidy, which included the following recommendations:
All women should be offered the option of aneuploidy screening or diagnostic testing for fetal genetic disorders regardless of maternal age.
In 2015, the public and professional policy committee of the European Society of Human Genetics and the social issues committee of the American Society of Human Genetics issued a joint statement on NIPS (also called noninvasive prenatal testing [NIPT]). Relevant recommendations are as follows:
In 2016, the National Society of Genetic Counselors (NSGC) published a position statement on prenatal cell-free DNA screening, which states: The National Society of Genetic Counselors supports prenatal cell-free DNA (cfDNA) screening, also known as NIPT or NIPS,* as an option for pregnant patients. Because cfDNA screening cannot definitively diagnose or rule out genetic conditions, qualified providers should communicate the benefits and limitations of cfDNA screening to patients prior to testing. Many factors influence cfDNA screening performance, therefore it may not be the most appropriate option for every pregnancy.
Prior to undergoing cfDNA screening, patients should have the opportunity to meet with qualified prenatal care providers who can facilitate an individualized discussion of patients’ values and needs, including the option to decline all screening or proceed directly to diagnostic testing. Clinicians with expertise in prenatal screening, such as genetic counselors, should provide post-test genetic counseling to patients with increased-risk screening results. Diagnostic testing should be offered to patients with increased-risk results to facilitate informed decision making.
In 2013, the American College of Medical Genetics and Genomics (ACMG) published a statement on noninvasive prenatal screening for fetal aneuploidy that addresses challenges in incorporating noninvasive testing into clinical practice. Limitations identified include that chromosomal abnormalities such as unbalanced translocations, deletions and duplications, single-gene 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. ACMG also stated that pretest and posttest counseling should be performed by trained personnel.
In 2016, the American College of Medical Genetics and Genomics (ACMG) published updated position statement regarding noninvasive prenatal screening for fetal aneuploidy.
Should noninvasive prenatal screening (NIPS) be offered to all patients including those at low or average risk?
Should NIPS be used to screen for autosomal aneuploidies other than Patau, Edwards, and Down Syndromes?
Should NIPS be offered to screen for sex chromosome aneuploidies?
Should NIPS be offered for detection of copy number variation (CNV)?
Special Considerations - multiple gestation and/or donor oocytes
In 2015, the International Society for Prenatal Diagnosis published a position statement on prenatal diagnosis of chromosomal abnormalities, an update of their 2013 statement.34,35 (Note that a number of the authors of the 2015 report had financial links to industry.) Following is the summary of recommendations:
Noninvasive prenatal screening using cell-free fetal DNA of maternal plasma for fetal aneuploidy trisomy 21 may be considerred medically necessary in women with a singleton pregnancy.
Concurrent noninvasive prenatal screening using cell free fetal DNA of maternal plasma for fetal aneuploidy trisomy 13 and/or 18 may be considered medically necessary in women with a singleton pregnancy and who are eligible and undergoing noninvasive prenatal screening using cell free fetal DNA of maternal plasma for trisomy 21.
Noninvasive prenatal screening using cell-free fetal DNA of maternal plasma for trisomy 13 and/or 18, other than in the situation specified above, is considered investigational.
Based on review of the peer reviewed medical literature, there are few studies and data on the diagnostic performance detecting T13 and/or T18 aneuploidies, due to small number of cases. The available data based on meta-analysis have suggested that diagnostic performance for detecting these aneuploidies is not has high as it is for detection of T21 and there is a higher rate of false positives. The evidence is insufficient to determine the effects of this testing on net health outcomes.
Noninvasive prenatal screening using cell free fetal DNA of maternal plasma for fetal aneuploidy trisomy 21, 18, and 13 in women with a twin or multiple gestation pregnancy is considered investigational.
Based on review of the peer reviewed medical literature there is little published data about the performance of cell-free fetal DNA for screening for fetal aneuploidies in twins or multiple gestations. For individuals who have a twin or multiple gestation pregnancy who receive noninvasive prenatal screening for aneuploidies using cell-free fetal DNA, the evidence includes observational studies and systematic reviews. The total number of cases of aneuploidy identified in these studies are small and are insufficient to draw conclusions about clinical validity. There is a lack of direct evidence of clinical utility, and a chain of evidence cannot be constructed due to insufficient evidence on clinical validity. Further studies are needed. The evidence is insufficient to determine the effects of this testing on net health outcomes in twin and multiple gestation pregnancies.
Noninvasive prenatal screening using cell free fetal DNA of maternal plasma for fetal sex chromosome aneuploidies is considered investigational.
Based on review of the peer reviewed medical literature, the evidence for noninvasive prenatal screening using cell free DNA to detect sex chromosome aneuploidies in pregnancy includes several observational studies mainly in high-risk pregnancies and systematic reviews. Meta-analyses of available data suggests high sensitivities and specificities, but the small number of cases makes definitive conclusions difficult. The evidence is insufficient to determine the effects of this testing on net health outcomes.
Noninvasive prenatal screening using cell-free fetal DNA of maternal plasma for microdeletions is considered investigational.
Based on the review of the peer reviewed medical literature, for individuals who receive noninvasive prenatal screening for microdeletions using cell-free fetal DNA, the evidence includes several observational and retrospective studies. The available studies on clinical validity have limitations (e.g. missing data on confirmatory testing, false negatives), and the added benefit of noninvasive prenatal screening using cell-free fetal DNA compared with current approaches is unclear. The clinical utility of noninvasive prenatal screening using cell-free fetal DNA for microdeletions has not been evaluated in published studies. A joint practice bulletin by the American College of Obstetricians and Gynecologists (ACOG) and the Society of Maternal-Fetal Medicine (SMFM) states cell-free DNA screening tests for microdeletions have not been validated clinically and are not recommended at this time. The evidence is insufficient to determine the effects of this testing on net health outcomes.
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