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Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy?
Clinical Opinion
www.AJOG .org
GENETICS
Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy? Michael T. Mennuti, MD; Athena M. Cherry, PhD; Jennifer J. D. Morrissette, PhD; Lorraine Dugoff, MD
C
ell-free DNA (cfDNA) testing on maternal blood samples recently has been introduced in obstetrics practice as a method for fetal aneuploidy screening.1 This testing examines cfDNA fragments circulating in the maternal plasma that originate primarily from cells of the mother and to a lesser extent from placental cells. The DNA fragments from particular chromosomes are identified by their nucleic acid sequence with a process that is known as massively parallel shotgun sequencing.2,3 Assuming that the maternal cells are euploid, quantitative analysis of the cfDNA fragments is used to predict whether the cells of the pregnancy have the normal or abnormal copy number of specific chromosomes. Recently, one laboratory has introduced the use of parental singlenucleotide polymorphisms as a means of predicting the copy number of specific chromosomes in the placental cells.4 Validation studies in high-risk patients who have undergone invasive diagnostic testing have shown a sensitivity of >98% for detection of trisomy 21 and trisomy 18, with remarkably low false-positive rates (<0.5%) but somewhat lower sensitivity for trisomy From the Department of Obstetrics and Gynecology (Drs Mennuti and Dugoff), Prenatal Cytogenetic Laboratory (Dr Mennuti); Department of Pathology and Laboratory Medicine and Clinical Cancer Cytogenetics (Dr Morrissette), Perelman School of Medicine, University of Pennsylvania, Philadelphia PA; and the Department of Pathology and Cytogenetics Laboratory, Stanford Hospital and Clinics, Stanford University School of Medicine, Palo Alto, CA (Dr Cherry). Received Dec. 10, 2012; revised March 5, 2013; accepted March 19, 2013. The authors report no conflict of interest. Reprints not available from the authors. 0002-9378/$36.00 ª 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2013.03.027
Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making. Key words: cfDNA, chromosome, fetus, positive predictive value, testing
13.5-10 Laboratories that developed the tests and clinical investigators who studied the test performance propose that positive results should be confirmed by invasive prenatal diagnosis before important parental decisions are made regarding the pregnancy. This recommendation is important because the positive predictive value of the test is imperfect (ie, some portion of pregnancies with an abnormal result will not be affected). Although the test methods demonstrate excellent discrimination between euploid pregnancies and those with trisomy 13, 18, or 21, the necessity of establishing a quantitative cutoff inevitably will result in some false-positive and false-negative results. We are aware of only one report of prospective performance of this testing in routine clinical practice.11 Recently, 8 cases in which there were discordant results between an abnormal cfDNA test and the normal cytogenetic testing of the pregnancy have come to our attention.
The cfDNA testing in these cases was not performed in the same laboratory. Massively parallel shotgun sequencing was used to identify the chromosomal origin of cfDNA fragments in all 8 cases. The discordance between the cfDNA testing and the cytogenetic tests dramatically underscores the importance of offering invasive diagnostic testing after an abnormal cfDNA test result. We describe these cases to call attention to the urgent need for studies to understand the possible causes of discordant results so that this information may be used in the development of clinical guidelines for evaluation of similar cases.
Case 1 A 40-year-old woman (G3P1011) had first- and second-trimester serum integrated screening with adjusted risks for Down syndrome of 1:2200, trisomy 18 of 1:10,000, and open neural tube defect of 1:6000. The patient requested a cfDNA
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test at 16 weeks 5 days’ gestation. The result was reported as negative for trisomy 21 but showed an increased representation of chromosome 18 material. At 18.5 weeks’ gestation, the patient had a normal fetal anatomic survey and opted for amniocentesis. Amniocentesis was reported to demonstrate a 46,XY chromosome constitution in 15 metaphases from 12 colonies. Studies of DNA from parental blood samples and from the amniotic cells confirmed biparental inheritance at 3 informative loci on chromosome 18. Ultrasound scans that were performed at 27.6 and 34.6 weeks’ gestation showed normal interval fetal growth. At 41 weeks’ gestation, the patient delivered a healthy male infant who weighed 6 lbs 11 oz. The infant had a normal clinical examination and an uneventful hospital course and was discharged at 2 days of age. Samples for chromosome testing were not obtained on the infant or placenta.
Case 2 A 34-year-old woman (G2P0010) with a maternal age of 35 years at her projected estimated delivery date opted for cfDNA testing at 11 weeks’ gestation. The result was negative for trisomy 21 but showed an increased representation of chromosome 18 material. Amniocentesis that was performed at 16 weeks’ gestation revealed a normal 46,XY chromosome analysis, and the amniotic fluid alphafetoprotein was normal. Fetal anatomic survey by ultrasound scanning was normal. The patient has not yet delivered at the time of this report. Case 3 A 20-year-old primigravid woman had first-trimester aneuploidy screening with adjusted risk for trisomy 21 of 1:10,000 and trisomy 18 of 1:10,000. The first-trimester screening included beta human chorionic gonadotropin (hCG), pregnancy-associated plasma proteineA (PAPP-A), maternal age, and nuchal translucency (NT). No comment regarding visualization of the nasal bone was provided. The patient subsequently had second-trimester maternal serum alpha-protein with adjusted risk of open neural tube defect of 1:1200. A fetal
anatomic survey was normal at 19.3 weeks’ gestation, and fetal biometry lagged 6 days behind the first-trimester ultrasound scan. At 23.3 weeks’ gestation, there was a progressive lag in fetal growth that was noted by ultrasound scanning with an estimate fetal weight at the 12th percentile. A hypoplastic nasal bone and echogenic bowel were noted. The patient declined amniocentesis for chromosome analysis but opted to have cfDNA testing. The cfDNA showed an increased representation of chromosome 18 material. Amniocentesis was performed, and fluorescence in situ hybridization (FISH) was negative for trisomy 18. At 28.4 weeks’ gestation, reversed end diastolic velocity on umbilical artery Doppler velocimetry prompted delivery of a male infant who weighed 570 g (<5th percentile for the gestational age) with an otherwise normal physical examination that was consistent with his gestational age. Karyotypes and single-nucleotide polymorphism microarrays from the amniocentesis and neonatal blood samples were reported as normal male, 46,XY.
Case 4 A 41-year-old woman (G6P3023) had cfDNA testing performed at 11 weeks’ gestation as her initial screening for aneuploidy. Her cfDNA result reported a >99% risk for trisomy 18. Subsequently, first-trimester screening by beta hCG, PAPP-A, and NT was performed and resulted in a risk of 1:720 for trisomy 21 and 1:20,000 for trisomy 18. Information regarding the nasal bone was not provided. A chorionic villus biopsy was performed at 13.1 weeks’ gestation. Both direct and long-term cultures showed a normal male karyotype (46,XY). No abnormalities were noted on subsequent ultrasound scanning, and the pregnancy is continuing. Case 5 A 35-year-old woman (G1P0) had firsttrimester screening by beta hCG and NT measurement at 11 weeks’ gestation that reported a risk of 1:87 for trisomy 21 (hCG 1.48 multiples of the median; PAPP-A, 0.36 multiples of the median; NT, 1.6 mm). Information regarding the
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www.AJOG.org nasal bone was not provided. The cfDNA testing result was positive for trisomy 18. Amniocentesis was performed at 16 weeks’ gestation. FISH on amniotic fluid cells was normal, as was chromosome analysis of cultured amniotic cells that showed a normal female karyotype (46,XX). Subsequent ultrasound scanning showed normal interval growth, and the fetal anatomic evaluation appeared normal. The pregnancy is continuing at the time of this report.
Case 6 A 37-year-old woman (G2P1001) opted for cfDNA testing at 12.6 weeks’ gestation. Ultrasound scanning showed a singleton pregnancy with a crown-rump length measurement that was consistent with the estimated delivery date and a nuchal translucency measurement of 1.6 mm. The cfDNA showed an increased representation of chromosome 13 material. Amniocentesis was performed at 15 weeks’ gestation. Sixty-six cells from 15 different colonies and an additional 34 cells from amniotic cell subcultures were examined. All 100 cells that were examined were found to have a normal 46,XX chromosome constitution. The patient declined uniparental disomy testing and a microarray of the amniotic cells. Ultrasound scanning at 18 weeks’ gestation showed appropriate fetal growth and normal fetal anatomy. Ultrasound assessment at 28 weeks’ gestation is planned. Case 7 A 34-year-old (35 years old at her estimated delivery date) woman (G2P1) had cfDNA testing performed at 12.3 weeks’ gestation. The result was reported as positive for trisomy 13. Chorionic villi sampling (CVS) was performed at 13.3 weeks’ gestation. FISH was positive for trisomy 13 and male gender in 50 cells that were examined. The patient elected to terminate the pregnancy. The karyotype from the CVS culture subsequently was reported as 46XY in 40 cells from 3 independent cultures. Single-nucleotide polymorphism microarray analysis was reported as consistent with a normal male. Examination of a sample of tissue that was submitted
www.AJOG.org for chromosome testing after termination of the pregnancy showed chorionic villi and decidua. No identifiable fetal tissue was available for study in this portion of the sample. Interphase FISH showed 2 signals for chromosome 13; a 46,XY chromosome constitution was noted in all 100 metaphases that were examined.
Case 8 A 34-year-old woman (G2P1) had cfDNA testing that was positive for trisomy 13. Ultrasound scanning showed a single intrauterine pregnancy that was consistent with a gestational age of 13.6 weeks and a nuchal translucency that measured 1.7 mm. Anatomic evaluation of the fetus was normal for the first trimester, with the exception of what appeared to be mild dilation of the renal pelves. Direct preparation on CVS showed 46,XX,þ13,der(13;13)(q10;q10) in 5 of 5 metaphases that were examined. This cytogenetic finding would be expected to result in a cfDNA test result that was positive for trisomy 13 and a trisomy 13 phenotype in the fetus. Two independent long-term cultures from the CVS showed a 46,XX chromosome constitution in 15 of 15 metaphases that were examined. An additional 50 metaphases that were examined from the CVS cultures were euploid. Further review of the direct preparation slides showed the derivative (13;13) translocation chromosome in all metaphases that were examined. Amniocentesis was performed at 15 weeks’ gestation. All 9 primary colonies that were examined had a normal 46,XX chromosome constitution. An additional 41 metaphase from an amniotic cell culture flask also had a 46,XX chromosome constitution. Fetal anatomic evaluation by ultrasound scanning at the time of amniocentesis appeared normal. The pregnancy is continuing at the time of this report, and ultrasound follow-up is planned. Comment Although many of the patients that we described above have not yet delivered their pregnancies, for the purposes of this discussion we will refer to all them as
Genetics having received false-positive cfDNA test results inasmuch as the results were not confirmed by prenatal chromosome analysis, which has been the standard against which the testing has been validated clinically. Given the quantitative nature of the interpretation of cfDNA testing for aneuploidy detection and the low prevalence of trisomy 18 and trisomy 13, it should not be surprising to clinicians that they occasionally may encounter false-positive test results, perhaps without ever having experienced a true positive test result in practice. Without knowing the number of tests that were performed by the laboratories, it is not possible to estimate whether the number of cases with positive cfDNA test results in which the prenatal chromosome testing was not concordant exceed the number of falsepositive cfDNA test results that were expected, based on the published studies. If false-positive test results, in large part, are due to the quantitative nature of the test interpretation, then one would expect that careful analysis might demonstrate that a group of such cases would have test results closer to the cut-off for normal than might be observed for a group with true positive test results. Because it has been shown that the discrimination between normal and aneuploid pregnancies improves with increasing “fetal fraction” of cfDNA, it might also be relevant to know the relative “fetal fraction” in a group of cases with false-positive test results. An informative analysis of false-positive test results would require a much larger collection of cases with quantitative data that is not generally available in the laboratory reports that are issued to clinicians. A rate of false-positive test results that is not higher than expected and a careful study that would demonstrate that these occurrences are most likely a function of the quantitative nature of the test interpretation would reaffirm the importance of always offering invasive testing when there is an abnormal cfDNA test result. The identification of the limitations of the positive predictive value as the likely cause of testing results such as those observed in cases 1-6 would also reduce concern about sample
Clinical Opinion
errors or rare biologic mechanisms that result in false-positive test results. If, on the other hand, the false-positive test results do not appear to be based on the quantitative test interpretation, then other explanations should be sought. These could be important for the future clinical application of the test, for counseling, and for the assessment of patients with positive cfDNA results. Cases 7 and 8 raise important questions about other low probability biologic mechanisms for some false-positive cfDNA results. Inasmuch as the DNA that was measured in this testing is of placental origin, mosaicism for aneuploidy in the placenta is a possible explanation. Mosaicism in the placenta can reflect mosaicism throughout the pregnancy, which includes the fetus, or it can be limited to the placenta (known as confined placental mosaicism [CPM]). Other, much less likely biologic causes of false-positive test results might include the contribution of a vanishing aneuploid twin to the cfDNA in the maternal plasma, a copy number duplication of portion of a fetal chromosome too small to be detected by the standard cytogenetic testing, or a low percentage maternal mosaicism for an autosomal aneuploidy that may be either constitutional or acquired. Concern about maternal mosaicism for sex chromosome aneuploidy is likely to become relevant because gender and sex chromosome aneuploidy are reported increasingly by the laboratories that are performing this testing. Should clinicians and cytogeneticists be concerned that clinically relevant fetal or placental mosaicism may be the cause of what initially appears to be falsepositive cfDNA testing results? If so, when the initial analysis of the confirmatory testing demonstrates all normal cells, it would seem prudent to increase the number of colonies or cells to be examined to increase the confidence that there is not clinically relevant mosaicism. Guidelines for the “extensive work-up for mosaicism” generally are used when only a portion of cells in an amniotic fluid or CVS are aneuploid.12 These same guidelines might be used when CVS or amniotic fluid samples are
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being tested in such cases, even though aneuploid metaphases were not observed in the initial standard evaluation. Because CPM has been associated with fetal growth restriction in some reports, serial ultrasound scanning to evaluate fetal growth might also be considered if CPM is suspected.13-15 CPM may occur because of missegregation of chromosomes in mitosis of a normal cell, which results in a lineage of daughter cells receiving 45 or 47 chromosomes. Importantly, CPM may also be the result of “trisomy rescue” in which a cell with 47 chromosomes loses an extra chromosome during mitosis and a lineage of cells with a normal number of chromosomes results. Trisomy rescue poses an increased risk of uniparental disomy, which is a condition in which both members of a chromosome pair are inherited from one parent. Although uniparental disomy is an infrequent occurrence, it may result in phenotypic problems if there are imprinted genes on the specific chromosome that is involved or if there is a recessive mutation that becomes homozygous as a result of the uniparental disomy.16 Even though imprinted genes have not been identified on chromosomes 13 and 18, testing for uniparental disomy might be considered selectively in cases that are associated with false-positive cfDNA testing. The finding of uniparental disomy in such cases might be taken as indirect evidence of “trisomy rescue” that is associated with mosaicism as the cause of the abnormal cfDNA result. Invasive prenatal diagnostic testing may also demonstrate “pseudomosaicism,” which is most often considered an in vitro phenomenon in the laboratory, rather than a reflection of the true fetal or placental chromosome constitution. This perplexing problem occurs less frequently with amniocentesis than it does with CVS. For this reason the finding of mosaicism in CVS often leads to a recommendation for amniocentesis in an attempt to resolve the issue. Although low percentage mosaicism cannot be excluded with certainty, an adequate amniotic fluid study with no abnormal cells coupled with a normal ultrasound scanning is generally somewhat
reassuring regarding clinically significant mosaicism. On the other hand, confirmation of mosaicism by amniocentesis is often considered to reflect the fetal status. Thus, if CPM is identified as a relatively important cause of falsepositive cfDNA testing for certain aneuploidies, one might question whether amniocentesis would be preferred over CVS for diagnostic confirmation in these circumstances. Such a suggestion would have the undesirable effect of losing the benefit of obtaining early test results for these patients. Thus, we believe that suggesting that amniocentesis might be a better confirmatory test should not be considered without scientific evidence to support a clear association of CPM with specific false-positive cfDNA testing results. For several decades, screening for aneuploidy with the use of maternal serum markers and fetal ultrasound scanning has focused on increasing sensitivity while minimizing the number of invasive diagnostic tests.17 Over this time, the positive predictive value (ie, the proportion of positive test results that are true positives) has improved substantially but remains less than ideal. Obstetricians know this because they spend considerable time helping patients understand that a positive screening test does not necessarily mean an affected fetus and that invasive testing by chromosome analysis and possibly microarray is needed to make or to exclude a diagnosis of aneuploidy with a high degree of confidence. Why then might we be alarmed by the observation of falsepositive cfDNA results? The answer may be obvious. The high sensitivity of cfDNA testing and the low falsepositive rate has led some investigators to speculate about the possibility of cfDNA replacing diagnostic testing in the future.18,19 In a competitive environment, the laboratories have extended the testing to the rarer autosomal aneuploidies and recently to sex chromosome aneuploidies. Scientific publications, marketing materials, and clinical laboratory reports emphasize the high sensitivity of the testing but do not state the positive predictive value. It seems that, in the excitement about this
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www.AJOG.org important advance, clinicians may also have lost sight of the fact that, as we test for disorders with lower and lower prevalence, the balance between true positives and false positives shifts. Thus, a sense of alarm on learning about these cases may simply be a result of expectations that have been created for us. We believe that it is unfortunate that the laboratories that offer cfDNA testing have not requested that clinicians report false-positive or false-negative results to them and that they do not provide quantitative laboratory results that could be used in the retrospective analysis of these cases. Although the positive predictive value of cfDNA based on data in the published validation studies appears better than the other currently available methods for aneuploidy screening, it is imperfect and will be influenced by the prevalence of the disease. A published study of routine clinical use in a large population and a study of stored samples in a general population that has aneuploidy screening in the first trimester have shown detection rates that are similar to those in high-risk populations.11,20 It seems likely that these tests will be applied to lower risk populations in the future. If so, we should expect that a larger proportion of positive results will be false positives because of the low prevalence of these rare problems in the general population. For this reason, we believe that data from clinical practice regarding performance characteristics of this testing is needed urgently to avoid having the confidence of physicians and patients in this new testing undermined by additional anecdotal experiences. We discuss low probability biologic explanations and raise hypothetical questions to underscore the need for coordinated clinical and laboratory investigation of these cases. The results of such investigations will be important to inform the pretest and posttest counseling by clinicians who offer cfDNA testing. Also, the development of clinical practice guidelines for the evaluation of cases of potential false-positive cfDNA testing awaits evidence on which to base recommendations. In the meantime, clinicians and cytogeneticists will
www.AJOG.org necessarily individualize evaluation of these patients. We propose that the establishment of a registry for the purpose of gathering information about false-positive and false-negative cfDNA testing would be an important step to address the questions that we raise. Only more systematically gathered data, rather than anecdotal experiences, will help us decide whether to be alarmed by these experiences. Finally, rather than being alarmed by these reports, clinicians should use this experience to remind themselves to discuss the possibility of false-positive test results during pretest counseling for screening, which would include cfDNA testing, and as reinforcement to follow the recommendation to offer diagnostic tests to patients who have a positive screening test.5 ACKNOWLEDGMENTS We acknowledge Robert Debbs, DO, Kristin Kinsler, DO, and Harish Sehdev, MD, for providing information about their patients.
REFERENCES 1. Chiu RW, Lo YM. Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research. Clin Chem Lab Med 2013;51:197-2. 2. Chiu RW, Chan KC, Gao Y, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing
Genetics of DNA in maternal plasma. Proc Natl Acad Sci USA 2008;105:20458-63. 3. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008;105:16266-71. 4. Zimmerman B, Hill M, Gemelos G, et al. Noninvasive prenatal aneuploidy testing of chromosome 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci. Prenat Diagn 2012;32:1233-41. 5. American College of Obstetricians and Gynecologists. Noninvasive prenatal testing for fetal aneuploidy; ACOG committee opinion no. 545. Washington, DC: The College; 2012. 6. 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 2012;14:296-305. 7. Ashoor G, Syngelaki A, Wagner M, Birdir C, Nicolaides KH. Chromosome-selective sequencing of maternal plasma cell-free DNA for first-trimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;206:322.e1-5. 8. Sparks AB, Struble CA, Wang ET, Song K, Oliphant A. 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 2012;206: 319.e1-9. 9. Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ, Rava RP. Genomewide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol 2012;119:890-901. 10. Norton ME, Brar H, Weiss J, et al. Noninvasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;207:137.e1-8.
Clinical Opinion
11. Dan S, Wang W, Ren J, et al. Clinical application of massively parallel sequencingbased prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11 105 pregnancies with mixed risk factors. Prenat Diagn 2012;9:1-8. 12. Hsu LY, Benn PA. Revised guidelines for the diagnosis of mosaicism in amniocytes. Prenat Diagn 1999;19:1081-2. 13. Lestou VS, Kalousek DK. Confined placental mosaicism and intrauterine fetal growth. Arch Dis Child Fetal Neonatal Ed 1998;79:223-6. 14. Wolstenholme J, Rooney DE, Davison EV. Confined placental mosaicism, IUGR, and adverse pregnancy outcome: a controlled retrospective U.K. collaborative survey. Prenat Diagn 1994;14:345-61. 15. Stipoljev F, Latin V, Kos M, Miskovic B, Kurjak A. Correlation of confined placental mosaicism with fetal intrauterine growth retardation: a case control study of placentas at delivery. Fetal Diagn Ther 2001;16:4-9. 16. Kotzot D. Prenatal testing for uniparental disomy: indications and clinical relevance. Ultrasound Obstet Gynecol 2008;31:100-5. 17. American College of Obstetricians and Gynecologists. First-trimester screening for fetal aneuploidy; ACOG committee opinion no. 296. Washington, DC: The College; 2004. 18. Simpson JL. Is cell-free fetal DNA from maternal blood finally ready for prime time? Obstet Gynecol 2012;119:883-5. 19. Wright C, Quake SR, Bianchi D, Wald NJ. Community Corner: Opening the Pandora’s box of prenatal genetic testing. Nat Med 2011;17: 250-1. 20. Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G. Noninvasive prenatal testing for fetal trisomies in a routinely screened firsttrimester population. Am J Obstet Gynecol 2012;207:374.e1-6.
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GENETICS
Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy? Michael T. Mennuti, MD; Athena M. Cherry, PhD; Jennifer J. D. Morrissette, PhD; Lorraine Dugoff, MD
C
ell-free DNA (cfDNA) testing on maternal blood samples recently has been introduced in obstetrics practice as a method for fetal aneuploidy screening.1 This testing examines cfDNA fragments circulating in the maternal plasma that originate primarily from cells of the mother and to a lesser extent from placental cells. The DNA fragments from particular chromosomes are identified by their nucleic acid sequence with a process that is known as massively parallel shotgun sequencing.2,3 Assuming that the maternal cells are euploid, quantitative analysis of the cfDNA fragments is used to predict whether the cells of the pregnancy have the normal or abnormal copy number of specific chromosomes. Recently, one laboratory has introduced the use of parental singlenucleotide polymorphisms as a means of predicting the copy number of specific chromosomes in the placental cells.4 Validation studies in high-risk patients who have undergone invasive diagnostic testing have shown a sensitivity of >98% for detection of trisomy 21 and trisomy 18, with remarkably low false-positive rates (<0.5%) but somewhat lower sensitivity for trisomy From the Department of Obstetrics and Gynecology (Drs Mennuti and Dugoff), Prenatal Cytogenetic Laboratory (Dr Mennuti); Department of Pathology and Laboratory Medicine and Clinical Cancer Cytogenetics (Dr Morrissette), Perelman School of Medicine, University of Pennsylvania, Philadelphia PA; and the Department of Pathology and Cytogenetics Laboratory, Stanford Hospital and Clinics, Stanford University School of Medicine, Palo Alto, CA (Dr Cherry). Received Dec. 10, 2012; revised March 5, 2013; accepted March 19, 2013. The authors report no conflict of interest. Reprints not available from the authors. 0002-9378/$36.00 ª 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2013.03.027
Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making. Key words: cfDNA, chromosome, fetus, positive predictive value, testing
13.5-10 Laboratories that developed the tests and clinical investigators who studied the test performance propose that positive results should be confirmed by invasive prenatal diagnosis before important parental decisions are made regarding the pregnancy. This recommendation is important because the positive predictive value of the test is imperfect (ie, some portion of pregnancies with an abnormal result will not be affected). Although the test methods demonstrate excellent discrimination between euploid pregnancies and those with trisomy 13, 18, or 21, the necessity of establishing a quantitative cutoff inevitably will result in some false-positive and false-negative results. We are aware of only one report of prospective performance of this testing in routine clinical practice.11 Recently, 8 cases in which there were discordant results between an abnormal cfDNA test and the normal cytogenetic testing of the pregnancy have come to our attention.
The cfDNA testing in these cases was not performed in the same laboratory. Massively parallel shotgun sequencing was used to identify the chromosomal origin of cfDNA fragments in all 8 cases. The discordance between the cfDNA testing and the cytogenetic tests dramatically underscores the importance of offering invasive diagnostic testing after an abnormal cfDNA test result. We describe these cases to call attention to the urgent need for studies to understand the possible causes of discordant results so that this information may be used in the development of clinical guidelines for evaluation of similar cases.
Case 1 A 40-year-old woman (G3P1011) had first- and second-trimester serum integrated screening with adjusted risks for Down syndrome of 1:2200, trisomy 18 of 1:10,000, and open neural tube defect of 1:6000. The patient requested a cfDNA
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test at 16 weeks 5 days’ gestation. The result was reported as negative for trisomy 21 but showed an increased representation of chromosome 18 material. At 18.5 weeks’ gestation, the patient had a normal fetal anatomic survey and opted for amniocentesis. Amniocentesis was reported to demonstrate a 46,XY chromosome constitution in 15 metaphases from 12 colonies. Studies of DNA from parental blood samples and from the amniotic cells confirmed biparental inheritance at 3 informative loci on chromosome 18. Ultrasound scans that were performed at 27.6 and 34.6 weeks’ gestation showed normal interval fetal growth. At 41 weeks’ gestation, the patient delivered a healthy male infant who weighed 6 lbs 11 oz. The infant had a normal clinical examination and an uneventful hospital course and was discharged at 2 days of age. Samples for chromosome testing were not obtained on the infant or placenta.
Case 2 A 34-year-old woman (G2P0010) with a maternal age of 35 years at her projected estimated delivery date opted for cfDNA testing at 11 weeks’ gestation. The result was negative for trisomy 21 but showed an increased representation of chromosome 18 material. Amniocentesis that was performed at 16 weeks’ gestation revealed a normal 46,XY chromosome analysis, and the amniotic fluid alphafetoprotein was normal. Fetal anatomic survey by ultrasound scanning was normal. The patient has not yet delivered at the time of this report. Case 3 A 20-year-old primigravid woman had first-trimester aneuploidy screening with adjusted risk for trisomy 21 of 1:10,000 and trisomy 18 of 1:10,000. The first-trimester screening included beta human chorionic gonadotropin (hCG), pregnancy-associated plasma proteineA (PAPP-A), maternal age, and nuchal translucency (NT). No comment regarding visualization of the nasal bone was provided. The patient subsequently had second-trimester maternal serum alpha-protein with adjusted risk of open neural tube defect of 1:1200. A fetal
anatomic survey was normal at 19.3 weeks’ gestation, and fetal biometry lagged 6 days behind the first-trimester ultrasound scan. At 23.3 weeks’ gestation, there was a progressive lag in fetal growth that was noted by ultrasound scanning with an estimate fetal weight at the 12th percentile. A hypoplastic nasal bone and echogenic bowel were noted. The patient declined amniocentesis for chromosome analysis but opted to have cfDNA testing. The cfDNA showed an increased representation of chromosome 18 material. Amniocentesis was performed, and fluorescence in situ hybridization (FISH) was negative for trisomy 18. At 28.4 weeks’ gestation, reversed end diastolic velocity on umbilical artery Doppler velocimetry prompted delivery of a male infant who weighed 570 g (<5th percentile for the gestational age) with an otherwise normal physical examination that was consistent with his gestational age. Karyotypes and single-nucleotide polymorphism microarrays from the amniocentesis and neonatal blood samples were reported as normal male, 46,XY.
Case 4 A 41-year-old woman (G6P3023) had cfDNA testing performed at 11 weeks’ gestation as her initial screening for aneuploidy. Her cfDNA result reported a >99% risk for trisomy 18. Subsequently, first-trimester screening by beta hCG, PAPP-A, and NT was performed and resulted in a risk of 1:720 for trisomy 21 and 1:20,000 for trisomy 18. Information regarding the nasal bone was not provided. A chorionic villus biopsy was performed at 13.1 weeks’ gestation. Both direct and long-term cultures showed a normal male karyotype (46,XY). No abnormalities were noted on subsequent ultrasound scanning, and the pregnancy is continuing. Case 5 A 35-year-old woman (G1P0) had firsttrimester screening by beta hCG and NT measurement at 11 weeks’ gestation that reported a risk of 1:87 for trisomy 21 (hCG 1.48 multiples of the median; PAPP-A, 0.36 multiples of the median; NT, 1.6 mm). Information regarding the
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www.AJOG.org nasal bone was not provided. The cfDNA testing result was positive for trisomy 18. Amniocentesis was performed at 16 weeks’ gestation. FISH on amniotic fluid cells was normal, as was chromosome analysis of cultured amniotic cells that showed a normal female karyotype (46,XX). Subsequent ultrasound scanning showed normal interval growth, and the fetal anatomic evaluation appeared normal. The pregnancy is continuing at the time of this report.
Case 6 A 37-year-old woman (G2P1001) opted for cfDNA testing at 12.6 weeks’ gestation. Ultrasound scanning showed a singleton pregnancy with a crown-rump length measurement that was consistent with the estimated delivery date and a nuchal translucency measurement of 1.6 mm. The cfDNA showed an increased representation of chromosome 13 material. Amniocentesis was performed at 15 weeks’ gestation. Sixty-six cells from 15 different colonies and an additional 34 cells from amniotic cell subcultures were examined. All 100 cells that were examined were found to have a normal 46,XX chromosome constitution. The patient declined uniparental disomy testing and a microarray of the amniotic cells. Ultrasound scanning at 18 weeks’ gestation showed appropriate fetal growth and normal fetal anatomy. Ultrasound assessment at 28 weeks’ gestation is planned. Case 7 A 34-year-old (35 years old at her estimated delivery date) woman (G2P1) had cfDNA testing performed at 12.3 weeks’ gestation. The result was reported as positive for trisomy 13. Chorionic villi sampling (CVS) was performed at 13.3 weeks’ gestation. FISH was positive for trisomy 13 and male gender in 50 cells that were examined. The patient elected to terminate the pregnancy. The karyotype from the CVS culture subsequently was reported as 46XY in 40 cells from 3 independent cultures. Single-nucleotide polymorphism microarray analysis was reported as consistent with a normal male. Examination of a sample of tissue that was submitted
www.AJOG.org for chromosome testing after termination of the pregnancy showed chorionic villi and decidua. No identifiable fetal tissue was available for study in this portion of the sample. Interphase FISH showed 2 signals for chromosome 13; a 46,XY chromosome constitution was noted in all 100 metaphases that were examined.
Case 8 A 34-year-old woman (G2P1) had cfDNA testing that was positive for trisomy 13. Ultrasound scanning showed a single intrauterine pregnancy that was consistent with a gestational age of 13.6 weeks and a nuchal translucency that measured 1.7 mm. Anatomic evaluation of the fetus was normal for the first trimester, with the exception of what appeared to be mild dilation of the renal pelves. Direct preparation on CVS showed 46,XX,þ13,der(13;13)(q10;q10) in 5 of 5 metaphases that were examined. This cytogenetic finding would be expected to result in a cfDNA test result that was positive for trisomy 13 and a trisomy 13 phenotype in the fetus. Two independent long-term cultures from the CVS showed a 46,XX chromosome constitution in 15 of 15 metaphases that were examined. An additional 50 metaphases that were examined from the CVS cultures were euploid. Further review of the direct preparation slides showed the derivative (13;13) translocation chromosome in all metaphases that were examined. Amniocentesis was performed at 15 weeks’ gestation. All 9 primary colonies that were examined had a normal 46,XX chromosome constitution. An additional 41 metaphase from an amniotic cell culture flask also had a 46,XX chromosome constitution. Fetal anatomic evaluation by ultrasound scanning at the time of amniocentesis appeared normal. The pregnancy is continuing at the time of this report, and ultrasound follow-up is planned. Comment Although many of the patients that we described above have not yet delivered their pregnancies, for the purposes of this discussion we will refer to all them as
Genetics having received false-positive cfDNA test results inasmuch as the results were not confirmed by prenatal chromosome analysis, which has been the standard against which the testing has been validated clinically. Given the quantitative nature of the interpretation of cfDNA testing for aneuploidy detection and the low prevalence of trisomy 18 and trisomy 13, it should not be surprising to clinicians that they occasionally may encounter false-positive test results, perhaps without ever having experienced a true positive test result in practice. Without knowing the number of tests that were performed by the laboratories, it is not possible to estimate whether the number of cases with positive cfDNA test results in which the prenatal chromosome testing was not concordant exceed the number of falsepositive cfDNA test results that were expected, based on the published studies. If false-positive test results, in large part, are due to the quantitative nature of the test interpretation, then one would expect that careful analysis might demonstrate that a group of such cases would have test results closer to the cut-off for normal than might be observed for a group with true positive test results. Because it has been shown that the discrimination between normal and aneuploid pregnancies improves with increasing “fetal fraction” of cfDNA, it might also be relevant to know the relative “fetal fraction” in a group of cases with false-positive test results. An informative analysis of false-positive test results would require a much larger collection of cases with quantitative data that is not generally available in the laboratory reports that are issued to clinicians. A rate of false-positive test results that is not higher than expected and a careful study that would demonstrate that these occurrences are most likely a function of the quantitative nature of the test interpretation would reaffirm the importance of always offering invasive testing when there is an abnormal cfDNA test result. The identification of the limitations of the positive predictive value as the likely cause of testing results such as those observed in cases 1-6 would also reduce concern about sample
Clinical Opinion
errors or rare biologic mechanisms that result in false-positive test results. If, on the other hand, the false-positive test results do not appear to be based on the quantitative test interpretation, then other explanations should be sought. These could be important for the future clinical application of the test, for counseling, and for the assessment of patients with positive cfDNA results. Cases 7 and 8 raise important questions about other low probability biologic mechanisms for some false-positive cfDNA results. Inasmuch as the DNA that was measured in this testing is of placental origin, mosaicism for aneuploidy in the placenta is a possible explanation. Mosaicism in the placenta can reflect mosaicism throughout the pregnancy, which includes the fetus, or it can be limited to the placenta (known as confined placental mosaicism [CPM]). Other, much less likely biologic causes of false-positive test results might include the contribution of a vanishing aneuploid twin to the cfDNA in the maternal plasma, a copy number duplication of portion of a fetal chromosome too small to be detected by the standard cytogenetic testing, or a low percentage maternal mosaicism for an autosomal aneuploidy that may be either constitutional or acquired. Concern about maternal mosaicism for sex chromosome aneuploidy is likely to become relevant because gender and sex chromosome aneuploidy are reported increasingly by the laboratories that are performing this testing. Should clinicians and cytogeneticists be concerned that clinically relevant fetal or placental mosaicism may be the cause of what initially appears to be falsepositive cfDNA testing results? If so, when the initial analysis of the confirmatory testing demonstrates all normal cells, it would seem prudent to increase the number of colonies or cells to be examined to increase the confidence that there is not clinically relevant mosaicism. Guidelines for the “extensive work-up for mosaicism” generally are used when only a portion of cells in an amniotic fluid or CVS are aneuploid.12 These same guidelines might be used when CVS or amniotic fluid samples are
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Clinical Opinion
Genetics
being tested in such cases, even though aneuploid metaphases were not observed in the initial standard evaluation. Because CPM has been associated with fetal growth restriction in some reports, serial ultrasound scanning to evaluate fetal growth might also be considered if CPM is suspected.13-15 CPM may occur because of missegregation of chromosomes in mitosis of a normal cell, which results in a lineage of daughter cells receiving 45 or 47 chromosomes. Importantly, CPM may also be the result of “trisomy rescue” in which a cell with 47 chromosomes loses an extra chromosome during mitosis and a lineage of cells with a normal number of chromosomes results. Trisomy rescue poses an increased risk of uniparental disomy, which is a condition in which both members of a chromosome pair are inherited from one parent. Although uniparental disomy is an infrequent occurrence, it may result in phenotypic problems if there are imprinted genes on the specific chromosome that is involved or if there is a recessive mutation that becomes homozygous as a result of the uniparental disomy.16 Even though imprinted genes have not been identified on chromosomes 13 and 18, testing for uniparental disomy might be considered selectively in cases that are associated with false-positive cfDNA testing. The finding of uniparental disomy in such cases might be taken as indirect evidence of “trisomy rescue” that is associated with mosaicism as the cause of the abnormal cfDNA result. Invasive prenatal diagnostic testing may also demonstrate “pseudomosaicism,” which is most often considered an in vitro phenomenon in the laboratory, rather than a reflection of the true fetal or placental chromosome constitution. This perplexing problem occurs less frequently with amniocentesis than it does with CVS. For this reason the finding of mosaicism in CVS often leads to a recommendation for amniocentesis in an attempt to resolve the issue. Although low percentage mosaicism cannot be excluded with certainty, an adequate amniotic fluid study with no abnormal cells coupled with a normal ultrasound scanning is generally somewhat
reassuring regarding clinically significant mosaicism. On the other hand, confirmation of mosaicism by amniocentesis is often considered to reflect the fetal status. Thus, if CPM is identified as a relatively important cause of falsepositive cfDNA testing for certain aneuploidies, one might question whether amniocentesis would be preferred over CVS for diagnostic confirmation in these circumstances. Such a suggestion would have the undesirable effect of losing the benefit of obtaining early test results for these patients. Thus, we believe that suggesting that amniocentesis might be a better confirmatory test should not be considered without scientific evidence to support a clear association of CPM with specific false-positive cfDNA testing results. For several decades, screening for aneuploidy with the use of maternal serum markers and fetal ultrasound scanning has focused on increasing sensitivity while minimizing the number of invasive diagnostic tests.17 Over this time, the positive predictive value (ie, the proportion of positive test results that are true positives) has improved substantially but remains less than ideal. Obstetricians know this because they spend considerable time helping patients understand that a positive screening test does not necessarily mean an affected fetus and that invasive testing by chromosome analysis and possibly microarray is needed to make or to exclude a diagnosis of aneuploidy with a high degree of confidence. Why then might we be alarmed by the observation of falsepositive cfDNA results? The answer may be obvious. The high sensitivity of cfDNA testing and the low falsepositive rate has led some investigators to speculate about the possibility of cfDNA replacing diagnostic testing in the future.18,19 In a competitive environment, the laboratories have extended the testing to the rarer autosomal aneuploidies and recently to sex chromosome aneuploidies. Scientific publications, marketing materials, and clinical laboratory reports emphasize the high sensitivity of the testing but do not state the positive predictive value. It seems that, in the excitement about this
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www.AJOG.org important advance, clinicians may also have lost sight of the fact that, as we test for disorders with lower and lower prevalence, the balance between true positives and false positives shifts. Thus, a sense of alarm on learning about these cases may simply be a result of expectations that have been created for us. We believe that it is unfortunate that the laboratories that offer cfDNA testing have not requested that clinicians report false-positive or false-negative results to them and that they do not provide quantitative laboratory results that could be used in the retrospective analysis of these cases. Although the positive predictive value of cfDNA based on data in the published validation studies appears better than the other currently available methods for aneuploidy screening, it is imperfect and will be influenced by the prevalence of the disease. A published study of routine clinical use in a large population and a study of stored samples in a general population that has aneuploidy screening in the first trimester have shown detection rates that are similar to those in high-risk populations.11,20 It seems likely that these tests will be applied to lower risk populations in the future. If so, we should expect that a larger proportion of positive results will be false positives because of the low prevalence of these rare problems in the general population. For this reason, we believe that data from clinical practice regarding performance characteristics of this testing is needed urgently to avoid having the confidence of physicians and patients in this new testing undermined by additional anecdotal experiences. We discuss low probability biologic explanations and raise hypothetical questions to underscore the need for coordinated clinical and laboratory investigation of these cases. The results of such investigations will be important to inform the pretest and posttest counseling by clinicians who offer cfDNA testing. Also, the development of clinical practice guidelines for the evaluation of cases of potential false-positive cfDNA testing awaits evidence on which to base recommendations. In the meantime, clinicians and cytogeneticists will
www.AJOG.org necessarily individualize evaluation of these patients. We propose that the establishment of a registry for the purpose of gathering information about false-positive and false-negative cfDNA testing would be an important step to address the questions that we raise. Only more systematically gathered data, rather than anecdotal experiences, will help us decide whether to be alarmed by these experiences. Finally, rather than being alarmed by these reports, clinicians should use this experience to remind themselves to discuss the possibility of false-positive test results during pretest counseling for screening, which would include cfDNA testing, and as reinforcement to follow the recommendation to offer diagnostic tests to patients who have a positive screening test.5 ACKNOWLEDGMENTS We acknowledge Robert Debbs, DO, Kristin Kinsler, DO, and Harish Sehdev, MD, for providing information about their patients.
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Genetics of DNA in maternal plasma. Proc Natl Acad Sci USA 2008;105:20458-63. 3. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008;105:16266-71. 4. Zimmerman B, Hill M, Gemelos G, et al. Noninvasive prenatal aneuploidy testing of chromosome 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci. Prenat Diagn 2012;32:1233-41. 5. American College of Obstetricians and Gynecologists. Noninvasive prenatal testing for fetal aneuploidy; ACOG committee opinion no. 545. Washington, DC: The College; 2012. 6. 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 2012;14:296-305. 7. Ashoor G, Syngelaki A, Wagner M, Birdir C, Nicolaides KH. Chromosome-selective sequencing of maternal plasma cell-free DNA for first-trimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;206:322.e1-5. 8. Sparks AB, Struble CA, Wang ET, Song K, Oliphant A. 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 2012;206: 319.e1-9. 9. Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ, Rava RP. Genomewide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol 2012;119:890-901. 10. Norton ME, Brar H, Weiss J, et al. Noninvasive chromosomal evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;207:137.e1-8.
Clinical Opinion
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