Background
Since its commercial release in 2011 cell-free DNA screening has been rapidly adopted as a routine prenatal genetic test. However, little is known about its performance in actual clinical practice.
Objective
We sought to investigate factors associated with the accuracy of abnormal autosomal cell-free DNA results.
Study Design
We conducted a retrospective cohort study of 121 patients with abnormal cell-free DNA results from a referral maternal-fetal medicine practice from March 2013 through July 2015. Patients were included if cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, or microdeletions (if reported by the laboratory) were positive or nonreportable. The primary outcome was confirmed aneuploidy or microarray abnormality on either prenatal or postnatal karyotype or microarray. Secondary outcomes were identifiable associations with in vitro fertilization, twins, ultrasound findings, testing platform, and testing laboratory. Kruskal-Wallis or Fisher exact tests were used as appropriate.
Results
A total of 121 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, and/or microdeletions. In all, 105 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, and trisomy 13. Of these, 92 (87.6%) were positive and 13 (12.4%) were nonreportable. The results of the 92 positive cell-free DNA were for trisomy 21 (48, 52.2%), trisomy 18 (22, 23.9%), trisomy 13 (17, 18.5%), triploidy (2, 2.2%), and positive for >1 parameter (3, 3.3%). Overall, the positive predictive value of cell-free DNA was 73.5% (61/83; 95% confidence interval, 63–82%) for all trisomies (by chromosome: trisomy 21, 83.0% [39/47; 95% confidence interval, 69–92%], trisomy 18, 65.0% [13/20; 95% confidence interval, 41–84%], and trisomy 13, 43.8% [7/16; 95% confidence interval, 21–70%]). Abnormal cell-free DNA results were associated with positive serum screening (by group: trisomy 21 [17/48, 70.8%]; trisomy 18 [7/22, 77.8%]; trisomy 13 [3/17, 37.5%]; nonreportable [2/13, 16.7%]; P = .004), and abnormal first-trimester ultrasound (trisomy 21 [25/45, 55.6%]; trisomy 18 [13/20, 65%]; trisomy 13 [6/14, 42.9%]; nonreportable [1/13, 7.7%]; P = .003). There was no association between false-positive rates and testing platform, but there was a difference between the 4 laboratories ( P = .018). In all, 26 patients had positive (n = 9) or nonreportable (n = 17) microdeletion results. Seven of 9 screens positive for microdeletions underwent confirmatory testing; all were false positives.
Conclusion
The positive predictive value of 73.5% for cell-free DNA screening for autosomal aneuploidy is lower than reported. The positive predictive value for microdeletion testing was 0%. Diagnostic testing is needed to confirm abnormal cell-free DNA results for aneuploidy and microdeletions.
Introduction
Introduced in 2011 to clinical practice, cell-free fetal DNA (cfDNA) has changed the landscape of prenatal care, despite recent guidelines recommending that its use be limited to screening for the most common aneuploidies (trisomy [T]21, T18, T13) in singleton gestations. Numerous case-controlled studies emerged reporting high sensitivities and specificities for the most common aneuploidies with low false-positive rates. The highest sensitivity (>99%) and lowest false-positive rates (1%) are reported for T21, with slightly lower sensitivities for T18 (97-99%) and T13 (79-92%). While detection rates are impressive, it is notable that specific detection rates vary depending on the laboratory used. Moreover, these data are published from studies evaluating high-risk populations, and at the time of the study, validation was limited for the general population, although recent data are encouraging. In addition, few reports have examined outcomes for microdeletion testing, which is clinically reported despite the lack of validation or endorsement by professional societies.
CfDNA has become widely adopted into routine prenatal care, and studies have reviewed its impact on patient decision-making. However, most studies have focused on large, laboratory-funded validation trials. In this study, we aimed to review all abnormal cfDNA results from a single high-volume referral maternal-fetal medicine practice to evaluate the performance of cfDNA in current clinical practice. Given the frequency of our observed false-positive results, we hypothesized that the test performance of cfDNA in current clinical practice is not as high as previously published.
Materials and Methods
This is a retrospective cohort study of all patients with abnormal cfDNA results from a referral maternal-fetal medicine practice in Los Angeles, CA, from March 2013 through July 2015. The patient population is derived from local referrals for prenatal ultrasound (US) screening (first and second trimester), as well as consultations for high-risk pregnancies. In total, the referring physician groups manage approximately 3000 deliveries per year, of which >95% of patients are insured. The median maternal age of the referral base is 37.5 years, and approximately 50% are advanced maternal age. The ethnic breakdown of the referral population is: 60.0% Caucasian, 17.4% Asian, 11.6% Hispanic, 6.2% African American, and 4.9% other. The majority of patients have their prenatal genetic screening tests ordered by their primary obstetric provider prior to being seen at this center. Approval for this study was granted by the Institutional Review Board at the University of California, Los Angeles.
Patients were included if cfDNA screening results for chromosomes 21, 18, 13, or microdeletions (when reported) were abnormal, defined as positive or nonreportable (NR). Because the recommendation for clinical follow-up is the same as for a positive result, tests reported as “suspected positive” or “borderline” (which was the terminology used by one of the companies in early reports, indicating higher aneuploidy risk, but not meeting cutoffs for a positive result) were considered positive for the purpose of this study. A NR result for any chromosome or individual microdeletion was categorized as NR, even if all other parameters reported (any chromosome or microdeletion) were negative. A microdeletion screen was considered abnormal and included if at least 1 of the reported individual microdeletions had a positive or NR result. In this study, a NR microdeletion result refers to the result stating “risk unchanged, unable to further refine risk” on the study report.
Cases were categorized into the following groups for subgroup analysis: (1) T21 positive only; (2) T18 positive only; (3) T13 positive only; (4) triploidy; (5) >1 if positive for 1 of T21, T18, or T13, and in addition, either positive or NR for a second independent parameter (T21, T18, T13, microdeletion, or sex chromosome); (6) NR for T21, 18, or 13; (7) negative cfDNA results but discordant with confirmatory testing; (8) only microdeletion positive; and (9) only microdeletion unreportable. Screens positive for isolated sex chromosome aneuploidies were not included in this study. However, if the cfDNA test was positive or NR for an autosomal aneuploidy or microdeletion, an abnormal result for the sex chromosomes was considered as a second abnormality in the analysis. Each individual patient was counted as 1 case, even if the cfDNA test was repeated with a new clinical sample. For the analysis of factors associated with true- vs false-positive results, all positive cases for each chromosome (21, 18, or 13) with confirmed outcomes were analyzed independently, as well as in composite. For this portion of the analysis investigating factors associated with test accuracy, cases of triploidy were excluded, as these cases are rare and results may be confounded by atypical factors, such as maternal malignancy.
The following demographic information was abstracted from the medical record: maternal age, in vitro fertilization, cfDNA laboratory, and multiple gestation. In addition, the results of these additional screening tests were recorded if obtained: serum screening (first-trimester serum screening, integrated screening, or second-trimester quad screening), nuchal translucency (NT), and first- and/or second-trimester US findings. Positive serum screening was determined by the California Prenatal Screening Program age-based cutoffs for first- or second-trimester screening. In California, first-trimester screening combines serum analyte testing with first-trimester US. NT measurements were performed by sonographers and/or physicians certified by the NT Quality Review Program.
The primary outcome was concordance of cfDNA results with confirmatory diagnostic testing (by chorionic villus sampling or amniocentesis) or postnatal genetic evaluation. Standard karyotype analysis was performed by a single cytogenetics laboratory. Microarray analysis was recommended in cases of microdeletion-positive screens, and was performed if the patient granted consent. Neonatal outcomes were reported by the patient’s primary obstetric provider in follow-up. Results for T21, T18, and T13 were considered independently. Secondary outcomes were associations with maternal age as a continuous variable, advanced maternal age (≥35 years, as a dichotomous variable), in vitro fertilization, twins, US findings, testing platform, and laboratory. Pregnancy outcomes were also obtained (termination of pregnancy, spontaneous loss, normal delivery, or lost-to-follow-up). Positive predictive value (PPV) calculations were made based on the total number of patients with confirmatory testing with karyotype or microarray testing; as expected, some patients were either lost to follow-up or declined confirmatory testing, and so were not included in the final denominators.
Kruskal-Wallis (continuous variables) or Fisher exact test (categorical variables) was used as appropriate to calculate P values. Software (Stata 14; StataCorp LP, College Station, TX) was used for all statistical analysis.
Materials and Methods
This is a retrospective cohort study of all patients with abnormal cfDNA results from a referral maternal-fetal medicine practice in Los Angeles, CA, from March 2013 through July 2015. The patient population is derived from local referrals for prenatal ultrasound (US) screening (first and second trimester), as well as consultations for high-risk pregnancies. In total, the referring physician groups manage approximately 3000 deliveries per year, of which >95% of patients are insured. The median maternal age of the referral base is 37.5 years, and approximately 50% are advanced maternal age. The ethnic breakdown of the referral population is: 60.0% Caucasian, 17.4% Asian, 11.6% Hispanic, 6.2% African American, and 4.9% other. The majority of patients have their prenatal genetic screening tests ordered by their primary obstetric provider prior to being seen at this center. Approval for this study was granted by the Institutional Review Board at the University of California, Los Angeles.
Patients were included if cfDNA screening results for chromosomes 21, 18, 13, or microdeletions (when reported) were abnormal, defined as positive or nonreportable (NR). Because the recommendation for clinical follow-up is the same as for a positive result, tests reported as “suspected positive” or “borderline” (which was the terminology used by one of the companies in early reports, indicating higher aneuploidy risk, but not meeting cutoffs for a positive result) were considered positive for the purpose of this study. A NR result for any chromosome or individual microdeletion was categorized as NR, even if all other parameters reported (any chromosome or microdeletion) were negative. A microdeletion screen was considered abnormal and included if at least 1 of the reported individual microdeletions had a positive or NR result. In this study, a NR microdeletion result refers to the result stating “risk unchanged, unable to further refine risk” on the study report.
Cases were categorized into the following groups for subgroup analysis: (1) T21 positive only; (2) T18 positive only; (3) T13 positive only; (4) triploidy; (5) >1 if positive for 1 of T21, T18, or T13, and in addition, either positive or NR for a second independent parameter (T21, T18, T13, microdeletion, or sex chromosome); (6) NR for T21, 18, or 13; (7) negative cfDNA results but discordant with confirmatory testing; (8) only microdeletion positive; and (9) only microdeletion unreportable. Screens positive for isolated sex chromosome aneuploidies were not included in this study. However, if the cfDNA test was positive or NR for an autosomal aneuploidy or microdeletion, an abnormal result for the sex chromosomes was considered as a second abnormality in the analysis. Each individual patient was counted as 1 case, even if the cfDNA test was repeated with a new clinical sample. For the analysis of factors associated with true- vs false-positive results, all positive cases for each chromosome (21, 18, or 13) with confirmed outcomes were analyzed independently, as well as in composite. For this portion of the analysis investigating factors associated with test accuracy, cases of triploidy were excluded, as these cases are rare and results may be confounded by atypical factors, such as maternal malignancy.
The following demographic information was abstracted from the medical record: maternal age, in vitro fertilization, cfDNA laboratory, and multiple gestation. In addition, the results of these additional screening tests were recorded if obtained: serum screening (first-trimester serum screening, integrated screening, or second-trimester quad screening), nuchal translucency (NT), and first- and/or second-trimester US findings. Positive serum screening was determined by the California Prenatal Screening Program age-based cutoffs for first- or second-trimester screening. In California, first-trimester screening combines serum analyte testing with first-trimester US. NT measurements were performed by sonographers and/or physicians certified by the NT Quality Review Program.
The primary outcome was concordance of cfDNA results with confirmatory diagnostic testing (by chorionic villus sampling or amniocentesis) or postnatal genetic evaluation. Standard karyotype analysis was performed by a single cytogenetics laboratory. Microarray analysis was recommended in cases of microdeletion-positive screens, and was performed if the patient granted consent. Neonatal outcomes were reported by the patient’s primary obstetric provider in follow-up. Results for T21, T18, and T13 were considered independently. Secondary outcomes were associations with maternal age as a continuous variable, advanced maternal age (≥35 years, as a dichotomous variable), in vitro fertilization, twins, US findings, testing platform, and laboratory. Pregnancy outcomes were also obtained (termination of pregnancy, spontaneous loss, normal delivery, or lost-to-follow-up). Positive predictive value (PPV) calculations were made based on the total number of patients with confirmatory testing with karyotype or microarray testing; as expected, some patients were either lost to follow-up or declined confirmatory testing, and so were not included in the final denominators.
Kruskal-Wallis (continuous variables) or Fisher exact test (categorical variables) was used as appropriate to calculate P values. Software (Stata 14; StataCorp LP, College Station, TX) was used for all statistical analysis.
Results
During the study period, 121 patients had abnormal cfDNA testing. Abnormal cfDNA results for T21, T18, or T13 were found in 105 patients. Of these, 92 (87.6%) were positive and 13 (12.4%) were NR. An additional 16 patients had abnormal results for microdeletions only. The sequence of patient treatment and outcomes is shown in the Figure .
In the 105 patients with abnormal results for chromosomes 21, 18, or 13 on cfDNA screening ( Table 1 ), as expected the most common positive result was T21 (48/92, 52%), followed by T18 (22/92, 24%), and T13 (17/92, 19%). There were 2 cases of triploidy, and 3 cases in which >1 parameters were abnormal. Patients with a positive results for T21, T18, T13, or triploidy were more likely to be older ( P = .007) and of advanced maternal age (≥35 years, P = .017). When comparing between cfDNA result groups, there were significant differences noted in the distribution of test results over the 4 commercially available laboratories used ( P = .006). Although fewer tests were performed by single nucleotide polymorphism sequencing technology, single nucleotide polymorphism sequencing was more likely to give >1 positive or NR results than the other platforms ( P < .001).
| All | T21 | T18 | T13 | Triploidy | >1 a | NR | P | |
|---|---|---|---|---|---|---|---|---|
| Cases | 105 | 48 (45.7) | 22 (21.0) | 17 (16.2) | 2 (1.9) | 3 (2.9) | 13 (12.4) | |
| Age, y, median (range) | 38 (27–48) | 38 (29–47) | 39 (27–48) | 38 (30–45) | 42 (40–44) | 34 (31–37) | 35 (29–41) | .007 b |
| AMA | 87 (82.9) | 42 (87.5) | 20 (90.9) | 15 (88.2) | 2 (100) | 1 (33.3) | 7 (53.9) | .017 b |
| IVF | 17/103 (16.5) | 5/47 (10.6) | 3/21 (14.3) | 4 (23.5) | 2 (100) | 0 | 3 (23.1) | .066 |
| Twins | 10 (9.5) | 4 (8.3) | 1 (4.6) | 3 (17.7) | 1 (50.0) | 0 | 1 (7.7) | .307 |
| Laboratory | .006 b | |||||||
| 1 | 37 (35.2) | 17 (35.4) | 10 (45.5) | 7 (41.2) | 0 | 0 | 3 (23.1) | |
| 2 | 38 (36.2) | 22 (45.8) | 9 (40.9) | 5 (29.4) | 0 | 1 (33.3) | 1 (7.7) | |
| 3 | 28 (26.7) | 8 (16.7) | 2 (9.1) | 5 (29.4) | 2 (100) | 2 (66.7) | 9 (69.2) | |
| 4 | 2 (1.9) | 1 (2.1) | 1 (4.6) | 0 | 0 | 0 | 0 | |
| Platform c | <.001 b | |||||||
| MPS | 75 (71.4) | 39 (81.3) | 19 (86.4) | 12 (70.6) | 0 | 1 (33.3) | 4 (30.8) | |
| SNP | 28 (26.7) | 8 (16.7) | 2 (9.1) | 5 (29.4) | 2 (100) | 2 (66.7) | 9 (69.3) | |
| TS | 2 (1.9) | 1 (2.1) | 1 (4.6) | 0 | 0 | 0 | 0 |
a >1 Parameter was abnormal (eg, trisomy and microdeletion; trisomy and sex chromosome; trisomy and NR)
b Statistically significant ( P < .05)
c MPS is used by laboratories 1 and 2; SNP sequencing/microarray by laboratory 3, TS by laboratory 4.
Screening and pregnancy outcomes stratified by cfDNA results are shown in Table 2 . Overall, 56 of 105 subjects (53.3%) underwent a form of traditional serum screening in addition to cfDNA testing. A reported positive cfDNA result for T21, T18, or T13 was associated more frequently with both a positive serum screen result ( P = .004) and first-trimester US findings ( P = .003), when compared to patients with NR results. Positive T18 was more likely to have NT ≥ 3.5 mm ( P = .019) compared to the other results. Abnormal findings on first- and second-trimester US (if performed, n = 98 and 43, respectively) are listed in Table 3 . Cases with NR results were more likely to undergo repeat cfDNA screening ( P < .001), which was repeated from 1-2 times (described below).
| All | T21 | T18 | T13 | Triploidy | >1 a | NR | P | |
|---|---|---|---|---|---|---|---|---|
| Cases | 105 | 48 | 22 | 17 | 2 | 3 | 13 | |
| Serum screening | 56 | 24 | 9 | 8 | 2 | 1 | 12 | <.001 b |
| First trimester | 39/56 (69.6) | 23 (95.8) | 8 (88.9) | 4 (50.0) | 1 (50.0) | 1 (100) | 3 (25.0) | |
| Integrated | 11/56 (19.6) | 0 | 1 (11.1) | 2 (25.0) | 1 (50.0) | 0 | 7 (58.3) | |
| Quad | 6/56 (10.7) | 1 (4.2) | 0 | 2 (25.0) | 0 | 0 | 2 (16.7) | |
| Serum screen positive c | 29/56 (51.8) | 17 (70.8) | 7 (77.8) | 3 (37.5) | 0 | 0 | 2 (16.7) | .004 b |
| Abnormal US findings | ||||||||
| First-trimester US | 45/97 (46.4) | 25/45 (55.6) | 13/20 (65.0) | 6/14 (42.9) | 0/2 | 0/3 | 1/13 (7.7) | .003 b |
| NT ≥3.5 mm | 28/91 (30.8) | 13/41 (31.7) | 11/18 (61.1) | 3/14 (21.4) | 0/2 | 0/3 | 1/13 (7.7) | .019 b |
| Second-trimester US | 7/42 (16.7) | 3/13 (23.1) | 1/8 (12.5) | 2/10 (20) | 0/1 | 0/1 | 1/9 (11.1) | .963 |
| Repeat cfDNA | 11/105 (10.5) | 2 (4.2) | 0 | 0 | 2 (100) | 0/3 | 7 (53.9) | <.001 b |
| Diagnostic testing | 84/103 (81.6) | 43/47 (91.5) | 19/22 (86.4) | 15/17 (88.2) | 0/2 | 3/3 (100) | 4/13 (30.8) | <.001 b |
| CVS | 70/103 (68.0) | 38/47 (80.9) | 15/22 (68.2) | 12/17 (70.6) | NA | 3/3 | 2/12 (16.7) | |
| Amniocentesis | 14/103 (13.6) | 5/47 (10.6) | 4/22 (18.2) | 3/17 (17.7) | NA | 0 | 2/12 (16.7) | |
| Abnormal genetics | ||||||||
| Karyotype | 60/84 (71.4) | 37/43 (86.1) | 12/19 (63.2) | 8/15 (53.3) | NA | 2/3 (66.7) | 1/4 (25.0) | .010 b |
| Microarray | 2/6 (33.3) | 0/1 | 1/3 | 1/1 | NA | NA | 0/1 | >.999 |
| Pregnancy outcome | <.001 b | |||||||
| TOP | 62/105 (59.1) | 37/48 (77.1) | 14/22 (63.6) | 8/17 (47.1) | 0/2 | 2/3 (66.7) | 1/13 (7.7) | |
| Spontaneous loss | 5/105 (4.8) | 2/48 (4.2) | 1/22 (4.6) | 1/17 (5.9) | 0/2 | 0/3 | 1/13 (7.7) | |
| Delivered | 35/105 (33.3) | 9/48 (18.8) | 4/22 (18.2) | 8/17 (47.1) | 2/2 | 1/3 (33.3) | 11/13 (84.6) | |
| Lost to follow-up | 3/105 (2.9) | 0/48 | 3/22 (13.6) | 0/17 | 0/2 | 0/3 | 0/13 | |
| No confirmed outcome | 7 | 3 | 3 | 1 | 0 | 0 | 0 | |
| Confirmed aneuploidy d | 62/98 (63.3) | 38/45 (84.4) | 12/19 (63.2) | 9/16 (56.3) | 0/2 | 2/3 (66.7) | 1/13 (7.7) | <.001 b |
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