Why Cell-Free DNA-Based Noninvasive Prenatal Testing for Fetal Chromosome Anomalies Is Not Diagnostic

Introduction

Since the commercial introduction of cfDNA-based prenatal tests in 2011, a plethora of studies with designs of proof of concept, validation, case reports, large cohort studies, and meta-analyses have been published. Although cfDNA-based prenatal screening for trisomy 21, 18, and 13 has made big steps forward from the very early stages of its use, it soon became evident that false-positive and false-negative results might occur and that many of these have biological causes. Knowledge and understanding of the causes of a false result is essential to enable clinicians and genetic counselors to counsel the patients comprehensively and appropriately, both prior to a test as well as after having received the test result. This will ensure responsible application of an innovative prenatal testing method and allow evidence-based decision making for a mother and subsequently her fetus.

Diagnostic Tests and Screening Tests

The performance of a new test method is usually described by four characteristics: sensitivity, specificity, and positive and negative predictive value. The latter two depend on the prevalence of the disease in a given population. The combination of sensitivity and false-positive rate (1 − specificity) allows a test method to be classified as a screening- or a diagnostic test for a given prevalence and cutoff. Sensitivity is defined as the ability of a test to correctly identify patients with a disease. In contrast, specificity describes the ability of a test to identify persons without the disease. These two test variables are fundamentally different between a diagnostic test and a screening test: diagnostic tests require both sensitivity and specificity to be as close as possible to 100%, whereas screening tests are usually characterized by either a high sensitivity or a high specificity. cfDNA-based prenatal testing for trisomy 21 has both, a high sensitivity and a high specificity . The occurrence of false results though prevents it from being a diagnostic test. Most of these false results find their origin in the placenta or in the mother. They may have an irreversible impact on pregnancy management and decisions about pregnancy termination. cfDNA-based prenatal testing is still considered as a screening test implicating confirmation of all positive test results by an independent test method.

Sensitivity and Specificity of CELL-FREE DNA-Based Prenatal Testing and Their Consequences for Predictive Values

Several meta-analyses on cfDNA-based prenatal testing have been published to date . To illustrate the current performance of cfDNA-based prenatal tests, the results of Mackie et al. are shown here .

Table 1 shows the sensitivity and specificity of cfDNA-based prenatal tests for trisomy 13, 18, 21 and monosomy X, which for trisomy 21 reach levels close to 100%. The sensitivity is highest for trisomy 21 and decreases for trisomy 18, monosomy X, and trisomy 13. Neither the cfDNA NIPT method (whole genome or targeted sequencing; qPCR or microarray data were not included) nor the test population's a priori risk (high risk vs low risk) impacted test characteristics. Reasons for false results mentioned in individual studies were as follows: fetal fraction below a predefined cutoff, confined placental mosaicism (CPM), and maternal copy number variations (CNVs) were the most common explanations provided. Gil et al. , who only included studies reporting clinical outcome in more than 85% of cases, reported slightly higher detection rates: 99.7% for trisomy 21, 97.9% for trisomy 18, 99% for trisomy 13, and 95.8% for monosomy X.

To further illustrate the performance metrics, we assume a hypothetical general obstetric population of 100,000 patients with a trisomy 21 prevalence of 0.5%. This translates into 500 pregnant women carrying a fetus with trisomy 21. cfDNA-based prenatal testing, with its meta-analytical sensitivity of 99.4% identifies 497 of them, while three cases will be missed. From the 99,500 pregnancies without trisomy 21, ~ 100 will receive a false-positive result given the specificity of 99.9%. A positive predictive value (PPV) can be calculated with these values and shows how likely it is that a pregnant woman with a trisomy 21 positive cfDNA NIPT report actually carries a fetus with trisomy 21: given a disease prevalence of 0.5%, the PPV will be 83.2%. By comparison, in a higher risk population with a trisomy 21 prevalence of 3%, 3000 out of 100,000 patients carry a fetus with trisomy 21: 2982 trisomy 21 cases will be detected but 18 will be missed; 97,000 pregnant women are trisomy 21 negative, but 97 will receive a false-positive result. The PPV will be 96.8%. These hypothetical calculations demonstrate that PPVs depend on disease prevalence and that the personal a priori risk influences the significance of an abnormal cfDNA NIPT result for the individual patient. As trisomy negative pregnancies are always in excess in a pregnant population (representing a high nondisease prevalence) and meta-analytical specificity values of cfDNA-based NIPT reach almost 100% independent of the chromosome tested, the negative predictive value (NPV) is always close to 100% both in high- and low-risk populations ( Table 2 ).

False-Positive Results of CELL-FREE DNA-Based Prenatal Testing

The main reason why cfDNA-based prenatal testing is currently classified as a screening test is the occurrence of false-positive results. These have important implications for pregnancy management and parental decisions.