NIPT assay performance and PPV

* Five twin pregnancies were correctly classified as presence of Y. Two pregnancies were correctly classified as no presence of Y.

** Other cytogenetic results were XXXXX and XXYY.

To estimate the performance of the VeriSeq NIPT Solution v2 in twin pregnancies, in silico models based on observations from clinical samples were used to simulate populations of twin pregnancies. This simulation was consistent with the intended use population. The distribution of fetal fraction was determined from approximately 4,500 twin samples and compared to the distribution from approximately 120,000 singleton samples. The distribution of fetal fraction conditional on aneuploidy status was determined from singleton putative calls (1,044 trisomy 21, 307 trisomy 18, and 192 trisomy 13). Combining the two distributions allowed for inferences of aneuploidy detection in twins. Sets of dizygotic and monozygotic twins were simulated, and a weighted average representing their prevalence in the intended use population was taken (2 dizygotic: 1 monozygotic) to estimate sensitivity. For specificity, sets of unaffected twins were simulated.

The fraction of each simulated sample affected by the trisomy (ie, the affected fraction) was calculated differently for each sample category:

• For monozygotic twins, the affected fraction of each sample was set to 1.0 because, in this situation, the trisomy affects both twins.
• For dizygotic twins, it was assumed that only one twin was affected (to have both dizygotic twins be affected is extremely rare). Affected fraction values were simulated using the known distribution of fetal fraction ratios as determined from sex discordant clinical twin samples. A conservative approach was taken whereby it was assumed that the affected twin always had the lowest fetal fraction of the two twins. A correction factor was applied for fetal fractions being on average lower in trisomy 13 and 18 pregnancies.
• For unaffected twins, the affected fraction of each sample was set to zero.

For twins affected by either trisomy 18 or 13, the fetal fraction corresponding to the affected fraction of the sample was reduced. The reduction was proportional to the average reduction in fetal fraction observed in clinical data in trisomy 18 or 13 singletons versus euploid singletons.

Both the overall fetal fraction and the affected fraction of each simulated sample were then used to calculate an aneuploidy score using the standard VeriSeq NIPT Solution v2 algorithm. Sensitivity was calculated by determining how often the aneuploidy scores for the simulated affected twins were above the corresponding aneuploidy cutoff. Correspondingly, specificity was calculated by determining how often the aneuploidy scores for the simulated unaffected twins were below the corresponding aneuploidy cutoff (Table 6). 95% confidence intervals were estimated based on the number of real clinical twin samples in the original data set, which were classified as either affected or unaffected by the relevant trisomy.

To estimate chromosome Y sensitivity in twin samples, sets of XY/XY and XX/XY twins were simulated. A weighted average representing their prevalence in the intended use population was taken (1 XY/XY: 1 XX/XY). To estimate chromosome Y specificity in twins, a set of XX/XX twins was simulated. The overall fetal fraction values were simulated according to the known distribution of fetal fraction in clinical twin samples.

For XY/XY and XX/XY twins, corresponding chromosome Y scores were estimated using the known relationship between fetal fraction and chromosome Y scores in clinical singleton samples classified as male. For XX/XY twins only, affected (ie, male) fetal fraction values were simulated using the known distribution of fetal fraction ratios observed between twins from the same pregnancy, as determined from sex discordant clinical twin samples. A conservative approach was taken whereby the affected fraction was selected such that it corresponded to the smaller of the two twins. For each simulated XX/XY sample, the chromosome Y score was multiplied by the affected fraction.

For XX/XX twins, chromosome Y scores were sampled from those scores observed in clinical singleton samples classified as female. The chromosome Y score and the overall fetal fraction were then used to classify each simulated sample as chromosome Y present or chromosome Y absent using the standard VeriSeq NIPT Solution v2 algorithm.

Sensitivity was calculated by determining how often the simulated XY/XY or XX/XY twins were correctly classified as chromosome Y present. Specificity was calculated by determining how often the simulated XX/XX twins were correctly classified as chromosome Y absent. 95% confidence intervals were estimated based on the number of real clinical twin samples in the original data set that were classified as either chromosome Y present or chromosome Y absent.

Table 18 provides point estimates and estimated 95% confidence intervals for the sensitivity and specificity of VeriSeq NIPT Solution v2 to detect trisomy 21, 18, 13, and the presence of Y in a simulated population of twin pregnancies consistent with the intended use population. Confidence intervals were estimated based on the number of QC passing clinical twin samples classified as either affected or unaffected by the relevant trisomy. The sensitivity calculation assumes that two thirds of affected twin pregnancies are dizygotic with one affected twin, while one third of affected twin pregnancies are monozygotic with both twins affected.

The estimates listed in Table 6 pertain to twin pregnancies only. Due to even lower prevalence, data for higher- order pregnancies (triplets or higher) were insufficient to establish appropriate statistical models to estimate accuracy of aneuploidy detection.