This cohort study investigates the use of longitudinal data from ultradeep sequencing of cell-free DNA in Danish patients with colorectal cancer.

Objective To estimate performance of a single-nucleotide-polymorphism–based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations upon single venopuncture. Methods One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; one thousand fifty-one were within specifications, 518 (49.3%) low-risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female. Results Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, CI: 93.8–100%), trisomy 13 (12/12, CI: 73.5–100%), and fetal sex (358/358 female, CI:99.0–100%; 418/418 male, CI: 99.1–100%), 96.0% for trisomy 18 (24/25, CI: 79.7–99.9%), and 90% for monosomy X (9/10, CI: 55.5–99.8%). Specificity for trisomies 21 and 13 was 100% (905/905 [CI: 99.6–100%] and 953/953 [CI: 99.6–100%], respectively) and for trisomy 18 and monosomy X was 99.9% (938/939 [CI: 99.4–100%] and 953/954 [CI: 99.4–100%], respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (p<0.001, odds ratio: 9.2, CI: 4.4–19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations. Conclusions This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction.

Objective Develop a non-invasive prenatal test based on analysis of cell-free DNA in maternal blood to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y. Methods 166 samples from pregnant women, including eleven trisomy 21, three trisomy 18, two trisomy 13, two 45,X, and two 47,XXY samples were analyzed using an informatics-based method. Cell-free DNA from maternal blood was isolated and amplified using a multiplex PCR assay targeting 11,000 SNPs on chromosomes 13, 18, 21, X, and Y in a single reaction, then sequenced. A Bayesian-based Maximum Likelihood statistical method was applied to determine the chromosomal count of the five chromosomes interrogated in each sample, along with a sample-specific calculated accuracy for each test result. Results The algorithm correctly reported the chromosome copy number at all five chromosomes in 145 samples that passed a DNA quality test, for a total of 725/725 correct calls. The average calculated accuracy for these samples was 99.92%. Twenty-one samples did not pass the DNA quality test. Conclusions This informatics-based method non-invasively detected fetuses with trisomy 13, 18, and 21, 45,X, and 47,XXY with high sample-specific calculated accuracies for each individual chromosome and across all five chromosomes.