Juan-Sebastian Saldivar

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma

Whole‐genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes.

Non-Invasive Prenatal Chromosomal Aneuploidy Testing - Clinical Experience: 100,000 Clinical Samples

Objective As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. Study Design The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA–licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. Results NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. Conclusion NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

Detection of Fetal Subchromosomal Abnormalities by Sequencing Circulating Cell-Free DNA from Maternal Plasma

BACKGROUND The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. METHODS We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the events location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. RESULTS We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. CONCLUSIONS The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

Clinical outcome of subchromosomal events detected by whole‐genome noninvasive prenatal testing

A novel algorithm to identify fetal microdeletion events in maternal plasma has been developed and used in clinical laboratory‐based noninvasive prenatal testing. We used this approach to identify the subchromosomal events 5pdel, 22q11del, 15qdel, 1p36del, 4pdel, 11qdel, and 8qdel in routine testing. We describe the clinical outcomes of those samples identified with these subchromosomal events.

Factors affecting levels of circulating cell‐free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing

Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction.

Uterine leiomyoma confounding a noninvasive prenatal test result

Sequenom Laboratories, San Diego, CA, USA Department of Obstetrics and Gynecology, Saitama Medical University Hospital, Saitama, Japan Perinatal Medical Center, Saitama Medical Center, Jichi Medical University, Saitama, Japan GeneTech Inc., Tokyo, Japan Sequenom Laboratories, Grand Rapids, MI, USA Department of Obstetrics, Gynecology and Reproductive Medicine, Michigan State University, East Lansing, MI, USA *Correspondence to: Nilesh G. Dharajiya. E-mail: ndharajiya@sequenom.com These authors contributed equally to this article.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Noninvasive prenatal testing for microdeletion syndromes and expanded trisomies: proceed with caution.

To the Editor: Drs. Vora and O’Brien indicate that noninvasive prenatal testing for chromosomal microdeletions has not been sufficiently validated. Although the subtitle, “Proceed With Caution,” appears as unassailable advice, some statements vacillate between incorrect and misleading. The validation of the MaterniT21 PLUS test was conducted as mandated by the College of American Pathologists (CAP), which has deemed regulatory status under Clinical Laboratory Improvement Amendments (CLIA), as promulgated by the Department of Health and Human Services. Laboratories such as ours that are CLIAcertified and CAP-accredited undergo a rigorous credentialing process covering all aspects of laboratory medicine. Furthermore, our laboratory currently offers testing in high-risk pregnancies, consistent with American College of Obstetricians and Gynecologists, Society for Maternal-Fetal Medicine, National Society of Genetic Couselors, and International Society for Prenatal Diagnosis guidelines. The low prevalence of these microdeletions, making a statistically powered clinical study extremely challenging, keeps the positive predictive value low, which is why our test favors specificity by design and is reported as an additional finding (a model following American College of Medical Genetics guidelines for reporting incidental sequencing findings). These relevant microdeletions were selected based on clinician and key opinion leader input. Detecting trisomy 16 and 22 provides an opportunity for a couple to mentally prepare for a miscarriage, but more importantly answers their question, “Why?” Clinicians must understand the technical limitations of any test, and we agree that the current standard of care for confirmatory diagnoses is microarray analysis. However, if we see a clear indication one is present, with clinician and patient assent, it is in their best interest to know this information. Fetal fraction and deletion size are known factors in detecting microdeletions, and these were accounted for in designing our test (manuscript in review). Our blinded validation studies were performed independently at both Sequenom Laboratories’ CLIA and CAP locations. Two-hundred sixty-four samples with documented duplication and deletion lesions or enriched genomic mixtures were analyzed. Insertions and deletions of more than 2 Mb were detected with sensitivity and specificity of 100% and 99.33%, respectively. We thank the authors for the opportunity to educate our colleagues about the regulatory standards of laboratory medicine and thought processes we incorporated into this innovative and clinically useful test to the obstetrics and gynecology community. Users of the MaterniT21 PLUS test may rest assured that this test has proceeded appropriately through the yellow light of caution and into the green light of availability.

Incidental Detection of Maternal Neoplasia in Noninvasive Prenatal Testing

BACKGROUND Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy. METHODS NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples. RESULTS In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification. CONCLUSIONS In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.

Abstract 421: Non reportable results - maternal neoplasm can alter results from non-invasive prenatal testing (NIPT)

Cell free DNA is a powerful new analyte for the detection of chromosomal abnormalities of fetuses from the plasma of pregnant women. In non-invasive prenatal testing (NIPT), cfDNA is contributed into the plasma by the placenta. In rare cases, these results are distorted by additional sources of cfDNA in the maternal bloodstream. For example, organ transplantation can severely affect the cfDNA levels and lead to uninterpretable results. Another source of cfDNA that can lead to similar difficulties and ultimately to a non-reportable NIPT results, can be a maternal neoplasm. In our clinical laboratory, we have processed samples from over 450,000 pregnant patients. In cases of uninterpretable findings due to aberrant genomic profiles, potential reasons for non-reportable results were discussed with the caregiving physician. When feedback about these cases was available, it was collected in a database. In summary, we observed non-reportable NIPT results with aberrant genomic profile for 55 cases. A set of 43 had sufficient information available to allow inclusion in this summary. In 40 cases, a maternal neoplasm was confirmed (18 malignant, 20 benign, and two with imaging but without pathological confirmation). These observational results support further investigation by prospective controlled trials. Citation Format: Daniel Grosu, Nilesh G. Dharajiya, Ron M. McCullough, Youting Sun, Juan-Sebastian Saldivar, Dirk van den Boom, Mathias Ehrich. Non reportable results - maternal neoplasm can alter results from non-invasive prenatal testing (NIPT). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 421.