Ron McCullough

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.

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.

The Suitability of Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry in a Laboratory Developed Test Using Cystic Fibrosis Carrier Screening as a Model

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific, single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by authority laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together, these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets.

Genome-wide cfDNA screening: clinical laboratory experience with the first 10,000 cases

PurposeInvasive diagnostic prenatal testing can provide the most comprehensive information about the genetic status of a fetus. Noninvasive prenatal screening methods, especially when using cell-free DNA (cfDNA), are often limited to reporting only on trisomies 21, 18, and 13 and sex chromosome aneuploidies. This can leave a significant number of chromosomal and subchromosomal copy-number variations undetected. In 2015, we launched a new genome-wide cfDNA screening test that has the potential to narrow this detection gap.MethodsHere, we review the results from the first 10,000 cases submitted to the Sequenom clinical laboratory for genome-wide cfDNA screening.ResultsThe high-risk indication for this cohort differed compared with standard cfDNA screening. More samples were submitted with ultrasound indications (25% compared with 13% for standard cfDNA screening) and fewer for advanced maternal age (51% for genome-wide screening versus 68% for standard cfDNA screening). A total of 554 positive calls were made, of which 164 were detectable only via genome-wide analysis.ConclusionThis reports indicates a difference in utilization compared with standard cfDNA screening, where positivity rates are higher and a large subset of positive calls could not have been made using standard cfDNA screening.

Incidental Detection of Maternal Neoplasia in Noninvasive Prenatal Testing

BACKGROUNDnNoninvasive 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.nnnMETHODSnNIPT 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.nnnRESULTSnIn 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.nnnCONCLUSIONSnIn 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.

Impact of Co-Twin Demise on Circulating Cell-Free DNA (ccfDNA) Testing [10R]

INTRODUCTION: Sequenom Laboratories clinical validation data show high sensitivity and specificity in multifetal gestations. To date more than 16,000 such samples, have been analyzed in clinical practice. A small subset of NIPT results were reported as discordant with fetal outcome. Upon investigation co-twin demise could be identified as a possible cause for the discrepant results. Here we review these co-twin demise discordant results. METHODS: ccfDNA testing using massively parallel sequencing (MPS) on maternal blood was performed by Sequenom Laboratories. RESULTS: We report 37 discrepant NIPT results, 31 accompanied by ultrasound documented co-twin demise and six strongly suspected co-twin demise based on reported clinical findings. The majority of discrepancies were for fetal sex (26). Eleven patients with a co-twin demise were discrepant for trisomies 13, 16, 18, 21, 22, and 1p36 deletion. In two of these cases, one trisomy 21 and one 1p36 deletion, the genetic profile has stabilized at 12 and 15 weeks after the original result. CONCLUSION: Our cases demonstrate that residual ccfDNA from a demised twin may impact NIPT results by way of false positive or discrepant fetal sex results. Many variables confound the issue, such as embryo/fetus reabsorption rate, maternal ccfDNA clearance, and gestational age at demise and at NIPT testing. Evidence of loss is commonly seen in NIPT results 8 weeks or more after demise, illustrating the importance of reporting this clinical information to the testing laboratory. With an increased incidence of fetal mortality in multiple gestations, this biological factor is an important differential in discrepant NIPT cases.

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.