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September 28, 2022

Discovery of Cell-Free Fetal DNA in Maternal Blood and Development of Noninvasive Prenatal Testing: 2022 Lasker-DeBakey Clinical Medical Research Award

Author Affiliations
  • 1Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
JAMA. 2022;328(13):1293-1294. doi:10.1001/jama.2022.14982

The 2022 Lasker-DeBakey Clinical Medical Research Award has been presented to Y. M. Dennis Lo, DM, DPhil, for the discovery of fetal DNA in maternal blood, leading to noninvasive prenatal testing for Down syndrome.

Prenatal testing is an established part of modern obstetrics care. However, conventional methods for obtaining fetal tissues for prenatal testing, such as amniocentesis, are invasive and present a small but definite risk to the fetus. Since the 1960s, there have been numerous attempts to use fetal nucleated cells that have entered into maternal blood for prenatal testing. However, the rarity of such cells has impeded this approach from becoming a clinical reality. Because cell-free tumor DNA has been detected in the plasma of patients with cancer, and because there are similarities between a tumor and the placenta implanted and growing inside the uterus, our research group found cell-free fetal DNA in the plasma and serum of pregnant women.1 A genetic marker on the Y chromosome that was present only in male fetuses was used as a marker of fetal DNA in maternal plasma and serum.

My colleagues and I1 also demonstrated that the fractional concentrations of fetal DNA in maternal plasma had mean values of 3.4% in samples collected from gestational weeks 11 to 17 and 6.2% in those collected during the late third trimester.2 These concentrations were several orders of magnitude higher than the fractional concentrations of fetal nucleated cells in maternal blood. The data also demonstrated a higher fractional concentration of fetal DNA in maternal plasma than in serum, thus leading to a preference of plasma as the sample type for subsequent development of noninvasive prenatal testing (NIPT). Altogether, these data provided guidance for the operational parameters of NIPT in terms of the concentration ranges in which such tests could operate and the gestational ages at which such tests would be usable.

Certain fetal nucleated cell types have been reported to persist in the maternal circulation for years after delivery of the baby. Hence, further studies investigated whether such concerns might also be relevant for cell-free DNA. Through the serial analysis of maternal blood after cesarean delivery, our research group3 showed that cell-free fetal DNA was undetectable by 2 hours after delivery, with a clearance half-life of 16 minutes. These data indicated that there should be no concern about cell-free fetal DNA persisting from one pregnancy to the next and thereby adversely affecting the accuracy of NIPT performed in the subsequent pregnancy. Such rapid kinetics of circulating DNA has also been observed in other diagnostic applications of plasma DNA (eg, for monitoring a cancer patient after treatment).

The first applications of cell-free fetal DNA for prenatal testing used genetic markers that the fetus had inherited from its father, but which were absent in the maternal genome. Examples included the Y chromosome mentioned earlier (for prenatal investigation of sex-linked genetic disorders) and the RHD gene (for fetal blood group genotyping in RhD-negative pregnant women).4 Such tests were rapidly adopted by a number of laboratories, such as those in the UK and the Netherlands.

Testing for fetal chromosomal aneuploidies is the most common reason pregnant women seek prenatal testing. However, owing to the relatively low positive predictive value of technologies such as nuchal translucency and maternal serum biochemical screening, most women with positive screen results who are referred for amniocentesis have euploid fetuses; therefore, it would be useful if cell-free fetal DNA could be used for developing a test with a much higher positive predictive value. Because maternal plasma contains a mixture of nucleic acids originating from the mother and the fetus, our research group had initially attempted to develop approaches for targeting the fetal portion of such molecules, using fetal epigenetic changes such as DNA methylation and messenger RNA (mRNA) expressed from the placenta. However, these approaches had significant technical disadvantages, such as the destructive nature of many methylation detection methods and the lability of mRNA, and consequently were not practical then.

An alternative approach is to analyze DNA molecules within maternal plasma without separating them into the fetal and maternal components. The key to this approach is the development of a quantitative platform with a precision that is able to detect minute changes in the ratio of genomic sequences from different chromosomes (eg, chromosome 21 [in the context of trisomy 21 detection] vs another chromosome) of the fetus even if the magnitude of such changes has been diluted by an excess of maternal DNA. Accordingly, our research group and others demonstrated that the use of digital polymerase chain reaction (PCR) was one way in which such precise measurements could be achieved.5,6 Because digital PCR counts only a small subset of plasma DNA molecules targeted by the PCR primers, most of the valuable information carried in the plasma DNA could be considered wasted. In an effort to overcome this shortcoming, we and others demonstrated that the use of information stored in maternal plasma could be maximized through the performance of massively parallel sequencing to sequence millions of plasma DNA molecules at random.7,8 We then performed the first large-scale clinical trial of this technology and achieved a sensitivity of 100% and specificity of 97.9% for the prenatal detection of trisomy 21.9 The high sensitivity and specificity of NIPT for fetal trisomy 21 detection has since been confirmed in multiple clinical studies. The technology was introduced in the latter half of 2011, was subsequently endorsed by multiple professional bodies, and is now used in dozens of countries. NIPT has been extended to other chromosomal aneuploidies, including trisomy 18, trisomy 13, and sex chromosomal aneuploidies, and even to subchromosomal aberrations.

To explore the “limit” of NIPT, we developed in 2010 an approach for sequencing the fetal genome, using maternal blood.10 This work has provided the necessary tools for extending NIPT to multiple types of single-gene diseases (eg, β-thalassemia, congenital adrenal hyperplasia). Apart from its diagnostic implications, this work has also generated detailed information about the size distribution of circulating fetal and maternal DNA. In particular, a 2004 study by our group demonstrated that cell-free fetal DNA had a shorter size distribution than circulating maternal DNA. Our 2010 report also highlighted the relationship between nucleosomal structures and cell-free DNA fragmentation, thus laying the foundation for the subsequent development of cell-free DNA fragmentomics.

The global adoption of NIPT has accelerated research into other diagnostic applications of plasma DNA, now generally referred to as liquid biopsies. The ability of plasma DNA sequencing to interrogate the fetal genome has prompted us and others to develop similar methods for investigating the circulating tumor genome. With advances in sequencing technologies and increasing knowledge of genomics, some of the technologies that we had previously investigated that had not been used clinically for NIPT have finally found their application in cancer screening. One example is the use of DNA methylation markers scattered across the genome in the screening and localization of cancer in multicancer early-detection tests. Another field that liquid biopsies have affected is transplantation. Hence, we discovered in 1998 that donor-derived DNA could be detected in the plasma of patients who had received transplantation. Because circulating DNA is a marker of cell death, subsequent work by our group and others has shown that measurement of donor-derived DNA in bodily fluids can be used to monitor posttransplantation rejection. In summary, cell-free DNA in bodily fluids is a treasure trove for molecular diagnostics and has opened up a noninvasive window into human health.

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Article Information

Corresponding Author: Y. M. Dennis Lo, DM, DPhil, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China (loym@cuhk.edu.hk).

Published Online: September 28, 2022. doi:10.1001/jama.2022.14982

Conflict of Interest Disclosures: Dr Lo reported receiving grants from the Innovation and Technology Commission, Hong Kong SAR government, and Li Ka Shing Foundation; serving as a board member for Take2 and DRA Limited; serving on the advisory boards for Grail and Decheng Capital; holding multiple patents or patent applications in diagnostic applications of cell-free DNA, with royalties paid from Illumina, Sequenom, Grail, Xcelom, Take2, and DRA Limited; and having a recently expired collaborative research agreement with Grail.

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Lo  YMD, Tein  MS, Lau  TK,  et al.  Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.   Am J Hum Genet. 1998;62(4):768-775. doi:10.1086/301800PubMedGoogle ScholarCrossref
Lo  YMD, Zhang  J, Leung  TN, Lau  TK, Chang  AM, Hjelm  NM.  Rapid clearance of fetal DNA from maternal plasma.   Am J Hum Genet. 1999;64(1):218-224. doi:10.1086/302205PubMedGoogle ScholarCrossref
Lo  YMD, Hjelm  NM, Fidler  C,  et al.  Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.   N Engl J Med. 1998;339(24):1734-1738. doi:10.1056/NEJM199812103392402PubMedGoogle ScholarCrossref
Lo  YMD, Lun  FM, Chan  KC,  et al.  Digital PCR for the molecular detection of fetal chromosomal aneuploidy.   Proc Natl Acad Sci U S A. 2007;104(32):13116-13121. doi:10.1073/pnas.0705765104PubMedGoogle ScholarCrossref
Fan  HC, Quake  SR.  Detection of aneuploidy with digital polymerase chain reaction.   Anal Chem. 2007;79(19):7576-7579. doi:10.1021/ac0709394PubMedGoogle ScholarCrossref
Chiu  RWK, Chan  KC, Gao  Y,  et al.  Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma.   Proc Natl Acad Sci U S A. 2008;105(51):20458-20463. doi:10.1073/pnas.0810641105PubMedGoogle ScholarCrossref
Fan  HC, Blumenfeld  YJ, Chitkara  U, Hudgins  L, Quake  SR.  Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.   Proc Natl Acad Sci U S A. 2008;105(42):16266-16271. doi:10.1073/pnas.0808319105PubMedGoogle ScholarCrossref
Chiu  RWK, Akolekar  R, Zheng  YW,  et al.  Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.   BMJ. 2011;342:c7401. doi:10.1136/bmj.c7401PubMedGoogle ScholarCrossref
Lo  YMD, Chan  KC, Sun  H,  et al.  Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus.   Sci Transl Med. 2010;2(61):61ra91. doi:10.1126/scitranslmed.3001720PubMedGoogle ScholarCrossref
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