Case 1:20-cv-01644-RGA Document 1-14 Filed 12/03/20 Page 1 of 4 PageID #: 851
`Case 1:20-cv-01644-RGA Document 1-14 Filed 12/03/20 Page 1 of 4 PageID #: 851
`
`
`
`
`
`EXHIBIT 14
`
`
`
`
`
`EXHIBIT 14
`
`
`
`
`
`
`

`

`Case 1:20-cv-01644-RGA Document 1-14 Filed 12/03/20 Page 2 of 4 PageID #: 852
`
`EDITORIALS
`
`Editorials represent the opinions
`of the authors and THE JOURNAL and not those of
`the American Medical Association.
`
`Cell-Free Fetal DNA in Maternal Blood
`Evolving Clinical Applications
`Joe Leigh Simpson, MD
`Farideh Bischoff, PhD
`
`IN THIS ISSUE OF THE JOURNAL, THE FINDINGS REPORTED
`
`in the study by Dhallan and colleagues1 on enhancing
`recovery of cell-free DNA in maternal blood have ma-
`jor clinical implications. Developing a reliable, trans-
`portable technology for cell-free DNA analysis impacts 2 cru-
`cial areas—prenatal genetic diagnosis and cancer detection
`and surveillance. In prenatal genetic diagnosis, detecting a
`fetal abnormality without an invasive procedure (or with
`fewer invasive procedures) is a major advantage. Likewise
`in cancer surveillance (eg, in patients with leukemia), moni-
`toring treatment without having to perform a bone mar-
`row aspiration for karyotype also would be of great benefit.
`Prenatal genetic diagnosis has been available in the United
`States since 1968, when chromosomal abnormalities and meta-
`bolic traits proved detectable by analysis of amniotic fluid cells
`obtained by amniocentesis. The most common indication for
`prenatal genetic testing is maternal age older than 35 years;
`other indications include prior trisomic offspring, balanced
`parental chromosomal rearrangements, or increased risk for
`a mendelian trait. The standard approach is to offer women
`either second-trimester amniocentesis or first-trimester cho-
`rionic villus sampling, which are comparable or nearly com-
`parable in safety. The high diagnostic accuracy of these tech-
`niques must be balanced against the risks of undergoing an
`invasive procedure.2 Estimates for procedure-related fetal loss
`following amniocentesis range from 1 per 200 to 1 in 400 to
`500.3-5 Nonetheless, because all invasive procedures have some
`risk, considerable effort has gone into developing noninva-
`sive means of prenatal diagnosis.
`Second-trimester maternal serum analyte screening (for
`alpha fetoprotein, unconjugated estriol, human chorionic
`gonadotropin [hCG], and inhibin A) can identify perhaps
`70% to 75% of fetuses with Down syndrome, based on iden-
`tifying those 5% of pregnant women having risk equal to
`that of a 35-year-old.6 In Europe, maternal serum analyte
`screening is commonly offered to all pregnant women,
`whereas in the United States it tends to be offered rou-
`tinely only to women younger than 35 years; older women
`initially are offered an invasive procedure.
`
`See also pp 1114 and 1127.
`
`First-trimester noninvasive screening for trisomy 21 is also
`available, using ultrasound measurement of nuchal trans-
`lucency, as well as measurement of levels of maternal se-
`rum pregnancy-associated plasma protein A, and maternal
`serum hCG. A recent collaborative study from the US Na-
`tional Institute of Child Health and Human Development
`(NICHD) has shown sensitivity as high or higher in the first
`trimester as in the second trimester.7 Screening sequen-
`tially in both the first and the second trimester has been ad-
`vocated and indeed should have the highest detection rate.
`However, this approach requires withholding results in the
`first trimester and is limited by the risk of losing patients to
`follow-up. For instance, in the UK Serum, Urine and Ultra-
`sound Screening Screening Study (SURUSS) cohort of 47053
`patients, 15278 did not return for second-trimester test-
`ing.8 Thus, sequential (integrated) screening seems un-
`likely to be the most practical approach, and first-trimester
`noninvasive screening will probably become the more com-
`mon strategy.
`Despite these promising noninvasive approaches, sensi-
`tivity does not approach the 100% possible with an inva-
`sive procedure. Thus, the search continues for noninvasive
`methods independent of or complementary to maternal
`serum analytes or ultrasound. One such method involves
`detection and analysis of fetal cells recovered in maternal
`blood. In 1991 and 1992, our group demonstrated that
`fetal trisomy 21 and 18 could be detected by fluorescent in
`situ hybridization (FISH) using chromosome-specific
`probes on flow-sorted intact fetal cells.9-11 One intact fetal
`cell is estimated to be present per cubic centimeter of
`maternal blood. The collaborative NICHD trial demon-
`strated an aneuploidy detection rate of 74%.12 Unfortu-
`nately, robust, reproducible, intact fetal cell recovery has
`proved difficult; therefore, this cannot yet be recom-
`mended routinely.
`While methods for analysis of intact fetal cells continue
`to improve, correlative approaches are also being pursued.
`
`Author Affiliations: Departments of Obstetrics and Gynecology (Drs Simpson and
`Bischoff ) and Molecular and Human Genetics (Dr Simpson), Baylor College of Medi-
`cine, Houston, Tex.
`Financial Disclosures: Drs Simpson and Bischoff have received research support
`from the National Institute of Child Health and Human Development and main-
`tain contractual relationships with Biosciences Inc, San Diego, Calif, and Ikonysis
`Inc, New Haven, Conn.
`Corresponding Author: Joe Leigh Simpson, MD, Department of Obstetrics and
`Gynecology, Baylor College of Medicine, 6550 Fannin, Suite 901A, Houston, TX
`77030 (jsimpson@bcm.tmc.edu).
`
`©2004 American Medical Association. All rights reserved.
`
`(Reprinted) JAMA, March 3, 2004—Vol 291, No. 9 1135
`
`Downloaded From: https://jamanetwork.com/ by Gwen Brons on 05/20/2020
`
`

`

`Case 1:20-cv-01644-RGA Document 1-14 Filed 12/03/20 Page 3 of 4 PageID #: 853
`
`EDITORIALS
`
`In 1998, Lo et al13 demonstrated cell-free fetal DNA in plasma
`from healthy pregnant women, using quantification through
`polymerase chain reaction (PCR). Surprisingly high con-
`centrations of fetal DNA—nearly 5% of total maternal DNA—
`were detected in plasma of pregnant women. Mean fetal DNA
`was 25.4 genome equivalents (GE) per milliliter in early preg-
`nancy, increasing to 292.2 GE/mL in late pregnancy. Such
`large amounts of cell-free DNA cannot simply be the result
`of degradation of the rare fetal cell. Presumably, cell-free
`fetal DNA is derived from the placenta, and indeed mRNA
`for fetal hCG and for human placenta lactogen is recover-
`able in maternal plasma.14 That cell-free fetal DNA is con-
`sistently detected in maternal blood during pregnancy raises
`diagnostic potential.
`Initial diagnostic application of cell-free DNA involved
`detection of fetuses who had inherited a mutant allele from
`an affected father. If the father has a DNA sequence that the
`mother lacks, presence of that sequence in maternal blood
`must be of fetal origin; thus, the fetus has inherited the mu-
`tant paternal allele. Detection of a paternally transmitted au-
`tosomal dominant trait (ie, a fetus with hemoglobin Lep-
`ore disease) was reported initially by Camaschella et al.15
`Analysis of cell-free DNA is potentially valuable in manag-
`ing Rh(D) isoimmunization. Rh-negative women (geno-
`type d/d) lack the Rh(D) D antigen as result of a deletion.
`Mothers may develop antibodies (anti-D) if exposed to Rh(D)
`erythrocytes. If antibodies are present and the father is het-
`erozygous (D/d), there is 50% chance of transmitting the D
`allele, leading to isoimmunization. Presence of the D allele
`in maternal blood can be detected and has been shown to
`indicate that the fetus has inherited the D allele from its fa-
`ther.16,17 This information can be invaluable early in preg-
`nancy. A converse strategy becomes applicable later in preg-
`nancy. Rather than all Rh-negative (d/d) women having to
`receive Rh immune globin at 27 weeks’ gestation (the cur-
`rent recommendation), only those women whose fetuses are
`definitively Rh(D) might be treated. Again, the Rh status
`could be determined by studying maternal blood for the (pa-
`ternal) D allele.
`Analysis of cell-free fetal DNA has other clinical applica-
`tions. Total cell-free fetal DNA levels are increased 2-fold
`when the fetus has trisomy 21, even though the fetal DNA
`need not be derived from a gene locus on chromosome 21.18
`Cell-free fetal DNA could thus serve as an additional (and
`perhaps independent) maternal serum analyte for aneu-
`ploidy screening.
`Analysis of cell-free DNA also may be applicable in moni-
`toring complications of pregnancy. Concentrations of cell-
`free DNA correlate with gestational age and are low in the
`first trimester but increase in the second and third trimes-
`ters.13 A sharp increase in fetal DNA levels in maternal plasma
`during the last 8 weeks of pregnancy has been demon-
`strated, presumably indicating breakdown of the maternal
`fetal interface and placental barrier.19 Any pathological pro-
`cess that disturbs the placenta should be accompanied by
`
`increased levels of cell-free fetal DNA in maternal blood. Cell-
`free DNA has already been shown to be increased in preg-
`nancies complicated by preeclampsia.20 This increase should
`become evident earlier in pregnancy than other physical or
`biological disturbances.
`Cancer detection and surveillance is the second general
`area in which cell-free DNA analysis is likely to become ap-
`plicable clinically. Nucleic acids (DNA and RNA) in plasma
`were first observed more than 50 years ago. However, the
`source, fate, and usefulness of this DNA was not deter-
`mined until tumor-specific extracellular DNA fragments
`could be studied in the plasma of cancer patients. In 1989,
`Stroun et al21 indirectly verified this by demonstrating that
`the DNA found in the plasma of cancer patients displayed
`neoplastic characteristics, such as DNA strand instability.
`Subsequent groups confirmed the principle through muta-
`tion analysis, loss of heterozygosity, and microsatellite test-
`ing, which correlated cell-free DNA from the blood of pa-
`tients with cancer with the DNA from their primary tumor.
`Anker et al22 showed that more than 80% of the cell-free DNA
`in plasma of patients with colorectal cancer was represen-
`tative of the tumor DNA, judged by specific mutation in the
`K-ras oncogene. Mutations of p53 and APC also have been
`detected.23,24
`All of these observations raise tantalizing diagnostic pros-
`pects. If cancer-derived cell-free DNA can be robustly ana-
`lyzed, and if detection is possible with only a few genome
`equivalents per milliliter of blood, a sea change in the cur-
`rent approach to monitoring cancers could occur. Detec-
`tion or surveillance might no longer depend on imaging tech-
`nology (eg, magnetic resonance imaging) or surgery. Rather,
`less expensive, more convenient, and perhaps even more sen-
`sitive cell-free DNA analysis could be used to identify and
`analyze cancer-specific sequences.
`Thus, the findings of Dhallan et al demonstrating an in-
`creased percentage of cell-free DNA in maternal blood hold
`tremendous promise. In their study, the percentage of cell-
`free DNA was increased in 7 of 10 matched samples: 20.2%
`in treated vs 7.7% in untreated samples. The improved re-
`covery shown with use of formaldehyde is plausible. Three
`sources of circulating DNA have been hypothesized: (1) dy-
`ing cells (necrotic or apoptotic), (2) active DNA secretion,
`and (3) terminal differentiation. Apoptosis seems the most
`likely source. If so, circulating cell-free DNA should exist
`in the form of apoptotic bodies or nucleosomes. Irrespec-
`tive of the type of bound vesicles in which fetal DNA re-
`sides, treatment with formaldehyde should have a salutary
`effect in its stabilization. Formaldehyde-tested plasma may
`specifically prove more resilient to variations introduced by
`delayed processing time, varying centrifugation speeds, and
`storage conditions.
`The report by Dhallan et al also raises several other im-
`portant questions. For instance, if cell-free DNA methods
`were to be incorporated clinically, would treatment of blood
`with formaldehyde actually be needed? Given the rela-
`
`1136 JAMA, March 3, 2004—Vol 291, No. 9 (Reprinted)
`
`©2004 American Medical Association. All rights reserved.
`
`Downloaded From: https://jamanetwork.com/ by Gwen Brons on 05/20/2020
`
`

`

`Case 1:20-cv-01644-RGA Document 1-14 Filed 12/03/20 Page 4 of 4 PageID #: 854
`
`EDITORIALS
`
`tively large amounts of fetal DNA present in the maternal
`plasma, are methods to stabilize cell-free DNA gratuitous?
`These approaches are, in fact, necessary because the amount
`of fetal DNA in early pregnancy, or the amount of cell-free
`circulating DNA derived from early neoplasia, will remain
`low compared with background levels. Whenever target DNA
`(ie, fetal or neoplastic) is present in low copy numbers (eg,
`1-3 copies/mL), quantitative PCR is at the limit of its sen-
`sitivity; thus, detecting the target becomes hazardous, es-
`pecially given the necessity to minimize false-negative re-
`sults in cancer surveillance. In the current study, the authors
`used conventional PCR techniques and serial dilutions of
`each sample to quantify fetal Y-specific DNA sequences, the
`Y simply serving as a surrogate for interloping DNA of any
`type. DNA proved detected if as few as 3 fetal copies per
`milliliter of blood exist.
`The report by Dhallan et al is an important step in im-
`proving detection of cell-free DNA. Further refinements in
`techniques should maximize recovery of cell-free DNA and
`facilitate practical application. With prospective studies fo-
`cusing on clinical applications of these findings, profound
`clinical implications could emerge for prenatal diagnosis and
`cancer surveillance.
`
`REFERENCES
`1. Dhallan R, Au W-C, Mattagajasingh S, et al. Methods to increase the percent-
`age of free fetal DNA recovered from the maternal circulation. JAMA. 2004;291:
`1114-1119.
`2. Simpson JL, Elias S. Genetics in Obstetrics and Gynecology. 3rd ed. Philadel-
`phia, Pa: WB Saunders; 2003:345-369.
`3. Armstrong A, Cohen AW, Bombard AT, et al. Comparison of amniocentesis-
`related loss rates between obstetrician-gynecologists and perinatologists [ab-
`stract]. Obstet Gynecol. 2002;99:65S.
`4. Tongsong T, Wanapirak C, Sirivatanapa P, Piyamongkol W, Sirichotiyakul S,
`Yampochai A. Amniocentesis-related fetal loss: a cohort study. Obstet Gynecol.
`1998;92:64-67.
`5. Eddleman K, Berkowitz R, Kharbutli Y, et al. Pregnancy loss rates after midtri-
`mester amniocentesis: the FASTER trial [abstract]. Am J Obstet Gynecol. 2003;
`189:S111.
`6. Wald NJ, Watt HC, Hackshaw AK. Integrated screening for Down’s syndrome
`
`on the basis of tests performed during the first and second trimesters. N Engl J
`Med. 1999;341:461-467.
`7. Wapner R, Thom E, Simpson JL, et al. First-trimester screening for trisomies 21
`and 18. N Engl J Med. 2003;349:1405-1413.
`8. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L, Mackinson AM. First
`and second trimester antenatal screening for Down’s syndrome: the results of the
`Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen. 2003;10:
`56-104.
`9. Price JO, Elias S, Wachtel SS, et al. Prenatal diagnosis with fetal cells isolated
`from maternal blood by multiparameter flow cytometry. Am J Obstet Gynecol.
`1991;165:1731-1737.
`10. Simpson JL, Elias S. Isolating fetal cells from maternal blood: advances in pre-
`natal diagnosis through molecular technology. JAMA. 1993;270:2357-2361.
`11. Elias S, Price J, Dockter M, et al. First trimester prenatal diagnosis of trisomy
`21 in fetal cells from maternal blood. Lancet. 1992;340:1033.
`12. Bianchi DW, Simpson JL, Jackson LG, et al, National Institute of Child Health
`and Development Fetal Cell Isolation Study. Fetal gender and aneuploidy detec-
`tion using fetal cells in maternal blood: analysis of NIFTY I data. Prenat Diagn. 2002;
`22:609-615.
`13. Lo YM, 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;62:768-775.
`14. Ng EK, Tsui NB, Lau TK, et al. mRNA of placental origin is readily detectable
`in maternal plasma. Proc Natl Acad Sci U S A. 2003;100:4748-4753.
`15. Camaschella C, Alfarano A, Gottardi E, et al. Prenatal diagnosis of fetal he-
`moglobin Lepore-Boston disease on maternal peripheral blood. Blood. 1990;75:
`2102-2106.
`16. Bischoff FZ, Nguyen DD, Marquez-Do D, Moise KJ, Simpson JL, Elias S. Non-
`invasive determination of fetal RhD status using fetal DNA in maternal serum and
`PCR. J Soc Gynecol Investig. 1999;6:64-69.
`17. Lo YM, Hjelm NM, Fidler C, et al. Prenatal diagnosis of fetal RhD status by
`molecular analysis of maternal plasma. N Engl J Med. 1998;339:1734-1738.
`18. Lo YM, Lau TK, Zhang J, et al. Increased fetal DNA concentrations in the plasma
`of pregnant women carrying fetuses with trisomy 21. Clin Chem. 1999;45:1747-
`1751.
`19. Bianchi DW. Fetomaternal cell trafficking: a new cause of disease? Am J Med
`Genet. 2000;91:22-28.
`20. Holzgreve W, Hahn S. Novel molecular biological approaches for the diag-
`nosis of preeclampsia. Clin Chem. 1999;45:451-452.
`21. Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M. Neoplastic
`characteristics of the DNA found in the plasma of cancer patients. Oncology. 1989;
`46:318-322.
`22. Anker P, Lefort F, Vasioukhin V, et al. K-ras mutations are found in DNA ex-
`tracted from the plasma of patients with colorectal cancer. Gastroenterology. 1997;
`112:1114-1120.
`23. Mayall F, Jacobson G, Wilkins R, Chang B. Mutations of p53 gene can be de-
`tected in the plasma of patients with large bowel carcinoma. J Clin Pathol. 1998;
`51:611-613.
`24. Gocke CD, Benko FA, Kopreski MS, McGarrity TJ. p53 and APC mutations
`are detectable in the plasma and serum of patients with colorectal cancer (CRC)
`or adenomas. Ann N Y Acad Sci. 2000;906:44-50.
`
`©2004 American Medical Association. All rights reserved.
`
`(Reprinted) JAMA, March 3, 2004—Vol 291, No. 9 1137
`
`Downloaded From: https://jamanetwork.com/ by Gwen Brons on 05/20/2020
`
`