About these slides Provided by the CAP as an aid to pathologists to facilitate discussion on the topic. Content has been reviewed by experts at the CAP, but does not necessarily reflect the official opinion of the College of American Pathologists. Non-CAP material with identified copyright source may only be copied or distributed under a license (permission) from the copyright holder, or under the doctrine of fair use. Version 1.0, rev. 7/1/2014

Prenatal Screening for Down Syndrome: Past, Present and Emerging Practices Short Presentation on Emerging Concept (SPEC)

Down syndrome screening The first test was the question ‘How old are you?’ If answered “35 or older”, the woman was offered invasive testing (amniocentesis or CVS) and diagnostic testing (karyotype). ‘Screen Positives’ located to the right of the red line at 35 years Detection rate 50% False positive rate 15% Figure Source: Canick JA, Palomaki GE. J Med Screen Jun;19(2):57-9.

Down syndrome screening Maternal age was inexpensive, reliable and available early in pregnancy, but had relatively low detection and high false positives Today, 2 nd trimester ‘quadruple’ testing is common ‘Screen Positives’ located to the left of the red line at risk of 1:270 Detection rate 80% False positive rate 5% Figure Source: Canick JA, Palomaki GE. J Med Screen Jun;19(2):57-9.

Down syndrome screening 1 st trimester ‘combined’ testing has similar performance to ‘quadruple testing’ ‘combined’ + ‘quadruple’ = an ‘integrated’ test ‘Screen Positives’ located to the left of the red line at risk of 1:100 Detection rate 90% False positive rate 2% Figure Source: Canick JA, Palomaki GE. J Med Screen Jun;19(2):57-9.

Prenatal screening in the US in 2012 Type of testLabsMedian NNumber (%) 1 st trimester 343, ,692 (19) 2 nd trimester1222,5381,770,024 (60) Integrated 304, ,416 (21) All1233,6602,963,592 (100) Palomaki GE et al., Archives Path Lab Med 2013 Represents about 70% of all US pregnancies

Current status Combinations of maternal age and multiple markers –Serum markers (AFP, uE3, hCG, PAPP-A, inhibin-A) –Ultrasound markers (nuchal translucency) But, these tests –are complex –still miss 10-20% of Down syndrome cases –lead to offering diagnostic tests to 2-6% of women –can only identify Down syndrome and to some extent, trisomy 18 and trisomy 13 Would like even better performance for a wider range of prenatal disorders

Circulating cell free (ccf) DNA First reported in 1997 by Dr. Dennis Lo, Chinese University of Hong Kong (Lancet, 350:485) Used Y chromosome probes in maternal plasma to identify male fetuses Both maternal and fetal (actually placental) DNA are found in maternal circulation DNA in small fragments (150 to 200 bp) Reliably represents the entire genome of the mother and fetus Fetal ccfDNA quickly cleared after birth Ratio of fetal to total ccf DNA is 10% (range <4% to 40%)

Current methodologies The current commercial laboratory-developed tests (LDT) in the US can be divided by (not FDA cleared/approved) –Sequencing methodology: any fragment sequenced (Shotgun) versus selectively amplified sequences (Targeted) –Interpretation methodology: comparing observed percentage of aligned fragments from chromosome of interest to expected (Counting) versus modeling observed SNP genotype to specific models (Genotyping)

External clinical validation studies AllTrisomy 21 StudyFalse Positive Rate (%) No-callsDown Syndrome Detection Rate (%) No-call Palomaki /1,471 (0.2) 13/1,697 (0.8)209/212 (98.6)0 Ashoor / 300 (0 ) 1/ 400 (0.7) 50/ 50 (100)0 Bianchi / 311 (0 ) 23/ 532 (4.3) 89/ 89 (100)1 Norton /2,887 (0.1)148/3,228 (4.6) 81/ 81 (100)3 Nicolaides / 204 (0 ) 13/ 242 (5.4) 25/ 25 (100)2 All 4/4,173 (0.1)454/457 (99.3)6 Technology is advancing and ‘real world’ experience is being gained. The performance of current commercial testing may differ

Down syndrome screening ccf DNA testing of maternal plasma Tests involve next generation sequencing (NGS) ‘Screen Positives’ located to the left of the red line at risk of 1:100 Detection rate 99% False positive rate 0.2% Figure Source: Canick JA, Palomaki GE. J Med Screen Jun;19(2):57-9.

Practice guidelines Sequencing of cell free DNA is considered sensitive and specific for the common trisomies, such that Multiple professional organizations have written practice guidelines relevant to ccfDNA plasma testing for common trisomies American Congress of Obstetricians and Gynecologists (ACOG) American College of Medical Genetics and Genomics (ACMG) International Society of Prenatal Diagnosis (ISPD) National Society of Genetic Counselors (NSGC) Society of Obstetricians and Gynecologists of Canada (SOGC)

Practice guidelines Testing for chromosomes 13, 18, and 21 should be offered to ‘high risk’ pregnancies Testing should not be offered to the general pregnancy population (‘low risk’) until more information is available Positive results followed up by offer of invasive testing –ccfDNA currently is still is considered a screening test Patient and provider education is important Insufficient data for twins

“High Risk” Pregnancies Maternal age 35 years or older at delivery Fetal ultrasonographic findings indicating an increased risk of aneuploidy History of a prior pregnancy with a trisomy Positive test result for aneuploidy, including first trimester, sequential, or integrated screen, or a quadruple screen Parental balanced robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21 American College of Obstetricians and Gynecologists Committee on Genetics (2012). Committee Opinion No. 545: Noninvasive prenatal testing for fetal aneuploidy. Obstet Gynecol, 120(6):

Limitations Data is currently only sufficient to warrant use for aneuploidy of chromosomes 13, 18, 21, and sex chromosome aneuploidies Patients at risk for other genetic abnormalities, including single gene disorders, should be counseled to utilize other testing modalities Does not replace α-fetoprotein testing for neural tube defects Testing turn-around-time should be considered when testing later in pregnancy

ccfDNA testing in ‘High risk’ women 6,000 ‘High risk’ (1:19) 5,700 Euploid 300 DS 284 pos 3 fail 3 neg 11 pos 57 fail 5,632 neg 5,635 (93.9%) Routine care 1:1,900 (3:5,632) 295 (4.9%) Offer Dx testing 26:1 (284:11) 60 (1.0%) Offer Dx testing 1:19 (3:57) Figure Source: Palomaki, GE.

Commercial LDTs: further disorders Trisomy 18 and trisomy 13 –All commercial LDTs provide an interpretation –> 95% detection Sex chromosome aneuploidies –Most commercial LDTs provide –45X, 47XXY, 47XXX, 47,XYY and fetal sex –>90% detection Twin pregnancies –Two commercial LDT provide –Limited data Deletion/duplication syndromes –Available for one commercial LDT –Validation data not yet presented

Selected Resources Lo YMD, et al. Presence of fetal DNA in maternal plasma and serum. Lancet, 1997;350:485. Bianchi WE et al. Fetal gender and aneuploidy detection using fetal cell in maternal blood: Analysis of NIFTY data. Prenat Diagn 2002;22: Fan HC et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. PNAS, 2008;105: Chiu RWK et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel sequencing of NDA in maternal plasma. PNAS 2008; 105: Palomaki GE et al. DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. Genet Med, 2011;13:913. Bianchi DW et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol, 2013;119:890 Ashoor G et al. Chromosome-selective sequencing of maternal plasma cell-free DNA for first- trimester detection of trisomy 21 and 18. Am J Obstet Gynecol, 2012;206:322 e1-5. Norton ME et al. Non-Invasive Chromosome Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;207:137 e1-8. Nicolaides KH et al. Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X and Y. Prenat Diagn 2013;33:575-9.

Additional Free Resource for CAP Members NOTE: please remove this page before presenting. CAP Member Exclusive: CAP Pathology Resource Guides Focused on a specific hot-topic technology, these comprehensive guides highlights current resources, select journal articles, as well as CAP and non-CAP educational opportunities. And don’t miss the “Insights From Early Adopters” section in each guide to gain perspective from pioneering colleagues. AVAILABLE NOW: Molecular Pathology (single gene test, small panel) Genomic Analysis (large panel, exome, genome) Learn more: go to cap.org and type Pathology Resource Guides in the “search” field located at the top of your screen. “Extremely well done, of high practical and educational value.” “An outstanding overview of basic materials, including the technology and links to a number of individuals and centers that can assist.”