Prenatal Screening for Down Syndrome: Past, Present and

Emerging PracticesShort Presentation on Emerging Concept

(SPEC)

15 20 25 30 35 40 45 50

Euploid

DownSyndrome

Maternal age (completed years)

Euploid

DownSyndrome

1.30 1.35 1.40 1.45 1.50 1.55

Chromosome 21%

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).

15 20 25 30 35 40 45 50

Euploid

DownSyndrome

Maternal age (completed years)

‘Screen Positives’ located to theright of the red line at 35 years

Detection rate 50%False positive rate 15%

Figure Source: Canick JA, Palomaki GE. J Med Screen. 2012 Jun;19(2):57-9.

10 - 3 10 - 2 10 - 1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7

Euploid

DownSyndrome

Down's syndrome risk (1:n)

Down syndrome screening• Maternal age was inexpensive, reliable and

available early in pregnancy, but had relatively low detection and high false positives

• Today, 2nd trimester ‘quadruple’ testing is common

‘Screen Positives’ located to theleft 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. 2012 Jun;19(2):57-9.

10 - 3 10 - 2 10 - 1 10 0 10 1 102 103 104 105 10 6 10 7

Euploid

DownSyndrome

Down's syndrome risk (1:n)

Down syndrome screening• 1st trimester ‘combined’ testing has similar

performance to ‘quadruple testing’

• ‘combined’ + ‘quadruple’ = an ‘integrated’ test

‘Screen Positives’ located to theleft 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. 2012 Jun;19(2):57-9.

Prenatal screening in the US in 2012

Type of test Labs Median N Number (%)

1st trimester 34 3,000 565,692 (19)

2nd trimester 122 2,538 1,770,024 (60)

Integrated 30 4,176 583,416 (21)

All 123 3,660 2,963,592 (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

All Trisomy 21

Study False Positive Rate (%)

No-calls Down Syndrome Detection Rate (%)

No-call

Palomaki 2011 3/1,471 (0.2) 13/1,697 (0.8) 209/212 (98.6) 0

Ashoor 2012 0/ 300 (0 ) 1/ 400 (0.7) 50/ 50 (100) 0

Bianchi 2012 0/ 311 (0 ) 23/ 532 (4.3) 89/ 89 (100) 1

Norton 2012 1/2,887 (0.1) 148/3,228 (4.6) 81/ 81 (100) 3

Nicolaides 2013 0/ 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

Euploid

DownSyndrome

1.30 1.35 1.40 1.45 1.50 1.55

Chromosome 21%

Down syndrome screening• ccf DNA testing of maternal plasma

• Tests involve next generation sequencing (NGS)

‘Screen Positives’ located to theleft 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. 2012 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): 1532-1534.

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) 5,700Euploid

300DS

284pos

3fail

3neg

11pos

57fail

5,632neg

5,635 (93.9%)Routine care

1:1,900 (3:5,632)

295 (4.9%)Offer Dx testing26:1 (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;22:609-15.

• Fan HC et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. PNAS, 2008;105:16266.

• Chiu RWK et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel sequencing of NDA in maternal plasma. PNAS 2008; 105:20458.

• 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.

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