THE NEW GENETICS: PARADIGM SHIFTS IN PRENATAL DIAGNOSIS

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1 THE NEW GENETICS: PARADIGM SHIFTS IN PRENATAL DIAGNOSIS Jennifer Hoskovec, MS, CGC Assistant Professor Director, Prenatal Genetic Counseling Services Department of Ob/Gyn and Reproductive Sciences UT Health

2 OUTLINE Overview of standard screening and testing options for aneuploidy Non-Invasive Prenatal Testing for aneuploidy (NIPT) Chromosomal Microarray Analysis in the prenatal setting

3 Standard Screening and Testing Options for Invasive Prenatal Screening Fetal Aneuploidy Diagnosis Screening Options: First Trimester Screening Quadruple Marker Screen Integrated, Sequential, or Contingency Screens Anatomy Scan Benefit(s) Non-invasive = no risk Identifies women from low risk pool who are at increased risk Disadvantage(s) Risk calculation only False positive/negative Limited to trisomy 18, 13, 21 Timing, insurance coverage Patient anxiety Testing Options: Chorionic Villus Sampling (CVS) Amniocentesis Benefit(s) Diagnostic information on all aneuploidies Additional testing available such as microarray, PCR Disadvantage(s) Invasive, risk of pregnancy loss (1/300-1/500)

4 NON-INVASIVE PRENATAL NIPT TESTING

5 NIPT Available clinically since November 2011 in the United States Analyzes cell-free fetal DNA circulating in maternal blood: (cffdna) Placental and fetal-derived cells Possibly through the breakdown of fetal cells in circulation ~10-15% of cell-free DNA circulating in maternal blood is from the fetus Quantitative differences in chromosome fragments can identify fetuses with Down syndrome, trisomy 18, trisomy 13, and sex chromosome abnormalies Two different techniques MPSS DANSR + FORTE

6 MASSIVELY PARALLEL SHOTGUN SEQUENCING Simultaneously sequence millions of short segments from amplified DNA Hundreds of sequences generated in a single run Amplifies maternal and fetal DNA together Increases number of samples run Decreases cost Different platforms used Technique currently used by Sequenom and Verinata

7 NIPT WITH MPSS Unaffected Each diagrammatic fragment represents many thousands of sequenced fragments from chromosome 21. The quantitative overabundance of Trisomy 21 fragments in an affected pregnancy is significant and can be measured with high precision. Extra Chromosome Fragments = Affected Unaffected Fetus Fetus with Trisomy 21 Slide adapted from Sequenom

8 DANSR + FORTE DANSR- Digital Analysis of Selected Regions Chromosome selective approach: selectively evaluates specific genomic fragments from cfdna Determines fraction of fetal cfdna in maternal plasma as well as the chromosome proportion by assaying polymorphic and nonpolymorphic loci FORTE- Fetal-fraction Optimized Risk of Trisomy Evaluation Algorithm that takes into account additional data: prior risk (based on maternal age and gestational age) as well as fetal fraction Enables determination of chromosome proportion and fetal fraction at same time Requires less DNA sequencing and can analyze ~750 patient samples per run Technique currently used by Ariosa

9 NIPT VALIDATION STUDIES NIPT has been validated by multiple groups: In high risk pregnancies AMA Abnormal serum screen Family or personal hx of child with aneuploidy Abnormal ultrasound suggestive of aneuploidy Between weeks gestation

10 25 twin pregnancies 17 normal pairs 5 with Down syndrome in one fetus of twin pair 2 with Down syndrome in both fetuses of twin pair 1 with trisomy 13 in one fetus of twin pair All pregnancies were correctly classified 25/25 confidence interval [59-100] Two triplet pregnancies studied Unaffected; correctly classified Authors note twin pregnancies have higher placental mass and therefore might have higher fetal fraction and thus better separation of affected and unaffected fetuses despite the presence of multiples.

11 2049 pregnant women undergoing routine screening at weeks gestation 86 pregnancies (4.3%) had karyotype via CVS or amniocentesis 1963 pregnancies were phenotypically normal at birth (assumed euploid) Harmony risk scores available for 1949 (95.1%) pregnancies 46 (2.2%) had low fetal fraction 54 (2.6%) had assay failure Trisomy 21 Detected 8/8 cases, all having risk scores >99% Trisomy 18 Detected 2/3 cases, both having risk scores >99%, the third was an assay failure 1939 euploid pregnancies 1937 has risk scores of <1% (cutoff for low risk) 2 = risk scores for trisomy 18 were 9.8% and 11.7%

12 RAPID CLINICAL EVOLUTION Verinata Reporting sex chromosomes (Normal = XX, XY) and identification of sex chromosome aneuploidies (XXX, XXY, XYY, monosomy X) Sequenom Reporting absence or presence of Y chromosome material on all patients (99.4% accuracy quoted)

13 NIPT IN CLINICAL CARE Three separate groups have now shown high sensitivity and specificity with low false positive rate

14 NIPT Very high specificity and sensitivity Detection Rates Down syndrome: >99% (0.2% FPR) Trisomy 18: % ( 0.2% FPR) Trisomy 13: 79-92% (1.0% FPR) * Detection rates and FPR vary slightly between labs Results Typically reported as positive or negative Some labs distinguish between results close to or distant from the cut-off Results close to the cut-off would have less confidence Some labs classify those results as unclassifiable, some would place results on a continuum scale Confirmatory testing via CVS or amniocentesis is recommended for positive results

15 INCORPORATION INTO CLINICAL CARE Assume 100,000 women at high risk» 1:32 of affected:unaffected» Diagnostic testing cost of $1000/patient» Procedure loss rate of 1/200 Complete uptake of diagnostic testing: Detects 3000 cases Cost of $100 million 500 procedure-related losses Complete uptake of MPSS followed by diagnostic testing for positive results:» Detect 2958 cases (miss 42)» Cost of $3.9 million» 20 procedure-related losses Palomaki GE et al. Genet Med 2011

16 NIPT: LIMITATIONS Current limitations Validation Limited validation studies in low risk women Validation study in twins had only 25 sets Not validated in triplet or higher order multiples Not validated in pregnancies conceived with egg donors Not validated past 22 weeks gestation Cost and insurance coverage Does not include screening for ONTD

17 NIPT Specifics Laboratory (Test name) Technology Conditions Tested For Sensitivity Specificity Reporting Sequenom (MaterniT21Plus) MPSS Trisomy 21 Trisomy 18 Trisomy 13 T21 = 99.1% T18 = >99.9% T13 = 91.7% T21 = 99.9% T18 = 99.6% T13 = 99.7% Positive Negative Failure Verinata (Verify) MPSS Trisomy 21 Trisomy 18 Trisomy 13 Sex Chromosomes T21 = 100% T18 = 97.2% T13 = 78.6% 45X = 95% XXX, XXY, XYY = Limited data T21 = 100% T18 = 100% T13 = 100% 45X = 100% Positive Negative Aneuploidy suspected Failure Ariosa (Harmony) Partnered with Integrated Genetics (LabCorp) DANSR (assay) + FORTE (algorithm) Trisomy 21 Trisomy 18 Trisomy 13 T21 = 100% T18 = 97.4% T21 = 99.9% T18 = 99.9% Risk Ratio via algorithm 1/10,000 99/100 (0.5% results fell between the two extreme values)

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25 NONINVASIVE PRENATAL TESTING/NONINVASIVE PRENATAL DIAGNOSIS (NIPT/NIPD): The National Society of Genetic Counselors currently supports Noninvasive Prenatal Testing/Noninvasive Prenatal Diagnosis (NIPT/NIPD) as an option for patients whose pregnancies are considered to be at an increased risk for certain chromosome abnormalities. NSGC urges that NIPT/NIPD only be offered in the context of informed consent, education, and counseling by a qualified provider, such as a certified genetic counselor. Patients whose NIPT/NIPD results are abnormal, or who have other factors suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of standard confirmatory diagnostic testing. (Adopted February 18, 2012)

26 ACOG/SMFM COMMITTEE OPINION NUMBER 545 DECEMBER 2012 Noninvasive Prenatal Testing for Fetal Aneuploidy ABSTRACT: Noninvasive prenatal testing that uses cell free fetal DNA from the plasma of pregnant women offers tremendous potential as a screening tool for fetal aneuploidy. Cell free fetal DNA testing should be an informed patient choice after pretest counseling and should not be part of routine prenatal laboratory assessment. Cell free fetal DNA testing should not be offered to low -risk women or women with multiple gestations because it has not been sufficiently evaluated in these groups. A negative cell free fetal DNA test result does not ensure an unaffected pregnancy. A patient with a positive test result should be referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation of test results.

27 NIPT FUTURE DIRECTIONS Additional validation studies on use in low -risk population and multiple gestations Other chromosomal disorders and microdeletions/duplications Use for Mendelian disorders New technology may increase accuracy MeDiP: enriches for fetal-specific hypermethylated DNA regions Whole genome sequencing Within the next 10 years, the complete fetal genome will be successfully sequenced from maternal plasma Lo (Prenat Diagn 2010;30:702-3)

28 SUMMARY So many options! Accurate and balanced discussion of options with patient is very important Benefits Limitations Risks Assist the patient in making informed, autonomous decision Be sensitive to personal nature of decision Family values Religious beliefs Family and life situations Concerns about having a child with an abnormality Concerns about risk of miscarriage

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31 PRENATAL DIAGNOSTIC TESTING CVS and amniocentesis Routine karyotype FISH Aneuploidy FISH (13, 18, 21, X, Y) Site specific FISH for deletion syndromes (22q11.2) Chromosomal Microarray Analysis

32 CHROMOSOMAL MICROARRAY ANALYSIS CMA platforms use thousands of DNA probes spread across the genome to detect gains and losses of genetic material. Extracted DNA from the patient (fetus) is compared with a reference (normal) genome. Allows identification of abnormal copy number changes (gains and losses). Aneuploidy Duplications and deletions too small to be seen by conventional cytogenetics Limitations: Cannot detect balanced chromosome rearrangements (identifies dosage differences, not positional differences) Cannot identify triploidy Possible Pitfalls: Identification of a copy variant of unknown significance (~1.5%) Requires parental bloods for comparison Possible out of pocket expense to the patient

33 Overview of CMA Process 1 Patient Control Hybridization to Array Mix CMA Methodology Laser Scanner 2 3 Data analysis FISH confirmation del 22:q11.21

34 EXAMPLE- NORMAL RESULTS

35 EXAMPLE- TRISOMY 21

36 CHROMOSOMAL MICROARRAY ANALYSIS IN PRENATAL CLINICAL PRACTICE Each individual syndrome incidence low Ex: 22q11.2 deletion syndrome, AKA DiGeorge syndrome (22q11.2 del; >95% detection; 1 in incidence) Ex: Williams Syndrome (7q11 del; 95% detection; 1 in 10,000 incidence) Ex: Prader Willi Syndrome (15q11 del; 70% detection; 1 in 25,000 incidence) Detailed detection potential for CMA version 6.3 oligo at BCM: Likelihood of finding a clinically relevant information not identified on routine karyotype ( N ENGL J MED 367;23 Dec 6, 2012) 1.7% of women referred for routine indications (AMA, pos screen, etc) with normal ultrasound and karyotype had a clinically significant finding on CMA 6% of women with abnormal ultrasound findings and normal karyotype had a clinically significant finding on CMA

37 QUESTIONS

38 REFERENCES Lo et al (1997) Presence of fetal DNA in maternal plasma and serum. Lancet Finning et al (2002) Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service. Transfusion Bianchi DW (2004) Circulating fetal DNA: its origin and diagnostic potential- a review. Ding et al (2004) MS analysis of single-nucleotide differences in circulating nucleic acids: application to non -invasive prenatal diagnosis. PNAS Gautier et al (2005) Fetal RhD genotyping by maternal serum analysis: a two-year experience. AJOG Scheffer et al (2011) Noninvasive fetal blood group genotyping if rhesus D, c, E, and of K in alloimmunised pregnant women: evaluation of a 7-year clinical experience. BJOG Chiu et al (2011), Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ

39 REFERENCES Palomaki et al (2011), DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genetics in Medicine Palomaki et al (2012), DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genetics in Medicine Bianchi et al (2012) Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstetrics and Gynecology Sparks et al (2012) Non-invasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. AJOG Ashoor et al (2012) Chromosome-selective sequencing of maternal plasma cell - free DNA for first-trimester detection of trisomy 21 and trisomy 18. AJOG Colah et al (2011) Invasive and non-invasive approaches for prenatal diagnosis of haemoglobinopathies: experiences from India. Indian J Med Res Norton et al (2012) Non Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. AJOG. Canick et al (2012) DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenat Diagnosis

40 REFERENCES Benn et al (2011) Prenatal detection of Down syndrome using massively parallel shotgun sequencing : a rapid response position statement from a committee on behalf of the board of the international society for prenatal diagnosis. NSGC (2012) Position statement on noninvasive prenatal testing/noninvasive prenatal diagnosis. Sehnert et al (2011) Optimal detection of fetal chromosomal abnormalities by massively parallel DNA sequencing of cell -free fetal DNA from maternal blood. Clinical Chemistry Ladha, S (2012) A new era of non-invasive prenatal genetic diagnosis: exploiting fetal epigenetic differences. Clin Genet Devaney et al (2011) Noninvasive fetal sex determination using cell -free fetal DNA: a systematic review and meta -analysis. JAMA Hill et al (2011) Non-invasive prenatal determination of fetal sex: translating research into clinical practice. Clin Genet Geifman-Holtzman et al (2006) Diagnostic accuracy of noninvasive fetal Rh genotyping from maternal blood- a meta-analysis. AJOG Lo, Y (1994) Non-invasive prenatal diagnosis using fetal cells in maternal blood. J Clin Pathol Benn et al (2012) Non-invasive prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly. Ultrasound Obstet Gynecol

41 REFERENCES Nicolaides et al (2012) Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. AJOG Wapner et al (212) Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis. NEJM