Is there over-enthusiasm for Non-Invasive Testing? Frank A. Chervenak, MD Shari E. Gelber, MD, PhD Tracy B. Grossman, MD MSc Laurence B. McCullough, Ph.D. Fix text University of Hawaii December 14-15 2018 Honolulu, Hawaii

Logarithmic axis Moore’s law (gordon Moore) co founder of intel. Doubling every year of the number of transistors in an integrated circuit. Allowed for development of digital electronics. Became a self fullfilling prophecy Genetics is now on a more rapid move forward

How do Tests Get Conveyed to the Public? First testing that has been industry driven….

What is cfDNA? Pieces of DNA from the fetal tissue end up in maternal serum Most labs need a certain percentage of fetal DNA found in the serum to report a result Lab is able to separate maternal DNA from fetal DNA using various techniques Usually by 9-10 weeks there will be enough circulated fetal DNA in maternal serum to report a result

RISK DIAGNOSIS SCREENING TESTING The term NIPT conflates screening and testing SCREENING TESTING

Aneuploidy Screening vs Testing Invasive Testing Risk of carrying fetus with a common aneuploidy (TS 13, 18, 21) Presence or absence of abnormalities in chromosome number Deletions and Duplications Evaluation of specific genetic disorders

Characteristics of a Screening Test Condition should be an important health problem There should be a test for the condition Facilities for diagnosis and treatment should be available The test should be acceptable to the population The natural history of the disease should be adequately understood The total cost of finding a case should be economically balanced in relation to medical expenditure as a whole Put in reference? https://www.ncbi.nlm.nih.gov/books/NBK138555/ Do you want reminders or do you want to read this?

Characteristics of a Screening Test Case-finding should be a continuous process Test used should be sensitive There should be a treatment for the condition There should be a latent or asymptomatic stage of the disease There should be an agreed policy on who to treat Remember to say: There is an intervention and people do benefit from counseling. This isn’t just about termination of pregnancy. Patients who know the fetus have TS21 may get a fetal echo, additional monitoring. Patients with TS 18 may chose not to have a cesarean for fetal indications….

Important to remember other benefits to screening than termination of pregnancy Insert respect for autonomy slide.

More information – can help patient decide on next steps, such as: Patient autonomy More information – can help patient decide on next steps, such as: Additional testing (amniocentesis, CVS) Counseling by specialists (pediatric cardiologists, neonatologists, pediatric surgeons) Patient has option of continuation of pregnancy or termination

Examples of Screening Tests for Aneuploidy AGE AFP Ultrasound Triple Quad Penta 1st Trimester risk assessment (NT+biomarkers) Integrated Sequential Contingent NIPT/S= cfDNA

NIPT/S= cfDNA is just another screening test Public perception is different because of marketing, overselling, and improved sensitivity for TS21. Potential to be used by patients without physician counseling / involvement.

Sensitivity of Aneuploidy Screens for TS21 Screening test Detection Rate Triple screen 69% Quad screen 81% NT alone 64-70% First trimester (NT+ biomarkers) 82-87% Integrated 96% Sequential Stepwise 95% Contingent 88-94% cfDNA 99% (in patients who receive result) Who doesn’t receive result: Overweight but also those with chromosome anomalies (DO YOU WANT THIS ON A SLIDE) Review table with FAC. Ultrasound. Ultrasound

Non Genetic Benefits of Screening Tests Quad screen Analytes associated with preeclampsia, growth restriction Screen for oNTD First trimester (NT+ biomarkers) 1. Independently associated with structural malformations 2. Analytes associated with preeclampsia, growth restriction. 3. Individual assessment of multifetal gestation cfDNA 1. Identification of fetal gender. 2. Identification of fetal rH status. TABLE cfDNA really genetic

Limitations of Screening Tests Benefit Quad screen Lower detection rate than combined tests. 2. Information provided late. 3. Requires accurate dating. First trimester (NT+ biomarkers) 1. Requires technical expertise cfDNA 1. PPV/NPV not clearly reported. 2. Higher false positive rate in lower risk patients. 3. Studies did not duplicate real world experience. (newest test) 4. No other clues for other conditions TABLE cfDNA really genetic

Large study comparing standard screening to cfDNA BUT 488 did not have cfDNA result and these were excluded. STUDY NOT ITT These patients were increased risk for aneuploidy! False positive rate lower with cfDNA 9 FP to pick up 38 TP False positive rate higher with SS. 854 FP to pick up 30 TP

cfDNA is the better test for TS21screening Standard screening is an excellent test .80-.90 = good .70-.80 = fair .60-.70 = poor .50-.60 = fail

Why do we care so much about TS21? Integrated Quad NT Contingent Sequential AGE TS21 is the most common disorder. BUT, the sum of all the disorders that are NOT TS21 is far greater than the risk of TS21 Why do we care so much about TS21? Why are we so focused on TS 21

cfDNA is the better test for TS21 screening but not for everything. (77.1)

Author’s comment: “For primary population screening, cfDNA provides lower DR than sequential screening if considering detection of all chromosomal abnormalities. cfDNA should not be adopted as primary screening without further evaluation of the implications for detection of all chromosomal abnormalities and how to best evaluate no results cases.”

Abnormal screening with normal karyotype is still a “high risk” condition Patients with invasive testing still had residual risk of >1% for pathogenic mutation or mutation with potential for clinical significance in setting of normal karyotype Risk with ultrasound anomaly = 6%

Why Not Just do Multiple Screening Tests? Increased false positive rate Incremental increase in detection rate How to deal with conflicting results? Cost

Abnormal Nuchal translucency result and normal cfDNA results Challenging to counsel these patients! Normal cfDNA and abnormal Nuchal Translucency (NT) Abnormal NT and patient doesn’t want amniocentesis or CVS – might choose to have cfDNA instead There are risks of amnio and CVS – pregnancy loss! Some patients may want to avoid these risks and would rather have cfDNA instead.

What does ACOG say? All pregnant women have option of screening or diagnostic testing Diagnostic testing is recommended with every abnormal nuchal translucency Diagnostic testing with amnio and CVS – recommended after every abnormal NT

Abnormal NT Increased nuchal translucency is associated with other abnormalities other than Trisomy 13, 18, 21 – Noonan syndrome Cardiac abnormalities

Examples of Abnormal NT: genetic results Abnormal NT is a condition, and not a diagnosis All of these fetuses had NTs of around 3.5mm. Among these four patients, one had normal genetic testing, one had Trisomy 21, one had a pathogenic copy number variant, and one had Noonan syndrome. Could you tell the difference if I didn’t give you the labels with each image? You wouldn’t know unless you did invasive testing!

What does SMFM say? Patients who decline invasive diagnostic testing may choose to have NIPS/T as an alternative There is still a role for ultrasound in first trimester – assess both nuchal translucency and for a basic anatomic survey – picks up anomalies earlier

Residual risk of genetic abnormality after normal cfDNA Retrospective study- patients with NT ≥ 3.0mm that also had genetic testing Compared the proportion of genetic abnormalities detectable with cfDNA vs other genetic abnormalities were made based on: NT size: (<3.5mm vs ≥ 3.5mm) Maternal age (< age 35, ≥ age 35) This study was done by one of my fellows at my institution. Over two years, pregnancies with nuchal translucency ≥ 3.0mm identified on ultrasounds performed at our institution were identified. Pregnancies with an abnormal nuchal translucency and genetic testing results were included, whether or not noninvasive prenatal testing was performed. Genetic testing included: karyotype, microarray, Noonan syndrome, and testing for other single gene disorders. This testing was done prenatally via CVS or amniocentesis, postnatally, or on products of conception after a termination of pregnancy. Abnormalities considered “detectable” by NIPT included: Trisomy 13, 18, 21, sex chromosome aneuploidy and triploidy. Results were stratified based on 2 criteria: Absolute NT value and maternal age Patients with NIPT and without NIPT performed were included in the study.

Results: What would cfDNA miss? 110 patients included 60 had genetic abnormalities 73.3% were “detectable” by cfDNA 26.7% were “not detectable” All abnormalities with NT < 3.4mm were detectable by cfDNA If NT ≥ 3.5mm, 71.4% of abnormalities were detectable by cfDNA For NT 3.5mm or greater, a significant proportion of abnormalities would NOT be detected (about 30%!)

Results: What would cfDNA miss? For patients with NT ≥3.5mm: ≥ age 35: 87.1% of abnormalities would be detected by cfDNA < age 35 only 52.0% (13 of 25) abnormalities would be detected by cfDNA Most abnormalities detected in patients over age 35, if NT was 3.5mm or greater BUT, for younger women, about half would be detected by cfDNA if NT was 3.5mm or greater – that’s not that many!

Conclusions of the study Normal cfDNA may be reassuring in those with mildly abnormal nuchal translucency But, a significant proportion of abnormalities in those with nuchal translucency ≥ 3.5mm would not be detected. This is particularly true in younger women, in whom cfDNA would miss about half of genetic abnormalities.

SMFM goes along with this as well – use NIPT for high risk women – (over age 35) – more useful for counseling

Role of ultrasound in women with cfDNA screening January 2017 – SMFM Consult series – addressed the question of role of ultrasound in women undergoing NIPS/T

SMFM Consult Series #42 SMFM states: In women who are considering cfDNA, first trimester NT assessment is beneficial – help choose between screening and diagnostic testing If abnormal nuchal translucency, or structural abnormalities – may choose diagnostic testing instead of screening But, if women already have a cfDNA result that is negative – first trimester nuchal translucency screening may slightly decrease residual risk of abnormalities. SMFM says: future research is needed to determine optimal screening approach in these patients In women with negative cfDNA: “first trimester NT screening may be little additional benefit as a screening test – due to how good cfDNA is at picking up the most common genetic abnormalities.”

Ultrasound in the era of cfDNA Study by Reiff, et al: 1739 patients with ultrasounds at 11-24 weeks, and with a negative cfDNA Abnormal ultrasound findings identified in 1 in 28 women Fetal anomaly identified in 1 in 290 Unexpected findings: seen on sono: Twins Fetal demise Structural abnormalities

Conclusions cfDNA has been aggressively marketed to physicians and patients as a test not a screen cfDNA is an incremental advance not a change cfDNA is the best screening test for TS21 This information comes at the expense of other information that may be more important to patients

Is there over-enthusiasm for Non-Invasive Testing? Frank A. Chervenak, MD Shari E. Gelber, MD, PhD Tracy B. Grossman, MD MSc Laurence B. McCullough, Ph.D. Fix text University of Hawaii December 14-15 2018 Honolulu, Hawaii