Aneuploidy Screening Ronald J' Librizzi, DO Chief, Maternal Fetal Medicine Virtua Health Clinical As

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Aneuploidy Screening Ronald J. Librizzi,
DOChief, Maternal Fetal MedicineVirtua
HealthClinical Associate Professor,
Ob/GynThomas Jefferson UniversitySchool of
Medicine
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PRENATAL SCREENING 28 YEAR HISTORY
1st Trimester Free Beta
ONTD Screening
NT
Free Beta
NB
Maximizing First Trimester Screening
1975 80 85 90 95 2000
hCG
Down Screening AFP Only
1st Trimester PAPP-A
1st Trimester Biochem NT
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Approaches to Prenatal Testing
Diagnostic Tests Screening
Tests Amniocentesis AFP CVS Quad
Screen Cordocentesis Ultrasound Ultrasound
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Characteristics of an Ideal Screening Test

• High sensitivity - Identifies a high percentage
  of affected individuals
• High specificity - Does not alarm a high
  percentage of unaffected individuals
• Positive early enough in gestation to allow
  intervention
• Easy and inexpensive to perform

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Prenatal Chromosome Screening

• ? Low risk patients
• ? 35 yrs, other chromo risks (FH and U/S)
• ? Biochemical screens
• ? U/S screen 1st or 2nd Trimester
• ? Risk cutoffs 35 yrs risk at specific
  gestation
• ? False Positive Rate 5

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• First Trimester Screening
• FreeBeta / PAPP-A / NT/ NB Screening
• 11-14 wk Fetal Ultrasound Exam
• CVS (Early Amniocentesis N/A)
• Second Trimester Screening
• AFP /or Ultrasound
• NT ? 3.0mm in 1st then Fetal Anatomy and Heart
  Ultrasound in 2nd

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NT General Population Screening

• ? 96,127 patients 10-14 wks
• ? DR 82.2 at 8.3 SPR (1300 cut off)
• ? DR 77 at 5 SPR
• ? All patients had 3 NTs, largest NT used
• ? Only 4.2 lost to follow-up
• ? True DR after 4 bias adjustment for 7 SABs
  between 12-16wks 78 at 8.3 (Spencer 2003),
  not 60.

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USA National FMF/SMFM/ NT Standard

• Standardized training, practical NT certificate,
  external audit, and NT certificate renewal
  required for 1st. Same Standard for gt 70
  countries
• 2. Over 500,000 NTs (USA UK) in database
• 3. Consistent NTs in 19 prospective studies of
  over 120,000 patients by Operator-Specific FMF
  certificate operators. More now with SMFM.
• Non-certified operators are inconsistent at 9-15
  week.
• SMFM has entered the Educational Certification

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A National Standard Must Be Maintained

• Everyone who does ultrasound should NOT do NT
  unless they have gone through the training for
  numerous reasons but mostly for consistency.
• Most labs for bloods (PAPP A and hCG will not
  accept specimen without certification number)

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Fetal Abnormalities At Birth

• 12 weeks NT Percentile
• 3mm 10
• 6mm 90

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Nuchal Translucency Advancing Gestation
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Anomalies in 4,116 Chromosomally Normal Fetuses
with Increased Nuchal Translucency gt 95th
centile at 10 -14 weeks of gestation

• n()
• Major cardiac defects
  43(27)
• (increases exponentially with NT)
• Talipes 15(9)
• Body stalk anomaly 10(6)
• Diaphrag. hernia
  9(6)
• Megacystis 8(5)
• Exomphalos 7(4)
• Akinesia deformity
  6(4)
• Ventriculomegaly 6(4)
• Anencephaly
  5(3)
• Encephalocele
  4(3)
• Multicystic kidney
  4(3)
• Spina bifida 4(3)
• Hydronephrosis
  3(2)

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1st Trimester Screening - USA

• National FMF Standard NT Training,
  Certificate and External Audit.
• Orlandi, et al., (1997)
• Krantz, et al., (2000) 10,581 pts
• NICHD-BUN Trial (2002) 8,514 pts
• CYRANO Nasal Bone Study
• NICHD FASTER Research 2004

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Which Biochemical Marker?
freeBeta/PAPP-A

• First Trimester
• ? High Free Beta hCG (2.0 MOM)
• ? Low Pregnancy-Associated Plasma Protein-A
• (PAPP-A) (0.4 MoM)
• ? NOT B-hCG (1.2 MOM) not associated with DS
• Second Trimester
• ? AFP for open spine and skull defects
• ? Free Beta hCG (2.8 MoM) or B-hCG (1.8 MOM)
• ? Estriol

AFP/freeBeta
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2nd Trimester Research

• NO PUBLISHED USA PROSPECTIVE OR NIH STUDIES TO
  SHOW BENEFIT, NO ACOG STATEMENTS.
• ? QUAD (AFP/ß-hCG/Estriol/Inhibin A)
• ? INTEGRATED (1st Quad) Can SPR be lowered by
  requiring 1st risks be withheld? No, not in
  clinical practice.
• ? Medical, Legal (HIPPA), ethical problems of
  withholding known ? risks (NT, NB) from
  physician/patient until 17-20 wks to add 4 more
  tests.
• ? Six markers double cost for lower DR and higher
  SPR
• than 1st.

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2nd Trimester Research

• ? FASTER Integrated Research - FIRST WORKS
  better than Integrated when all DS fetuses are
  counted.
• ? 1st prospective FMF clinical studies have
• ? detection rate (DR) and ? screen positive rate
  (SPR), so 1st obviates any benefit of
  Integrated.
• ? Integrated withholds advantages of 1st for no
  benefit. Is it an acceptable medical procedure?
• ? Impact of immediate results after NT!!!!!!

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Dried Blood Screening

• Eliminates broken tubes, biohazard, hemolysis,
  specimen degradation, and centrifugation
• Stabilizes protein markers
• Reduces Marker Variation (S.D.)
• Increases Separation between Distributions
• Patient and staff acceptance!!!!

• CUTS SCREEN POSITIVE RATES BY HALF (1/2)
• RAISES DETECTION RATES

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First Trimester ScreeningWhen?
DATING BY CRL
freeBeta / PAPP-A
24-84mm CRL 9w 0d - 13w 6d LMP
freeBeta / PAPP-A / NT
45-84mm CRL 11w 0d 13w 6d LMP
Not 10-14 wks
Immediate results
0 5 9 11 12
13 14
Weeks
(Adjusted for MA, GA, FH, Wt., Ethnicity)
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DS freeBeta/PAPP-A DR _at_ 5 SPR
USA Detection 63 at 4.5 vs. triple 60 at 7.6

• Study-FMF NT DS
  Weeks Detection ()
• Krantz, 1996 22 10-13 63
• Wald, 1996 77 8-13 62
• Berry, 1997 47 9-13 55
• Orlandi, 1997 11 9-13 61
• Haddow, 1998 48 9-15 60
• Wheeler Sinosich, 1998 17 9-12 67
• De Graaf, 1999 37 10-13 55
• Spencer, 1999 210 10-13 67
• Tsuckerman, 1999 31 8-13 69
• Krantz, 2000 50 9-13 63

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Why Combine freeBeta / PAPP-A / NT?

(Current USA data)
? NT ALONE DOUBLES SPR AND ? DR 10 ? ALWAYS
COMBINE NT, freeBeta, PAPP-A TO ? DR, ?
SPR, AND ? YIELD
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DS freeBeta / PAPP-A /NT DR at 5 SPR
USA Detection 91 _at_ 2.4 SPR
Study DS Weeks
SPR Detection ()
Brizot, 1994, 1995 80 10-13 5.0 89 Orland
i, 1997 11 9-13 5.0 87 De Graaf, 1999
37 10-13 5.0 85 De Biasio, 1999
13 10-13 3.3 85 Spencer, 1999
210 10-13 5.0 89 Spencer, 2000
7 10-13 5.5 86 Krantz, 2000
33 10-13 5.0 91 Schucter, 2002
14 11-13 5.0 86 Wapner, 2002
61 10-13 5.0 78.7 Bindra, 2002
82 11-13 6.8 92 Crossley, 2002
37 11-13 6.0 92 Spencer, 2003
25 10-13 5.2 92
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freeBeta/PAPP-A/ NT Krantz et al. Obstet Gynecol
96 207 (2000)

• 10,253 patients lt age 35 between 9-13 weeks
• 5,811 patients with NT at 10w4d - 13w3d
• SPR Detection Rate Yield
• Down syndrome 4.8 91 1/17
• Trisomy 18 1.2 97 1/17

Yield 1/17 vs. 1/80 for triple screen
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Maternal Age NT/FreeBeta / PAPP-A
(Krantz, et al, 2000)
Age(y) SPR Detection Rate () 24
3.2 88 25-29 3.7 88 30-34 5.3 90
35-39 10.3 91 40 24.7 97

• All Ages 4.8 91

? 35 YRS ROUNTINELY OFFER AMNIO ? Some day
we will eliminate this FEAR of gt35 years!!!!! We
are getting close but it may take a generation!!!!
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Second Trimester Screening

• ? AFP / B-hCG / UE3 (16w - 20w) (Triple)
• SPR
  DR Yield
• ONTD 5.0 80 1 / 42
• Down Syndrome 7.6 60 1 / 80
  
  
  
  Trisomy 18 0.5 60 1 / 16
• 12
• ? AFP / freeBeta hCG (13w 4d to 22w 3d)
• 
  SPR DR Yield
• ONTD 2.7 98 1 /
  25
• Down Syndrome 2.8 80 1 / 25
  
  
  
  Trisomy 18 0.1 70 1 / 6
• 5.6

? HIGHEST DR AND YIELD, LOWEST SPR ?
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DS SCREENING
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Down Syndrome Detection
Detection
93
80
63
60
2.4
4.4
2.9
7.6
First Trimester
SecondTrimester
A freeBeta/PAPP-A/NT (11w 0d - 13w 6d) B
freeBeta/PAPP-A (9w 0d - 13w 6d) C
AFP/freeBeta (13w 4d - 22w 3d) D Triple /
Quad (16w - 20w)
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Patients Prefer 1st

• All 6 published surveys show patients strongly
  prefer 1st.

(Monni G, et al. Lancet 1998) (Mulvey S and
Wallace, EM. Br J Obstet Gynecol 2000 107
1302-5)
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Should 2nd Be Moved To 1st?Cuckle Lith Prenat
Diag 19505 (1999)

• There is overwhelming evidence that screening
  for Down syndrome could be moved from the second
  to the first trimester of pregnancy. (The Royal
  College of Obstetricians and Gynaecologists,
  1997)
• The Time for First Trimester NT Has ComeChasen
  et al. J. Ultrasound Med 201147, 2001
• Given the scientific rigor and the results of
  the FMF database, in our view nuchal translucency
  should not be considered investigational but
  instead a highly reliable diagnostic screen when
  performed in expert hands.

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1st 2nd Trimester Screening Genetic
Counseling

• Nondirective counseling and obtain Consent
• Discuss 1st and 2nd screening in the 1st
  trimester
• Copy patients Consent to her physician to avoid
  automatic 2nd chromosomal screen
• Impact of IMMEDIATE RESULTS!!!!!
• SMFM supports 1ST (Feb,2004). ACOG to review in
  2004.

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Jan 2007 ACOG Bulletin

• If patients present in the first trimester, she
  should be offered a first trimester or combined
  screening
• If patients present in the second trimester, sh
  ould be offered second trimester screening

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Does a 2nd Trimester Follow-up Chromosome Screen
Help or Hurt?

• Because 1st ? PREVELENCE 91-97
• Yield will ?5 fold from 1/17 to 1/250
• 2nd Triple will ? SPR to 13 (2.4 1st 10.3
  2nd-BUN)
• 2nd AFP/freeBeta will ? SPR to 4.4 (2.4 1st
  2 2nd)

• A F/UP FOR SNP AND SPP PATIENTS CANNOT BE
  ACCURATE (DATABASE, PRIOR RISK) AND WILL CONFUSE.
• A 2nd F/UP IS NOT STANDARD OF CARE.
• PLAN FOR SNP AFTER 1st DO MSAFP OR U/S?????

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Dont do QUAD screen after patient has had FTS
results and risks!!!NOT the same as a planned
sequential or integrated

• What happens when the MSAFP that we normally
  order after FTS is inadvertently ordered as a
  QUAD screen? Whoops!!!!

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Here is what we do????

• Genetic Counseling

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Insurers Cover 1st

• 1. freeBeta / PAPP-A YES over 85
• 2. Second Trimester YES
• 3. NT YES, for ? risk patients, for consult
  referral. For low risk patients, use general ICD
  codes.
• 4. CPT Options
• ? 99242,3, or 4, consult
• ? 76815 (limited) or 76801-22 U/S with
  documentation (maternal/fetal, lt14wk)
• ? Laboratory bills for biochemistry

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• Hold on..

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Absence of Nasal Bone in Trisomy 21Fetuses at
11-14 Weeks of GestationLancet (2001) 3581665-67

• Simona Cicero, Patrizia Curcio, Aris
  Papageorghiou,
  Jiri Sonek, Kypros Nicolaides
• Harris Birthright Research Centre for Fetal
  Medicine,
  Kings College Hospital Medical School, London,
  UK
• Dept. of Obstetrics Gynecology
• Ohio State University, Ohio, USA

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Nasal Bone Assessment

• ? Absent and/or Short Nasal Bone is associated
  with T21, T18, and 58 Other Syndromes
• ? Proper NB measurement requires Standardization
  and Formal Training (similar to NT Certificate)
• ? Cicero, 2003 After training 100 NBs needed
  for proficiency
• ? Further Study of 1st trimester Short/ Absent
  NB is need for QC
• ? FASTER, 2004 NB without special
  training/QC, 9 of 9 DS missed vs. consistent
  high DR (70) for over 20,000 patients, with
  NB training/QC.

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First-Trimester Nasal Bone
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NT FreeBeta PAPP-A Nasal Bone Fetal
Medicine Foundation Data
vs. Integrated 1 FPR _at_85 DR (Wald, 1999)
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Prospective DS Screening

• DR SPR
• AFP / freeBeta (2nd) 80 2.9
• Triple (2nd) 60 7.6
• Integrated(2nd) 76/90/90 3.4/5/5.4
• 1st 93 2.4
• 1st NB 95 2
• 1st NB 94 1

(CAN,WALD,FASTER)
First PLUS NB has the highest DR and yield with
the lowest SPR
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First Trimester Advantages

• Earliest, most effective and safest risk
  assessment
• Highest Detection and Yield for chromosomal,
  heart (40), and many other anomalies at the
  lowest SPR of all screens
• Early anomaly and perinatal risk management
• Privacy for patient! Preferred by Patients!
• Less bonding at early GA
• Earlier, safer, and lower cost options
• Early resolution of patient anxiety
• Early Reassurance for 97.6 of Low Risk patients
• Meets ACOG guideline for national implementation
  by Operator - Specific certificate providers.
• Cost effective (?36), insurance coverage as
  acceptable medical procedure.

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2nd Trimester Research

• NO PUBLISHED USA PROSPECTIVE OR NIH STUDIES TO
  SHOW BENEFIT, NO ACOG STATEMENTS.
• ? QUAD (AFP/ß-hCG/Estriol/Inhibin A)
• ? INTEGRATED (1st Quad) Can SPR be lowered by
  requiring 1st risks be withheld? No, not in
  clinical practice.
• ? Medical, Legal (HIPAA), ethical problems of
  withholding known ? risks (NT, NB) from
  physician/patient until 17-20 wks to add 4 more
  tests.
• ? Six markers double cost for lower DR and higher
  SPR
• than 1st.

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2nd Trimester Research

• ? FASTER Integrated Research - FIRST WORKS
  better than Integrated when all DS fetuses are
  counted.
• ? 1st USA prospective FMF clinical studies have ?
  DR, ? SPR, so 1st obviates any benefit of
  Integrated.
• ? Integrated withholds advantages of 1st for no
  benefit. Is it an acceptable medical procedure?
  YES, so says the 2007 ACOG bulletin!!!!

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• Prenatal Screening Tests Comparison
• First Trimester Combined (1st) NT/Nasal Bone
  (NB)/freeBeta/PAPP-A _at_ 11-14 wks has 94
  detection rate (DR) _at_ 1 screen positive rate
  (SPR) or 97 _at_ lt5 SPR. 1st (NT/freeBeta/PAPP-A)
  has the highest DR (91) and yield (1/17) with
  the lowest SPR (2.4). 1st is the most
  effective, earliest and safest screening test.
  1st identifies other chromosomal anomalies, 40
  of heart defects and ? perinatal risks. 1st
  provides optimal pregnancy management, more
  options and ? cost.

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Prenatal Screening Tests Comparison
Sequential 1st, then a 2nd chromosomal screen
(2nd) w/o risk adjustment Lowest yield, Highest
SPR Not Recommended. A follow-up 2nd test is
not standard of care because the YIELD is ? 5
Fold, the SPR is over 13, and 2nd cant be
accurate due to ? DS incidence, revised prior
risk, and unadjusted 2nd. New Sequential
procedure offers results but lowers first
trimester Sensitivity
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• Prenatal Screening Tests Comparison
• Integrated Withholding (2nd) NT/PAPP-A _at_
  10-14wks and Quad _at_ 16-20 wks has 75-85 DR _at_
  2.8 SPR Lower DR and yield, Higher SPR than
  1st, therefore Integrated has No Benefit over
  1st. Can we justify withholding known ? risks
  until 17-21 wks for no documented benefit?
• ? FASTER published, but does not show benefit
  over 1st when all Down syndrome fetuses are
  counted however some will elect this. (my
  opinion)
• ? 1st studies with higher DS DR _at_ lower SPR have
  been published (Cicero 2003). (my opinion)
• ? Therefore, 1st has negated any integrated
  benefit while offering many new advantages. (my
  opinion)

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• ?Integrated Disadvantages (opinion)
• ? Loss of all 1st benefits by requiring known 1st
  risks be withheld from patients and physicians
  for 6-10wks to keep SPR low.
• ? Physicians cant withhold known 1st risks for
  6-10wks, so SPR rises no benefit.
• ? 1st results are not improved with follow up
  screen and increases SPR.
• ? 2-3 visits required, incomplete till 17-21 wks.
• ? A 3rd visit required for screen positive
  patients.
• ? Drop-Out 8-35 due to U/S reassurance, cost,
  time, and insurance lab preferences.
• ? Drop-Out patients are billed for NT/PAPP-A but
  dont receive an integrated test result.

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• ? Fetal and perinatal risks are withheld 6-10wks
  for no benefit.
• ? Patients and physicians should not be denied
  immediate 1st risks or reassurance (gt97).
  Integrated give a less specific risk.
• ? Increased patient anxiety.
• ? Loss of privacy and optimal pregnancy
  management options when you go into the second
  trimester.
• ? All 6 published surveys show patients strongly
  prefer First Trimester Screening.
• Triple and Quad (2nd) Lowest DR and yield,
  Highest SPR of available prenatal screening tests.

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What do we do???....

• This screening issue is so confusing to most
  patients, we offer all the testing but when
  asked, we give them our suggestion of doing FTS
  with NB as what we think is the best test.
• We offer Genetic counseling liberally many
  people are withholding this option until after
  they get results of screen. This adds to patient
  confusion.

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• Hold on again!

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Chromosomal Microarray Analysis (CMA)

• Single test includes all disorders detected by
  standard cytogenetics and multiple FISH tests
• Probes for virtually all known microdeletion/dupli
  cation syndromes and all telomeres are included
• Major advance in the diagnosis of patients with
  suspected genetic etiology
• Can expand prenatal diagnosis from karyotype to
  numerous other conditions caused by small
  deletions and duplications, not visible on
  routine chromosome analysis

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NON INVASIVE FISH TESTING

• Harvest fetal trophoblastic cells from cervix
  with cytobrush!!!!!
• Non-invasive
• Fish results quickly
• NOT A KARYOTYPE AS YET
• PRETTY COOL SCREENING TEST BUT.
• Controversial issues!!!!!

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Fetal cells and DNA in maternal circulation

• Ive been waiting for this since I was a resident
• 2000 Bianchi cells in peripheral maternal
  blood
• Nucleated RBC antigens for isolation
• Fetal DNA less labor intensive but need to be
  able to differentiate from maternal

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• WOW!!!
• Are we in exciting times or what..
• Thanks for your attention!!!