Director

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Director Italian Branch Cagliari Regional
Director for Europe
Director Ian Donald School for Invasive
Procedures
INVASIVE VS NON-INVASIVE PRENATAL DIAGNOSTIC
PROCEDURES Giovanni Monni
12th TURKISH GYNECOLOGY AND OBSTETRICS
CONGRESS Antalya, 15th 19th Maggio 2014
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DILEMMAS TO AVOID GENETIC DISORDERS IN THE
NEWBORNS

• Screening based on maternal age alone?
• First or second trimester ultrasound and
  biochemical screening?
• Prenatal invasive procedures?
• Standard karyotype? aCGH analysis?
• Preimplantation genetic diagnosis?
• Diagnostic ultrasonography (1st-2nd trimester)?
• Fetal cell free-fetal DNA (cff- DNA) in maternal
  blood (general or contingent) ?

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CHANGES IN THE APPROACH FOR INVASIVE PRENATAL
DIAGNOSIS IN 35,127 CASES AT A SINGLE CENTER FROM
1977 TO 2004

• DIAGNOSI SEMPRE Più PRECOCE

Monni, Fetal Diagn Ther 2006
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Number of amniocenteses and chorionic villus
samplings carried out in Denmark, 2000-2006
Ekelund, BMJ 2008
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Total Number of Diagnostic Procedures in England
(2003- 2012)
Morgan,UOG 2013
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UK national policy study ofaneuploidy
screening after the implementation of the
combined test

1. Reduction of false positive rate from 6 to 3
   without significant change of DR of Down Syndrome
2. Progressive reduction in the number of
   screen-positive cases
3. Significant reduction in the number of invasive
   prenatal diagnostic procedures

Morgan, Ultrsound Obstet Gynecol 2013
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UK NATIONAL POLICY STUDYANEUPLOIDY SCREENING
The odds of the fetus being affected after a
positive combined test in the first trimester
were much greater than were the odds based on
advanced maternal age alone (120 vs 175). So a
significantly higher probability of an invasive
test would confirm an abnormal fetal karyotype.
Morgan, Ultrsound Obstet Gynecol 2013
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REDUCTION IN THE FETAL NUMBER OF INVASIVE
PROCEDURES PERFORMED FOR PRENATAL KARYOTYPE
Redistribution of the proportion of procedures
performed by amnio and CVS- Denmark in 2006
CVS in 66 of cases- UK in 2003 Amnio/CVS
31 in 2011 Amnio/CVS 11
Monni, Zoppi, Ultrasound Obstet Gynecol 2013
Opinion
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FIRST TRIMESTER EUROPEAN NATIONAL POLICY FOR
PRENATAL DOWN SYNDROME SCREENING Denmark (BMJ
2008) and UK (UOG 2013) Studies
- Decrease in Fetal Loss due to a
reduction in invasive diagnostic procedures-
Earlier Diagnosis of Chromosomal
Aneuploidies
Monni, Zoppi, Ultrasound Obstet Gynecol 2013
Opinion
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FIRST TRIMESTER SCREENING AND INVERSION OF THE
PYRAMID OF PRENATAL CARE
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INVASIVE PRENATAL DIAGNOSIS TECHNIQUE OF 78
CHROMOSOMAL ABNORMALITIESOSPEDALE MICROCITEMICO-
CAGLIARI JANUARY 2011 DECEMBER 2011
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Distribution of number of fellows for CVS
training at the Ospedale Microcitemico -
Cagliari
Period No.
1983- 1996 42 28
1997- 2012 109 72
151 100
BEFORE NT SCREENING AFTER NT SCREENING
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FELLOWS TUTORED AT MICROCITEMICO HOSPITAL IN
CAGLIARI (No 151) Other Argentina, Azerbaijan,
Bosnia, Czech Republic, Canada, Japan, France,
Germany, India, Lebanon, Mongolia, Morocco,
Netherlands, Portugal, Romania, S. Arabia,
Slovenia, Spain, Sudan, Un. Arab Emirates,
Venezuela
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NEW LABORATORY TECHNIQUES

• Fluorescent in situ hybridization (FISH)
• Amplification of polymorphic chromosome-specific
  markers by polymerase chain reaction (PCR)
• Most laboratories offer a rapid test (PCR or
  FISH) to detect trisomy 21, 18, 13 and sex
  chromosome aneuploidies, as well as tissue
  culture to provide a full karyotype
• Array comparative genomic hybridization (a-CGH)
• in cases of multiple congenital abnormalities at
  ultrasound or for clinical diagnosis?

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Advantages of array Comparative Genomic
Hybridization (aCGH) or Chromosomal Microarray
Analysis (CMA)

• aCGH allows detection of smaller pathogenic
  chromosomal variants that are undetectable using
  standard cytogenetic analyses (G-band
  karyotyping)

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DISADVANTAGES OF ACGH

• aCGH does not allow detection of balanced
  chromosomal rearrangements triploidy and some
  instances of mosaicism
• The biggest challenge presented by aCGH is the
  detection of chromosomal variants of unknown
  clinical significance (VOUS)

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METHODS FOR ANALYSIS OF CELL-FREE (CF) DNA IN
MATERNAL BLOOD

• Shotgun massively parallel sequencing (s-MPS)
• Targeted massively parallel sequencing (t- MPS)
• Single nucleotide polymorphism (SNP) -based
  analysis

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CFDNA ANALYSIS FOR T21 A META-ANALYSIS (18
CITATIONS 2011- 2013)

• Individual studies
• Detection Rate (DR) ranges 94.4-100
• False Positive Rate (FPR) ranges 0- 2.05
• Pooled weighted
• DR 99.0 (95 CI 98.2- 99.6)
• FPR 0.08 (95 CI 0.03- 0.14)

Gil et Nicolaides, Fetal Diag Ther 2014
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CFDNA ANALYSIS FOR T21 A META-ANALYSIS
Gil et Nicolaides, Fetal Diag Ther 2014
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CFDNA ANALYSIS FOR T18, 13, MONOSOMY X A
META-ANALYSIS
Detection rate False positive rate
Trisomy 18 96.8 (95 CI 94.5- 98.4) 0.15 (95 CI 0.08- 0.25)
Trisomy 13 92.1 (95 CI 85.9- 96.7) 0.20 (95 CI 0.04- 0.46)
Monosomy X 88.6 (95 CI 83.0- 93.1) 0.12 (95 CI 0.05- 0.24)
The poor performance of cfDNA analysis in
screening for trisomy 13 and monosomy X could be
due to the highly variable amplification of
chromosome X and 13 because of a lower guanosine-
cytosine content
Gil et Nicolaides, Fetal Diag Ther 2014
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CFDNA ANALYSIS FOR SEX CHROMOSOME ANEUPLOIDIES
OTHER THAN MONOSOMY X

• Pooled weighted
• DR 93.8 (95 CI 85.9- 98.7)
• FPR 0.12 (95 CI 0.02- 0.28)

Gil et Nicolaides, Fetal Diag Ther 2014
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CFDNA ANALYSIS FOR TRIPLOIDY

• Diandric (paternal)
• Placenta enlarged and partially molar
• NT enlarged
• Free- beta hCG very high (10 times higher than
  normal)
• Digynic (maternal)
• Placenta very small
• Fetus severely growth restricted
• Normal NT
• Free- beta hCG and PAPP-A very low

The SNP method for cfDNA testing is the only one
at present that can detect triploidy because it
analyses allele distributions 4 out 8 cases of
diandric triploidy have been detected, and
suspicion raised for a case of diagynic triploidy
Utility of cfDNA as first-line method of
screening because identification of triploidy
would be beneficial (diandric triploidy can cause
maternal complications including early- onset
preeclamsia and choriocarcinoma)
Gil et Nicolaides, Fetal Diag Ther 2014
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LIMITATIONS OF CFDNA TESTING

• Failure to provide results
• Receiving results in 1- 2 weeks
• Cost

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FAILURE TO PROVIDE RESULTS

• In 1- 5 of cases no results is given after
  first sampling
• Problems with sample collection or with
  transportation to the laboratory (on repeat
  sampling result is obtained in about 100)
• Assay failure (on repeat sampling result is
  obtained in about 75)
• Low fetal fraction (on repeat sampling result is
  obtained in about 50) if it is a consequence of
  maternal obesity this problem is difficult to
  overcome

Gil et Nicolaides, Fetal Diag Ther 2014
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RECEIVING RESULTS IN 1- 2 WEEKS

• Average interval 10 calendar days
• In 95- 98 of cases a result available within 14
  days
• In 2 of cases a result may not be available in
  less than 3-4 weeks

Such delay may reverse the beneficial shift in
screening and diagnosis of aneuploidies from the
second to the first trimester
Gil et Nicolaides, Fetal Diag Ther 2014
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MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA
TESTING IN MATERNAL BLOOD

• Routine screening for whole population
• Contingent screening based on the result of first
  trimester combined test

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MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA
TESTING AS FIRST-LINE METHOD FOR ALL PREGNANCIES

• 10 weeks, maternal blood to all
• 12 weeks first trimester us
• Expected
• 99 DR of trisomy 21
• 95 DR of trisomy 18 and 13
• 1 Invasive testing rate

Gil et Nicolaides, Fetal Diag Ther 2014
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MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA
TESTING AS CONTINGENT SCREENING HIGH RISK GROUP

• Maternal blood in the high risk group (gt 1100)
• Expected
• 86 DR of tris. 21
• 89 DR of tris.18 /13
• 0.4 Invasive test. rate

cfDNA testing could not detect other aneuploidies
Gil et Nicolaides, Fetal Diag Ther 2014
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MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA
TESTING AS CONTINGENT SCREENING INTERMEDIATE
RISK GROUP

• Maternal blood in the Intermediate Risk Group
  (gt111lt12,500)
• Expected
• 97.6 DR of tris. 21
• 98.1 DR of tris. 18/13
• 0.8 Invasive test. rate

cfDNA testing could not detect other aneuploidies
Gil et Nicolaides, Fetal Diag Ther 2014
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PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC
DISEASE ACTUALLY VALIDATED USE

• Fetal sex determination (X-linked diseases in
  order to avoid invasive procedure in female
  fetuses) or for congenital adrenal hyperplasia
  (CAH) for therapeutic options
• RH blood group, D antigen
• Paternal inherited autosomal dominant diseases or
  de- novo after ultrasound suspicion
  (chondrodysplasias)

SIGU 2014, Document on the indications of use of
performing non-invasive prenatal research
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PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC
DISEASE NOT YET VALIDATED USE

• Autosomal recessive diseases
• X linked diseases
• Autosomal dominat diseases of maternal origin

SIGU 2014, Document on the indications of use of
performing non-invasive prenatal research
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MAIN FEATURES OF FREE DNA IN MATERNAL PLASMA

• Free DNA is always present in peripheral blood
  with a magnitude of between 145 and 201 bp
• Pregnancy causes an increase in the size of
  circulating DNA of maternal origin and a
  progressive increase in the concentration of
  Fetal DNA that is smaller
• The origin of circulating Fetal DNA in maternal
  plasma is due to placental apoptotic processes of
  the syncytium trophoblast
• The Fetal DNA is present since the 7th week of
  pregnancy and increases during pregnancy. In 10
  weeks increases to about 5 or 10 of the total
  circulating plasma DNA. The fraction of fetal
  tissue correlates negatively with the maternal
  weight
• The presence of Fetal DNA in maternal plasma is
  no longer detected two hours after giving birth.
  The average half-life of 16.3 minutes (range 4-30
  minutes)

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TAKE HOME MESSAGES (1)

• Maternal age should no longer be the sole
  criterium for set the parental choice of invasive
  prenatal diagnosis
• First trimester combined screening reduces the
  number of invasive prenatal diagnostic procedures
• After a positive combined test, a significantly
  high probability of an invasive test would
  confirm an abnormal fetal karyotype
• First trimester combined test induces reversing
  the traditional pyramid of prenatal care
• Educational organizations have faced new
  challenges in providing training for invasive
  procedures

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TAKE HOME MESSAGES (2)

• aCGH is not a substitute for conventional
  karyotyping
• 2) aCGH should be used for specific diagnostic
  purposes in selected pregnancies and not for
  general screening in all pregnancies

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TAKE HOME MESSAGES (3)
1) cff- DNA for NIPT has the role of a screening
test 2) Evidence from high risk population 3)
Necessity of implementation of cff- DNA in low
risk series 4) Genetic counselling is mandatory
before and after NIPT
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NON-INVASIVE PRENATAL TEST (NIPT)

• The expectations regarding cff-DNA for fetal
  genetic anomalies are very high because it may
  have the potential to change the landscape of
  prenatal diagnosis. However, to the
  disappointment of many, cff-DNA does not have the
  ability to function as a diagnostic test but is
  considered at present time as a
• super screening test.

Monni, Journal of Perinatal Medicine 2014