Interpretare i risultati NIPT: i mosaicismi feto-placentari e le implicazioni sulla scelta tra amniocentesi o villocentesi come test di conferma dopo un risultato NIPT ad alto rischio / Interpreting NIPT results: feto-placental mosaicism and the implication on choice between confirmatory amniocentesis or CVS after a positive cfDNA testing result (F. R. Grati)

1. Interpretare i risultati NIPT: i mosaicismi feto-placentari e leInterpretare i risultati NIPT: i mosaicismi feto-placentari e le
implicazioni sulla scelta tra amniocentesi o villocentesi comeimplicazioni sulla scelta tra amniocentesi o villocentesi come
test di conferma dopo un risultato NIPT ad alto rischiotest di conferma dopo un risultato NIPT ad alto rischio
Interpreting NIPT results: feto-placental mosaicism and theInterpreting NIPT results: feto-placental mosaicism and the
implication on choice between confirmatory amniocentesisimplication on choice between confirmatory amniocentesis
or CVS after a positive cfDNA testing resultor CVS after a positive cfDNA testing result
LA DIAGNOSI PRENATALE E GLI SCREENING PRENATALI: cosa sta cambiando?LA DIAGNOSI PRENATALE E GLI SCREENING PRENATALI: cosa sta cambiando?
TOMA lab, Busto Arsizio –TOMA lab, Busto Arsizio – 18 September 2015
Francesca Romana GRATI, Ph.D., ErCLGFrancesca Romana GRATI, Ph.D., ErCLG
R&D Director, TOMA Advanced Biomedical Assays, S.p.A.R&D Director, TOMA Advanced Biomedical Assays, S.p.A.
fgrati@tomalab.comfgrati@tomalab.com

2. OUTLINEOUTLINE
Describe the gold standard method for cytogeneticDescribe the gold standard method for cytogenetic
analysis of CVSanalysis of CVS
Introduce the issue of chromosomal mosaicism in CVIntroduce the issue of chromosomal mosaicism in CV
Discuss the issue of FP and FN (discrepant) cfDNADiscuss the issue of FP and FN (discrepant) cfDNA
testing results related to fetoplacental mosaicismstesting results related to fetoplacental mosaicisms
Discuss the implications of fetoplacental mosaicism onDiscuss the implications of fetoplacental mosaicism on
choice of confirmatory prenatal diagnostic procedurechoice of confirmatory prenatal diagnostic procedure
after a high risk cfDNA testing resultafter a high risk cfDNA testing result

3. Chromosomal mosaicism in fetoplacental unitChromosomal mosaicism in fetoplacental unit
Kalousek D, 1984
Chromosomal mosaicism: genetic constitution of an individual derived from aChromosomal mosaicism: genetic constitution of an individual derived from a
single zygote who has two or more populations of cells with distinct karyotypessingle zygote who has two or more populations of cells with distinct karyotypes
CONFINEDCONFINED
PLACENTALPLACENTAL
MOSAICISMMOSAICISM
(CPM)(CPM)
TRUE FETALTRUE FETAL
MOSAICISMMOSAICISM
(TFM)(TFM)

4. MOSAICISMS IN CVMOSAICISMS IN CV
CV mosaic Type 1 2 3
--------------------------------------------------------------------------------------------------------------------------
Abn. Plac. Tissue Cytotrophoblast Mesenchyme Cytotrophoblast
&
Mesenchyme
Simoni and Sirchia, Prenatal Diagnosis, 14:1185-1189 (1994)

5. Kalousek D, 1984
CONFINEDCONFINED
PLACENTALPLACENTAL
MOSAICISMMOSAICISM
(CPM)(CPM)
TRUE FETALTRUE FETAL
MOSAICISMMOSAICISM
(TFM)(TFM)
CONFIRMATORY AMNIOCENTESISCONFIRMATORY AMNIOCENTESIS

6. OVERALL RESULTS: CPM and TFM incidencesOVERALL RESULTS: CPM and TFM incidences
Grati FR, J. Clin. Med. 2014, 3, 809-837; Malvestiti F et al, Prenatal Diagnosis 2015; Simoni et al, 1983; Brambati and
Simoni, Lancet 1983; Grati FR, et al. Eur J Hum Genet 2006;14:282–8; Grati FR, et al. Prenat Diagn 2013;33:1–7.
CVS (2000-2014) 60347
Mosaicism on CVS 1317 (2,18%)
Follow up at amniocentesis 1001
TFM 131 (13,08%)
Direct
preparation
(24-48h)
Long Term
Culture, LTC
(5-7 days)
AMNIO AT TOMAAMNIO AT TOMA
N=60.347N=60.347 N=1.317 (Mos in CV 2%)N=1.317 (Mos in CV 2%)
N=1001N=1001
(TFM=13%)(TFM=13%)

7. OVERALL RESULTS: INCIDENCES OF THE DIFFERENT CPMs AND TFMsOVERALL RESULTS: INCIDENCES OF THE DIFFERENT CPMs AND TFMs
TYPE NATURE CYTOTROPHOBLAST MESENCHYME AMNIOCYTES RELATIVE FREQUENCIES
I CPM Abnormal Normal Normal 35,66% (357/1001)
II CPM Normal Abnormal Normal 41,25% (413/1001)
III CPM Abnormal Abnormal Normal 9,99% (100/1001)
IV TFM Abnormal Normal Abnormal 1,40% (14/1001)
V TFM Normal Abnormal Abnormal 5,69% (57/1001)
VI TFM Abnormal Abnormal Abnormal 5,99% (60/1001)
CPM type I and II were the most frequent types of mosaicismCPM type I and II were the most frequent types of mosaicism
TFMIV was the rarest one (1.4%)*TFMIV was the rarest one (1.4%)*
Grati FR, J. Clin. Med. 2014, 3, 809-837; Malvestiti F et al, Prenatal Diagnosis 2015; *Hahnemann JM and Vejerslev LO
Am J Med Genet 1997;70:179-187; *Battaglia et al. Prenat Diagn 2014;34:739–47.

8. CV mosaic Type 1 2 3
--------------------------------------------------------------------------------------------------------------------------
Abn. Plac. Tissue Cytotrophoblast Mesenchyme Cyto & Mese
--------------------------------------------------------------------------------------------------------------------------
Risk of fetal conf. 3.77% 12.12% 43.48%
RISK OF TFM DEPENDING ON THE DISTRIBUTION OF THE ABNORMALRISK OF TFM DEPENDING ON THE DISTRIBUTION OF THE ABNORMAL
CELL LINE IN THE PLACENTACELL LINE IN THE PLACENTA
Grati FR, J. Clin. Med. 2014, 3, 809-837; Malvestiti F et al, Prenatal Diagnosis 2015

9. TOMA LAB SURVEYTOMA LAB SURVEY
Grati FR, J. Clin. Med. 2014, 3, 809-837; Malvestiti F et al, Prenatal Diagnosis 2015
Probabilities of confirmation on amniocytes considering theProbabilities of confirmation on amniocytes considering the
different chromosome abnormalitiesdifferent chromosome abnormalities
Chromosome based differences in the frequency of TFM and CPM among theChromosome based differences in the frequency of TFM and CPM among the
different abnormalities and common aneuploidies targeted by NIPTdifferent abnormalities and common aneuploidies targeted by NIPT
100 100 98
90
83
74
66 66
0 0 2
10
17
26
34 34
0
10
20
30
40
50
60
70
80
90
100
Trisom
y
2(n=64)
Trisom
y
7
(n=64)
Trisom
y
13
(n=42)
Trisom
y
20
(n=29)
Trisom
y
18
(n=53)M
onosom
y
X
(n=114)47,XY/XX,+m
ar(n=65)
Trisom
y21
(n=56)
Type of mosaic chromosome abnormality
%
CPM cases %
TFM cases %

10. An important cause of discrepant results isAn important cause of discrepant results is
based on the fact that cffDNA derives mainly frombased on the fact that cffDNA derives mainly from
cytotrophoblast and that it can be involved incytotrophoblast and that it can be involved in
fetoplacental mosaicismsfetoplacental mosaicisms
NIPT DISCREPANT RESULTS AND CYTOTROPHOBLASTNIPT DISCREPANT RESULTS AND CYTOTROPHOBLAST
Tjoa ML, Am J Pathol 2006; Flori E et al, Hum Reprod 2004; Faas et al, Expert Opin Biol Ther 2012; Kalousek DK, J Med Genet 1999; Hahnemann
JM, Vejerslev LO. Am J Med Genet 1997;
All current statements underline that cfDNAAll current statements underline that cfDNA
testings don’t have to be presented as a diagnostictestings don’t have to be presented as a diagnostic
test for fetal aneuploidy: although rare, there aretest for fetal aneuploidy: although rare, there are
false positive/negative (discrepant) resultsfalse positive/negative (discrepant) results

11. Table 1: schematic representation of the different types of mosaics and the of the associated expected NIPS results
Type of mosaic
(Cyto)trophoblast
(direct preparation or short
term culture)
Mesenchyme
(long term culture)
Amniocytes NI PS result
CPM 1 abn n n FP
CPM 2 n abn n TN
CPM 3 abn abn n FP
TFM 4 abn n abn TP*
TFM 5 n abn abn FN
TFM 6 abn abn abn TP*
CPM=confined placental mosaicism; TFM=true fetal mosaicism;
Abn=abnormal; n= normal; FP= false positive; FN=false negative; TN=true negative;
TP* =true positive assuming a consistent percentage of the mosaic abnormal cell line in cytotrophoblast
CPM 1 & 3CPM 1 & 3 TFM 5TFM 5
NormalNormal
AbnormalAbnormal
n/an/a aa
AbnormalAbnormal
NormalNormal
Grati et al, Genet Med. 2014 Aug;16(8):620-4
CPM1 and 3 can be potential sources of FPCPM1 and 3 can be potential sources of FP
TFM5 can be a potential source of FN resultsTFM5 can be a potential source of FN results
Maternal
bloodstream
Maternal
bloodstream
NIPT DISCREPANT RESULTS AND CYTOTROPHOBLASTNIPT DISCREPANT RESULTS AND CYTOTROPHOBLAST

12. NormalNormal
AbnormalAbnormal
n/an/a
FP rate due to CPM type 1 & 3FP rate due to CPM type 1 & 3
CYTOGENETICSCYTOGENETICS
~800~800 NORMALNORMAL
FETUSESFETUSES
NIPSNIPS
1 FETUS DETECTED AS1 FETUS DETECTED AS
“ABNORMAL” DUE TO THE“ABNORMAL” DUE TO THE
PRESENCE OF CPM1/3PRESENCE OF CPM1/3
Grati et al, Genet Med. 2014 Aug;16(8):620-4
Maternal
bloodstream
Cumulative risk of FP rate for all chr abn targeted by cfDNA testingCumulative risk of FP rate for all chr abn targeted by cfDNA testing
(T13,18,21,MX)(T13,18,21,MX)

13. CYTOGENETICSCYTOGENETICS
~~70 ABNORMAL70 ABNORMAL
FETUSESFETUSES
NIPSNIPS
1 FETUS DETECTED AS1 FETUS DETECTED AS
“NORMAL” DUE TO THE“NORMAL” DUE TO THE
PRESENCE OF TFM5PRESENCE OF TFM5
aa
AbnormalAbnormal
NormalNormal
FN rate due to TFM type 5FN rate due to TFM type 5
Grati et al, Genet Med. 2014 Aug;16(8):620-4
Cumulative risk of FN rate for all chr abn targeted by cfDNA testingCumulative risk of FN rate for all chr abn targeted by cfDNA testing
(T13,18,21,MX)(T13,18,21,MX)
Maternal
bloodstream

14. Due to the possibility of a CPM1/3 aDue to the possibility of a CPM1/3 a
confirmatory iconfirmatory invasive diagnosticnvasive diagnostic
procedure is recommended after aprocedure is recommended after a
positive cfDNA testing resultpositive cfDNA testing result
Because cfDNA testing is frequentlyBecause cfDNA testing is frequently
performed in the first trimester, CVS isperformed in the first trimester, CVS is
frequently the invasive procedure methodfrequently the invasive procedure method
Mosaicism on CVSMosaicism on CVS  confirmatoryconfirmatory
amniocentesisamniocentesis
Confirmatory CVS or amnio after a positive cfDNA testing result?Confirmatory CVS or amnio after a positive cfDNA testing result?
CVS (11-13wg)CVS (11-13wg)
Type 1Type 1 Type 3Type 3
AMNIO
CPM 1 & 3CPM 1 & 3
NormalNormal
AbnormalAbnormal
n/an/a
Maternal
bloodstream
Grati FR, JCM 2014; Grati FR et al, Prenatal Diagnosis 2015; Malvestiti et al, 2015

15. Grati FR et al, Prenatal Diagnosis 2015
Confirmatory CVS or amnio after a positive cfDNA testing result?Confirmatory CVS or amnio after a positive cfDNA testing result?
Madonna del parto (Piero della Francesca, 1455-1465)Madonna del parto (Piero della Francesca, 1455-1465)
The best approach to confirm a high-risk cfDNA testing result is a source of controversyThe best approach to confirm a high-risk cfDNA testing result is a source of controversy

16. Grati FR et al, Prenatal Diagnosis 2015
Confirmatory CVS or amnio after a positive cfDNA testing result?Confirmatory CVS or amnio after a positive cfDNA testing result?
Amniocentesis obviatesAmniocentesis obviates
the potential risk ofthe potential risk of
fetoplacentalfetoplacental
discrepancies because cellsdiscrepancies because cells
used for karyotyping areused for karyotyping are
fetal and not placentalfetal and not placental
The early geneticThe early genetic
information providedinformation provided
by CVS can be a benefitby CVS can be a benefit
We tried to estimate the chromosome specific frequency in which aWe tried to estimate the chromosome specific frequency in which a
CVS performed after high-risk cfDNA result would require a follow-upCVS performed after high-risk cfDNA result would require a follow-up
amniocentesis due to the diagnosis of mosaicism on CVSamniocentesis due to the diagnosis of mosaicism on CVS
AFAFCVSCVS

17. Grati FR et al, Prenatal Diagnosis 2015
~900 mosaic cases with abnormal cytotrophoblast: CPM1/3-~900 mosaic cases with abnormal cytotrophoblast: CPM1/3-
TFM4/6TFM4/6
Total abnormal casesTotal abnormal cases
Formulas:Formulas:
 CPM1+CPM3+TFM4+TFM6/Total-(CPM2+TFM5)CPM1+CPM3+TFM4+TFM6/Total-(CPM2+TFM5)
 (TFM4+TFM6)/(CPM1+CPM3+TFM4+TFM6)(TFM4+TFM6)/(CPM1+CPM3+TFM4+TFM6)
 (CPM3+TFM6)/(CPM1+CPM3+TFM4+TFM6)(CPM3+TFM6)/(CPM1+CPM3+TFM4+TFM6)
 (CPM1+TFM4)/(CPM1+CPM3+TFM4+TFM6)(CPM1+TFM4)/(CPM1+CPM3+TFM4+TFM6)
Table 1: schematic representation of the different types of mosaics and the of the associated expected NIPS results
Type of mosaic
(Cyto)trophoblast
(direct preparation or short
term culture)
Mesenchyme
(long term culture)
Amniocytes NI PS result
CPM 1 abn n n FP
CPM 2 n abn n TN
CPM 3 abn abn n FP
TFM 4 abn n abn TP*
TFM 5 n abn abn FN
TFM 6 abn abn abn TP*
CPM=confined placental mosaicism; TFM=true fetal mosaicism;
Abn=abnormal; n= normal; FP= false positive; FN=false negative; TN=true negative;
TP* =true positive assuming a consistent percentage of the mosaic abnormal cell line in cytotrophoblast
Confirmatory CVS or amnio after a positive cfDNA testing result?Confirmatory CVS or amnio after a positive cfDNA testing result?

18. Type 3Type 3
Type 1Type 1
CVS (11-13wg)CVS (11-13wg)
Type 1 Type 3Type 1 Type 3
aa
AbnormalAbnormal
AF (16-18wg)AF (16-18wg)
MMoossaaiicc
Trisomy 21 Trisomy 18 Trisomy 13 Monosomy X
2 4 22 59
(58 (27-91)) (25(15-41)) (5(3-6)) (2(1-2))
% (1/ (95% CI)
Trisomy 21 Trisomy 18 Trisomy 13 Monosomy X
44 14 4 26
(2(2-4)) (7(3-25)) (27(5-152)) (4(3-6))
% (1/ (95% CI)
Trisomy 21 Trisomy 18 Trisomy 13 Monosomy X
78 40 NA 48
(1(1-2)) (3(1-9)) NA (2(1-4))
% (1/ (95% CI)
Trisomy 21 Trisomy 18 Trisomy 13 Monosomy X
11 NA 5 16
(9(2-50)) NA (20(4-113)) (6(3-13))
% (1/ (95% CI)
Probability of the fetus havingProbability of the fetus having
abnormal cells if a mosaic wasabnormal cells if a mosaic was
diagnosed in a CVS sample after a HRdiagnosed in a CVS sample after a HR
cfDNA testing resultcfDNA testing result
Probability of requiring anProbability of requiring an
amniocentesis if a CVS isamniocentesis if a CVS is
performed after a high-risk cfDNAperformed after a high-risk cfDNA
screening report (Type 1+3)screening report (Type 1+3)
Confirmatory CVS or amnio after a positive cfDNA testing result?Confirmatory CVS or amnio after a positive cfDNA testing result?
Grati FR et al, Prenatal Diagnosis 2015
 T21/18T21/18 may be reasonable to perform CVSmay be reasonable to perform CVS
 MXMX frequency of mosaicism quite high: prenatalfrequency of mosaicism quite high: prenatal
diagnosis should be deferred to amniocentesis evendiagnosis should be deferred to amniocentesis even
if there is a normal sonogramif there is a normal sonogram
 T13T13 frequency of mosaicism relatively highfrequency of mosaicism relatively high
 Reasonable offering an amniocentesis,Reasonable offering an amniocentesis,
specially if the sonogram is normalspecially if the sonogram is normal
 It makes sense to do a CVS if sonogram isIt makes sense to do a CVS if sonogram is
abnormalabnormal

19. If, after informed consent, a patient does elect CVSIf, after informed consent, a patient does elect CVS
following cfDNA testing,following cfDNA testing, both cytotrophoblast andboth cytotrophoblast and
mesenchyme karyotypesmesenchyme karyotypes are necessary to provide herare necessary to provide her
the most accurate risk assessment for fetal involvement.the most accurate risk assessment for fetal involvement.
There is prognostic value for fetal involvement onlyThere is prognostic value for fetal involvement only
in determining which placental cell type is affected byin determining which placental cell type is affected by
mosaicism and its distribution in the involved tissue/smosaicism and its distribution in the involved tissue/s
CONCLUSIONS & PRACTICAL INDICATIONSCONCLUSIONS & PRACTICAL INDICATIONS
Grati FR et al, Prenatal Diagnosis 2015
CHOOSING CVS…

20. Grati FR et al, PD 2015; Grati FR, JCM 2014
CONCLUSIONS & PRACTICAL INDICATIONSCONCLUSIONS & PRACTICAL INDICATIONS
CHOOSING AMNIO…
Any High Risk cfDNA testing result for aneuploidies shouldAny High Risk cfDNA testing result for aneuploidies should
be considered as POTENTIALLY derived from a mosaicbe considered as POTENTIALLY derived from a mosaic
cytotrophoblastcytotrophoblast
Quantitative fluorescent PCR (QF-PCR)Quantitative fluorescent PCR (QF-PCR) NOT INFORMATIVENOT INFORMATIVE
for:for:
• low level trisomy mosaicism (<20%)low level trisomy mosaicism (<20%)
• mosaic monosomy X (MX)mosaic monosomy X (MX)
Extended cytogenetic analysis and/or cell count onExtended cytogenetic analysis and/or cell count on
amniocytes after a standard karyotype is normal (Guidelines:amniocytes after a standard karyotype is normal (Guidelines:
confirmatory amnio after that a mosaic abnormality is found inconfirmatory amnio after that a mosaic abnormality is found in
CVS)CVS)

21. UN GRAZIE SPECIALE! THANK YOU!
Giuseppe SimoniGiuseppe Simoni
Susan GrossSusan Gross Komal BajajKomal BajajJose FerreiraJose Ferreira

22. GRAZIE! THANK YOU FOR YOUR ATTENTION!