Cell-free DNA test oltre le comuni aneuploidie

Francesca Romana GRATI, Ph.D., ErCLG
R&D Director
TOMA Advanced Biomedical Assays, S.p.A.
fgrati@tomalab.com
Cell-free DNA test oltre le comuni aneuploidie

Expansion of cfDNA testing menus beyond
T21,18,13
• Sex chromosome aneuploidies
• 22q11.2DS
• Other rare microdels/dups (non-22q)
• ‘Large’ partial imbalances
• Rare autosomal trisomies

Expansion of cfDNA testing menus beyond T21,18,13
Increased False Positive Rate of the applied test
Combined false positive rate
Increases with number of conditions screened
False positive rate = 21 + 18 + 13 + XX + XY + XO + XXY + XYY+ XXX + ...
Unnecessary anxiety and invasive testing
Complex counseling

CfDNA SCREENING FOR SCAs
Advantages
High prevalence (combined: 1/500 live births), constant at all
maternal ages
After Down syndrome, SCAs are the most frequent diagnosis at the time
of amniocentesis
Early hormone therapy may be beneficial
Better prognosis in prenatally diagnosed newborns with 47,XXY
Samango-Sprouse et al, Am J Med Genet C Semin Med Genet. 2015 Jun;169(2):150-7; Am J Med Genet A. 2013
Mar;161A(3):501-8; March of Dimes, December 2009. Retrieved January 2013 at
http://www.marchofdimes.com/baby/birthdefects_chromosomal.html.; Mennuti et al, Prenat Diagn. 2015 Oct;35(10):980-5.

CfDNA SCREENING FOR SCAs
Mennuti et al, Prenat Diagn. 2015 Oct;35(10):980-5.; Wang et al. Clinical Chemistry 2014; Grati et al, Genetics in Medicine
2014; Wang et al Clinical Chemistry 60:1 251–259 (2014); Petersen et al, Am J Obstet Gynecol 2017;217:691.e1-6; Meck et
al. Am J Obstet Gynecol 2015;213:214.e1-5; Reiss et al, Prenat Diagn. 2017 May;37(5):515-520.
Limits
Compared to T21, T18 and T13, SCAs have relatively few serious physical
abnormalities
Challenging genetic counseling:
Variable and unpredictable clinical outcome
Over-representation bias of individuals with more severe phenotypes
Maternal incidental findings
8.6% of positive cfDNA results for SCAs are due to a SCA in the maternal karyotype
Mosaic in fetus: 15% of 47,XXY, 10% of 47,XXX, 50% of 45,X
Vanishing twins may cause FP results
Higher FP rate and decreased PPV for MX should be anticipated during pretest
counseling

cfDNA testing performances: a meta-analysis
Gil et al, Ultrasound Obstet Gynecol. 2017 Apr 11. doi: 10.1002/uog.17484. [Epub ahead of
print]
DR FPR
n n n (95% CI) (95% CI)
99.7% 0.04%
(99.1-99.9) (0.02-0.08)
98.2% 0.05%
(95.5-99.2) (0.03-0.07)
99.0% 0.04%
(65.8-100) (0.02-0.07)
95.8% 0.14%
(70.3-99.5) (0.05-0.38)
100.0% 0.003%
(83.6-100) (0-0.07)
DR FPR
(95% CI) (95% CI)
100.0% 0%
(95.2-100) (0-0.003)
*peer-review studies reporting on clinical validation or implementation of maternal cfDNA testing in screening for
aneuploidies, in which data on pregnancy outcome were provided for more than 85% of the study population
(January 2011-31 December 2016)
Type of
aneuploidy
Twin pregnancies: weighted pooled
T21 8 24 1,111
45,X 23 36 7,677
other SCA 11 17 5,383
T18 25 560 212,019
T13 18 119 212,883
Type of
aneuploidy
number of
studies
trisomic
cases
non-trisomic
cases
Singleton pregnancies: weighted pooled
T21 30 1,963 225,032

CLINICAL STUDIES WITH cfDNA TEST FOR SCAs
Clinical studies systematically assessing fetal karyotype in all screen positive and
negative cases are not easy to perform
Rarity of individual SCAs (XYY)
High incidence of mosaic conditions for SCAs
Mild features at birth (e.g. 47,XXX or 47,XYY)
SCAs will not necessarily be apparent from observing the neonatal phenotype
Clinical sensitivity should be assessed with
Karyotype on apparently normal liveborns with a screen negative result
Long-term clinical follow-up
Performances and PPV have not been clearly established and NPV is unknown

REASONS FOR FALSE POSITIVE cfDNA RESULTS FOR SCAs
Feto-Placental Mosaicisms
(Vanishing) twin
Maternal imbalances
Fetal side Maternal side
Test characteristics (algorithm, analysis
depth, quality metrics…)

HIGH RISK RESULTS FOR SCAs
Choice of confirmatory invasive procedure (CVS or AFS)
In the context of the clinical outcome of the specific syndrome
In the context of U/S investigation
HR MX result in combination with an abnormal U/S scan is likely to be a NM MX in CV
Maternal testing could be privileged in high risk MX cases with advanced
maternal age
Detection of maternal mosMX does not exclude the presence of a SCA in the fetus
This finding does not circumvent the need to offer fetal diagnostic testing
Grati et al, Prenat Diagn. 2017 Oct;37(10):1017-1027; Reiss et al, Prenat Diagn.
2017 Mar 21. doi: 10.1002/pd.5039

GENETIC DEFECT OF 22q11.2DS AND cfDNA TECHNOLOGIES
GeneReviews® , Donna M McDonald-McGinn, MS, CGC, Beverly S Emanuel, PhD, and Elaine H
Zackai, MD, FACMG
85%
15% atypical
“nested”
deletions
BCR
Proximal
Central
Distal
SNP-based / DANSR
TCEA
MPSS (whole chr 22)

Dugoff et al, Prenat Diagn. 2016 Jun 22. doi: 10.1002/pd.4864; Grati et al, Prenat Diagn. 2015 May 11. doi:
10.1002/pd.4613; McDonald-McGinn DM et al, GeneReviews, 1993; McDonald-McGinn DM et al, Genet Med
2001;3:23–9; Bassett AS et al, J Pediatr 2011;159:332–9.e1; Cheung EN, et al, Genet Med. 2014;16:40-4.
Most common microdeletion in humans (1/1000)
Maternal age is not a risk factor (constant at all MA)
Up to 90% of individuals diagnosed with 22q11.2 deletion have a de novo
(new) mutation
Not detectable by routine screening or standard karyotyping
CfDNA screening can be done before Level II ultrasound with fetal
echocardiogram
Advantages of 22q11.2DS screening

Dugoff et al, Prenat Diagn. 2016 Jun 22. doi: 10.1002/pd.4864; Grati et al, Prenat Diagn. 2015 May 11. doi:
10.1002/pd.4613; McDonald-McGinn DM et al, GeneReviews, 1993; McDonald-McGinn DM et al, Genet Med
2001;3:23–9; Bassett AS et al, J Pediatr 2011;159:332–9.e1; Cheung EN, et al, Genet Med. 2014;16:40-4.
Prevention and therapeutic options:
Prevent the distressing ‘diagnostic odyssey’ due to variable expressivity
Allow preparation for delivery at a center capable of caring for the child
Improve neonatal management
Enable the detection of hypocalcemia, dysphagia and those congenital heart defects
which may go undetected prior to neonatal discharge
Prevent neonatal seizures secondary to unrecognized hypocalcemia leading to more
severe intellectual deficits
Offer PGD or prenatal diagnosis in future pregnancies for previously
undiagnosed mothers
Advantages of 22q11.2DS screening

Challenging pre-test counseling
Variable expressivity & unpredictable severity
Unknown clinical performances
Variable from one test to another one due to the variable design and analysis
depth
Residual risk for 22q11.2DS after a low risk result
Only a subset of 22q11 deletions (3Mb ± A-C and A-B) covering approx. 85-
95% of the overall deletions in affected patients
Follow up of HR results
Confirmatory invasive procedure with CMA after a HR result
Follow-up maternal CMA investigation if fetus is normal
Limitations of 22q11.2DS screening
Grati and Gross, manuscript under revision

Discordant results:
Biological reasons for discordant results
Insufficient FF%  FN
Maternal 22q11.2 del  FP
Vanishing twin  FP
Fetoplacental mosaicism  FP+FN
Maternal malignancy  FP
Grati and Gross, manuscript under revision; Dugoff et al, Prenat Diagn. 2016 Jun
22. doi: 10.1002/pd.4864

No results:
% is unknown and strictly dependent of type of technology (MPSS vs DANSR vs SNP-
based)
Possible causes:
Low FF%
Low cfDNA (maternal/fetal) quality
Insufficient # of informative SNPs (homozygozity stretces)
Other counting issues related to reference chromosomes?
Other unknown factors
Follow up of No results for 22q del:
No consensus on management options
US scan is first option that will decrease the residual risk
US scan might be silent also at later GA & serum analytes do not correlate
Invasive procedure or CMA at birth?? Still not enough evidence for an increased risk for
22q11.2DS
Grati and Gross, manuscript under revision

CfDNA TESTING FOR OTHER MICRODELS/DUPS (non-22q)
Yaron et al, Obstet Gynecol. 2015 Nov;126(5):1095-9; Grati FR, Ultrasound Obstet Gynecol. 2016 May 31. doi:
10.1002/uog.15975; Shaffer et al, PD 2012; Benn, Clin Genet. 2016 Jun 10. doi: 10.1111/cge.12818.
Restricted panel of submicroscopic CNVs (<5Mb)
15q11, 5p, 4p, 1p36 microdeletions
Specific recurrent CNVs with well-know highly penetrant genomic syndromes

High post-test residual risk for fetal pCNVs
Wapner et al, NEJM 2012; Yaron et al, Obstet Gynecol. 2015 Nov;126(5):1095-9; Grati FR, Ultrasound
Obstet Gynecol. 2016 May 31. doi: 10.1002/uog.15975
CNVs are common in the low risk population (1.7%)
Mixture different types of CNVs, but individually rare (<1/10000-1/50000)
They represent only a portion (~20%) of the overall pCNVs that can affect the fetus
All
possible
CNVs
(1.7%)
CNVs
targeted by
cfDNA test
Avoid false reassurance to patients! Consistent residual risk

CfDNA TESTING FOR MICRODELETIONS
10.1002/uog.15975; Shaffer et al, PD 2012; Benn, Clin Genet. 2016 Jun 10. doi: 10.1111/cge.12818; Martin et al
Clin Genet. 2017. doi: 10.1111/cge.13098; Helgeson et al, Prenat Diagn. 2015;35(10):999-1004
Limitations
Minimal data on general risk populations nor prospective data as to whether there
would be any tangible benefits with respect to outcome
Data on high risk population:
SNP-based: 31.7% overall PPV; overall FPR 0.07%
MPSS: overall PPV 60-100% when follow up was available; overall FPR 0.0017%.
Poorly validated tests because individually rare in the general population
Performances?
Minimal required FF% in relation to the size?
No result rate?
PPV in low risk/general population is expected to be very low (because of the rarity)

Genome-wide cfDNA testing
(large partial imbalances and RATs)
10.1002/uog.15975; Shaffer et al, PD 2012; Benn, Clin Genet. 2016 Jun 10. doi: 10.1111/cge.12818.
Genome-wide large microscopic CNVs:
≥7-10Mb (resolution of the standard karyotype)
deletions and duplications - large partial imbalances (PIs)
rare autosomal trisomies (RATs)
across the whole genome

CfDNA testing for rare chromosomal conditions
(RATs and large partial imbalances)
Trophoblast is the main source of ‘fetal’ circulating cfDNA fragments
Projected predictions of GW-cfDNA testing performance were based on
cytotrophoblast karyotype
If “Fetal” cf-DNA and “direct” CVS preparation are equivalent, what can we expect
to see for expanded NIPS and what are the pitfalls of offering this testing?
Benn and Grati, Ultrasound Obstet Gynecol. 2018 Jan 24. doi: 10.1002/uog.19014

CfDNA testing for rare chromosomal conditions (RATs
and large partial imbalances)
100,000 all-risk population - first
trimester GW-cfDNA testing
Bal abns/Triploid/polyploid
Not detectable by cfDNA
test
RATs and other segmental imbalances
497 (0.47%) RAT +
(cytotrophoblast
with RAT)
300 (0.3%) Other +
(cytotrophoblast with
partial imbalance)
99,227 (99.23%) NL
karyotype/CMA
72 (0.07%)
Non-mos
abn; fetal
demise
7 Abn fetal
ktype; TFM
214 NL fetal
ktype; Risk for
significant UPD
183 NL fetal
ktype (CPM for
other RATs)
22 NL fetal
ktype
(CPM16);
Risk for t16
related
placental
dysfunction
Amnio – 425 (0.43%)
Variable and
unpredictable
phenotype
~2 abnormal U/S
~2 confirmed in
fetus or
newborn?
Very small risk
for recessive
single gene
disorder due to
UPD.
Low and
unpredictable
risk for placental
dysfunction
Risk for IUGR,
preeclampsia,
and fetal abn
~4 with UPD (2%);
~210 without UPD
Low and
unpredictable risk
for placental
dysfunction
t21, t18, t13, SCA Microdeletions/
duplication

LARGE PARTIAL IMBALANCES DETECTABLE WITH GW-
cfDNA TESTING
Supernumerary marker chromosomes
47,XX,+mar
Derivative of unbalanced reciprocal translocations
46,XY,der(3)t(3;7)(p25;q21)mat
Partial deletions
46,XX,del(8)(p23.1p23.2)
Derivatives of unbalanced inversions
46,XX,rec(5)dup(5q)inv(5)(p15.2q32)
Dicentric chromosomes
47,XX,+dic(15;15)(q11.1;q11.1)

FOLLOW-UP OF A HIGH RISK GW-cfDNA TESTING
RESULT FOR LARGE PARTIAL IMBALANCES
Invasive procedure + CMA
Phenotype-genotype correlations are not always predictable
with certainty also after CMA and detailed US scan
VOUS, incidental findings and secondary findings

Interpretation of Copy Number Variation
Wapner et al, NEJM 2012; Kearney et al. Genet Med 2011;13(7):680–685.
2-4% VOUS

100,000 all-risk population - first
trimester GW-cfDNA testing
Bal abns/Triploid/polyploid
Not detectable by cfDNA
test
RATs and other segmental imbalances
497 (0.47%) RAT +
(cytotrophoblast
with RAT)
82 non-mos
Abn ktype
300 (0.3%) Other +
(cytotrophoblast with
partial imbalance)
99,227 (99.23%) NL
karyotype/CMA
72 (0.07%)
Non-mos
abn; fetal
demise
7 Abn fetal
ktype; TFM
214 NL fetal
ktype; Risk for
significant UPD
183 NL fetal
ktype (CPM for
other RATs)
22 NL fetal
ktype
(CPM16);
Risk for t16
related
placental
dysfunction
172 NL fetal
ktype; CPM
46 Abn
fetal
ktype;
TFM
Amnio – 425 (0.43%) Amnio
Variable and
unpredictable
phenotype
Abnormal
>50% with
abnormal U/S
scan
Variable and
unpredictable
phenotype
~2 abnormal U/S
~2 confirmed in
fetus or
newborn?
Very small risk
for recessive
single gene
disorder due to
UPD.
Low and
unpredictable
risk for placental
dysfunction
Risk for IUGR,
preeclampsia,
and fetal abn
No significant risk
for UPD/recessive
monogenic
disorders.
Minimal
(unquantifiable)
risk for placental
dysfunction/insuff
iciency
~4 with UPD (2%);
~210 without UPD
Low and
unpredictable risk
for placental
dysfunction
t21, t18, t13, SCA Microdeletions/
duplication

Additional screen-positive rate of GW-cfDNA test beyond T21, 18, 13 and SCAs: 0.8%
(0.5%RATs+0.3%PI)
0.1% associated with an unambiguous diagnosis of fetal abnormality due to non-mosaic
chromosome imbalance or clinically significant UPD – clinically actionable?
0.1% associated with early fetal loss (non-mosaic or mosaic RAT) - clinically not actionable
0.6% associated with a mosaic partial imbalance or SMC with a highly variable risk and
some degree of uncertainty for the residual risk of a fetal abnormality (including that for
recessive disorders), IUGR or pregnancy complications – unclear clinical utility
The main reason for the phenotype uncertainty is mosaicism

Limitations of the study
Additional sources of FP & FN results usually associated with cfDNA testing
were not considered
Sources of FPs
Vanished twins: up to 0.5% of women screened?
Maternal CNVs: particularly when evaluating smaller imbalances
Maternal cancer (“dysploidy”): perhaps 0.008% of all women screened
Sources of FNs
Low level mosaics in cytotrophoblast – TFM4 and 6 (especially with low fetal fraction)
Smaller partial imbalances (especially with low fetal fraction)
True mosaicism in the fetus not represented in cytotrophoblasts (>0.01%) (TFM5) (FNR for
PI: ~1/40)

Projections vs clinical experience
Pescia et al. 2017 Genet Med. 19:169-175; Ehrich et al. 2017 Genet Med 19;1332-7; Pertile et al. 2017. Sci Transl Med. 9,eaan1240;
Fiorentino et al., 2017. Prenat Diagn. 37;593-601; Van Opstal et al. Genet Med 2017 eprint ahead of publication; Brady et al, Clin
Genet. 2016, 89(5):523-30; Porat et al, AJOG, Volume 218, Issue 1, Supplement, Page S58; Benn and Grati, Ultrasound Obstet Gynecol.
2018 Jan 24. doi: 10.1002/uog.19014
Combined test selects for placental insufficiency (PAPPA and hCG), and t16, t9
Study N RAT rate Most common Other findings Clinical follow-up Proportions
Unambiguous(RAT+PI)*:0.06 % 7.50%
Miscarriage (RAT+PI)**:0.1% 12.50%
Ambiguous(RAT+PI)***:0.64% 80.00%
Brady et al,
2016
4,000 (a)
15
(0.38%)
T7 (3),
t1,8,9,10,15,16,20,22
(1) ,partt18(2),m20,
m22 (1)
Largedel or dup not
reported; 4 no follow-up,
7 unconfirmed on AF
Confirmed:1 UPD15,1
mosT16,1 part t18; m20
and m22
NA
Pesciaet al.
2017
6,388
50
(0.78%)
t7 (16); t8 (8); t22 (4);
t16 (3); t17 (3)…
Some small CNVs
detected
NA
Ehrich et al.
2017
10,000 (b) 80 (0.8%)
t16 (15),t7 (11), t3
(10),…
Other del or dup (PI) in
0.84%
NA
Unambiguous(RAT only)*: 6/89817 (0.007%) 2.00%
Miscarriage (RAT only)**: 24/89817 (0.03%) 7.80%
Ambiguous(RAT only)***: 17/89817 (0.02%) 5.60%
Unknown (RAT only): 259/89817 (0.29%) 84.60%
Unambiguous(RAT+PI)*: 1/12078 (0.008%) 3.6%
Miscarriage (RAT+PI)**: 7/12078 (0.06%) 25%
Ambiguous(RAT+PI)***: 20/12078 (0.17%) 71.40%
Unambiguous(RAT+PI)*:17/2527 (0.67%) 42.50%
Miscarriage(RAT+PI)**:0/2527 0%
Ambiguous(RAT+PI)***:21/2527 (0.83%) 52.50%
Unknown:2/2527 (0.08%) 5%
Unambiguous(RAT+PI)*: 2/671 (0.3%) 18.20%
Miscarriage (RAT+PI)**: 4/671 (0.6%) 36.40%
Ambiguous(RAT+PI)***: 5/671 (0.75%) 45.50%
10
(0.49%)
most7,t7,t8,t10,t14,
t15,t16 (2),t22 (2)
1 case of 4p15.2-15.1 del
(PI: 0.15%)
Porat et al,
2018
671 (f)
12078 (d)
Fiorentino et al.
2017
VanOpstal et
al.,2017
2527(e)
Bennand Grati,
2018
>40,000^ 0.47%
t16,CPM,mosaics,
homogeneous
causingfetal loss
Large del or dup(PI) in
0.3%
Pertile et al.
2017
89817 ©
28
(1.11%)
t16 (9),t7 (6),t9 (3),
t8 (3),…
306
(0.34%)
t7 (67); t15 (44); t16
(37); t22 (28); t3
(22)….
“Dysploid”: 16 (0.02%)
Large del or dup (PI): 11
(0.09%) (2 casesexcluded
asdetected in pregnancies
with known abn u/s)
t22 (5); t7 (4); t15
(4)…
17
(0.14%)
Largedel or dup(PI):12
(0.47%)

LEVEL OF EVIDENCE FOR CLINICAL UTILITY
Grati FR and Benn P, Reply to Fiorentino et al Prenatal Diagnosis 2017, 37, 1050–1052
Before providing Genomewide tests we need:
• Unbiased assessment of sensitivity and specificity for clinically significant
actionable conditions
• Follow-up studies on all cases with RATs detected by cf-DNA analysis
• Definition of the patient management paths for women with positive
findings
Clinical utility is of key importance and this requires much more than
demonstrating that some additional imbalances are detectable.

CfDNA testing chromosome targets
Is the dynamics of cfDNA testing genetic contents
driven by technological possibilities or by clinical
evidences?

Susan Gross
Komal BajajJose Ferreira
Peter Benn

Cell-free DNA test oltre le comuni aneuploidie

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