BREVE PREMESSA: Negli ultimi anni si sono sviluppate tecnologie altamente sofisticate che consentono di valutare il rischio per condizioni cromosomiche fetali. L'ampio ventaglio di opzioni oramai disponibili nell'ambito degli screening non invasivi pone numerosi quesiti su quale tecnologia utilizzare e le problematiche specifiche connesse alla tecnologia. Durante questa mezza giornata di aggiornamento verranno dunque presentate in modo semplificato le basi molecolari delle differenti tecnologie coi vantaggi e vantaggi correlati, quali test sono disponibili e il loro livello di certificazione in relazione alla normativa europea inerente alla marchiatura CE-IVD, quali sono le cause di risultati discordanti, dei ‘no results’ e la gestione dei casi con risultato ad alto rischio, no result e discordanze OBIETTIVI FORMATIVI: • Descrivere le differenti tecnologie disponibili coi relativi vantaggi e svantaggi; • Presentare le cause biologiche dei risultati discordanti mediante cfDNA test; • Illustrare le diverse cause di ‘no result’ e le implicazioni sulle performances del test; • Descrivere l’utilità delle certificazioni, validazioni dei cfDNA test e dei controlli esterni di qualità; • Discutere circa l’utilità clinica dei contenuti aggiuntivi oltre alle trisomie 21,18,13; • Discutere circa il follow-up e il management dei risultati ad alto rischio, dei no results e dei risultati discordanti.

2. YOU ARE ALL INVITED TO ATTEND THE
ISPD ANNUAL MEETING!!!

3. Francesca Romana GRATI, Ph.D., ErCLG
R&D Director
TOMA Advanced Biomedical Assays, S.p.A.
fgrati@tomalab.com
Panorama delle diverse tecnologie: cosa è
successo dal 2011 a oggi nell’ambito del cfDNA
test per le aneuploidie fetali

4. OUTLINE
General principle of cfDNA tests
Description of genome-wide and targeted technologies
Analysis depth and breadth of chromosome targets
Fetal fraction measurement
level of validation, reliability, accuracy and consistency
Pros and cons of genome-wide and targeted technologies
CfDNA testing spreading process
Overview of different cfDNA tests: CE-IVD, home-brew, home-made, RUO, CLIA,
CAP, targeted, genome-wide

5. OUTLINE
General principle of cfDNA tests
Description of genome-wide and targeted technologies
Analysis depth and breadth of chromosome targets
Fetal fraction measurement
level of validation, reliability, accuracy and consistency
Pros and cons of genome-wide and targeted technologies
CfDNA testing spreading process
Overview of different cfDNA tests: CE-IVD, home-brew, home-made, RUO, CLIA,
CAP, targeted, genome-wide

6. How do different cfDNA technologies work?
Fetal trisomy
risk score
DANSR
SNP-based
Targeted NGS
MPSS-1
MPSS-2
MPSS-3
Clinicians are asked to chose a technology and NOT anymore a test!

7. General principle
TOTAL CELL
FREE DNA
CIRCULATING
IN THE
MATERNAL
PLASMA

8. General principle
TOTAL CELL
FREE DNA
CIRCULATING
IN THE
MATERNAL
PLASMA
10% fetal fraction
In average at 10 sdg*
90% maternal fraction
*Ashoor et al, Ultrasound Obstet Gynecol 2013;41:26–32.

9. Assessment of fetal aneuploidy with cell-free DNA
Reference euploid
conceptus/counts
*Ashoor et al, Ultrasound Obstet Gynecol 2013;41:26–32.
Test pregnancy
without T21
Chr21 Disomy: expected ratio 1:1 (r=1)fetal fraction
maternal fraction

10. Assessment of fetal aneuploidy with cell-free DNA
fetal fraction
maternal fraction
*Ashoor et al, Ultrasound Obstet Gynecol 2013;41:26–32.
Trisomy 21
10% fetal fraction Counts ratio increases from r=1 to r=1.05
5% increment of the FF
Reference euploid
conceptus/counts
Test pregnancy
WITH T21

11. The higher the fetal fraction, the higher is the count ratio value and the more reliable
is the aneuploidy detection
FF increment
+ 10% at
20%FF
+ 20% at
40%FF
Reference
euploid counts
R=1.20
R= ≈1
R=1.10
+ 5% at
10%FF
R=1.05
R=1.02
+ 2% at
4%FF
<2% at
<4%FF
Pregnancies with Trisomy 21

12. Importance of Fetal Fraction measurement
To reliably distinguish affected from unaffected
pregnancies, cfDNA tests require a minimum amount of
fetal DNA in the maternal circulation – typically ~4%

13. OUTLINE
General principle of cfDNA tests
Description of genome-wide and targeted technologies
Analysis depth and breadth of chromosome targets
Fetal fraction measurement
level of validation, reliability, accuracy and consistency
Pros and cons of genome-wide and targeted technologies
CfDNA testing spreading process
Overview of different cfDNA tests: CE-IVD, home-brew, home-made, RUO, CLIA,
CAP, targeted, genome-wide

14. SEVERAL cfDNA TESTS HAVE BEEN DEVELOPED
3 different testing strategies:
NGS-based counting technologies:
Massively parallel shotgun sequencing (MPSS) - majority of assays
Targeted sequencing
Digital analysis of selected regions (DANSR)
Targeted SNP-counting
TARGETED
GENOMEWIDE
OR WHOLE
GENOME

15. Chr 21, 18, 13, X & Y cfDNA
Other Chr cfDNA
Unmapped cfDNA
Origin of cfDNA in maternal bloodstream
Genome-wide Technology: sequencing by NGS of maternal and fetal cfDNA fragments belonging
from all chromosomes – up to ~25 Million of cfDNA fragments
MPSS
Sehnert et al. Clin Chem. 2011 Jul;57(7):1042-9; Jensen et al. PLoS One. 2013;8(3):e57381;
Lau TK et al. Ultrasound Obstet Gynecol. 2014;43(3):254-264.

16. SEQUENCING OF cfDNA FRAGMENTS
Sequencing of the first nucleotides of each circulating fragment (usually from 25 to 36 base pairs, bp)
Variable # depending on the type of test
Chiu et al, PNAS, 2008, 105, 20458–20463; Fan et al, 16266–16271, PNAS, 2008, 105
Sequencing
Sequencing

17. ALLIGNMENT OF THE SEQUENCE OF cfDNA FRAGMENTS
Chiu et al, PNAS, 2008, 105, 20458–20463; Fan et al, 16266–16271, PNAS, 2008, 105
Human Reference Genome
Determination of the chromosome origin of each individual sequenced fragment by mapping it against a
Human Reference Genome using an algorithm (approx. 50-60% remains unmapped)

18. COUNTING OF ALLIGNED SEQUENCES WITH MPSS
Chiu et al, PNAS, 2008, 105, 20458–20463; Fan et al, 16266–16271, PNAS, 2008, 105
COUNTS
Z-score analyis  fetal trisomy risk score

19. DATA ANALYSIS IN MPSS: Z-SCORE
Palomaki et al, Genet Med 2011; 13: 913–920; Canick, et al. Prenat Diagn 2013, 33, 1–8
cfDNA results by MPSS are expressed in ‘z-score’ values
Distribution of Z-scores:
euploid fetuses: distribution is Gaussian, with a mean of 0 and a SD of 1
trisomic fetuses: the mean of the Z-score > 0 and increases with increasing fetal fraction

20. DATA GENERATION WITH MPSS
Counting data are generated for ALL CHROMOSOMES (122+X/Y)
Dosage results for chromosomes 13,18,21 (X/Y) are provided by applying a bioinformatic filter
hiding the ‘off-target’ chromosomes
Dosage results for ‘off-target’ chromosomes can be provided unlocking the bioinformatic filter
BIOINFORMATIC FILTER

21. MPSS ANALYSIS SETTING - MULTIPLEXING
Analysis in multiplexing: several samples can be sequenced in parallel in one reaction
All ‘barcoded’ samples mixed in a unique sequencing ‘tube’
Given a total fixed amount of sequences available in one reaction, the # of cfDNA fragments that can
be sequenced for each sample depends on the number of sample input
Analytical conditions of the samples may change depending on the number of sample input
The higher the sample input the lower the analysis depth
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
NGS Cartridge
NGS Cartridge
Chiu et al, BMJ 2011;342:c7401, doi:10.1136/bmj.c7401

22. FACTORS INFLUENCING ANALYSIS DEPTH WITH MPSS
TECHNICAL FACTORS #reads/sample
Size of NGS reagent cartridge
MPSS Labs do not have the same settings
# sample input
May vary from lab to lab (24-48-96)
Concentration of the samples analysed in parallel
With multiplexing samples compete for a fixed amount of NGS reagents
Samples with higher concentration may deprive reagents to others reducing
reads available for the remaining samples
Process of concentration normalization is crucial

23. FACTORS INFLUENCING ANALYSIS DEPTH WITH MPSS
BIOLOGICAL FACTORS #reads/chromosome
Fetal fraction level
The higher the FF, the higher the number of fragments deriving from each
fetal chromosome and the higher the accuracy of fetal aneuploidy detection
Size of the analysed chromosome/region
Smaller chromosomes/regions (chr21, 22q11.2, …) need a higher
#reads/sample to improve clinical performances

24. TARGETED cfDNA TESTING TECHNOLOGIES
Targeted Sequencing
Targeted SNP-counting
Digital analysis of selected regions (DANSR)

25. DATA GENERATION WITH TARGETED cfDNA TESTS
Data are generated for chromosomes of interest only (13,18,21 ± X/Y ±
subset of microdels)
Dosage results for ‘off-target’ chromosomes cannot be obtained unless
specific assays analyzing fragments belonging from other chromosomes are
added

26. Chr 21, 18, 13, X & Y cfDNA
Other Chr cfDNA
Unmapped cfDNA
Origin of cfDNA in maternal bloodstream
Koumbaris et al, Clin Chem 62:6, 848-855, 2016, Sparks AB et al., Am J Obstet Gynecol. 2012 Apr;206(4):319.e1-9; Sparks
AB et al., Prenat Diagn. 2012 Jan;32(1):3-9; Zimmermann et al, Prenat Diagn. 2012 Dec;32(13):1233-41.
GENERAL PRINCIPLE OF TARGETED METHODS
Analysis focused on chromosomes of clinical interest  Analysis of 1 million of cfDNA fragments
Targeted capture/analysis with specific molecular probes of cfDNA fragments belonging from
chromosomes of clinical interest and reference chromosomes

27. ANALYSIS SETTINGS OF TARGETED METHODS – PROBE CAPTURE&AMPLIFICATION
Total CfDNA
Chr21
Chr18
Chr13
Chr1
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
Chr8
Chr9 Chr10
Chr11
Chr12
Chr14
Chr15
Chr16
Chr17
Chr19
Chr20
Chr22
ChrX
ChrY
Capture of the specific
cfDNA fragments
(chr 13,18,21)
Probe sequence complementary
to the cfDNA fragment of interest
Chr21
complementary
probe
Chr18
complementary
probe
Chr13
complementary
probe
Amplification
Many probes are used in parallel to analyse
multiple loci for each chromosome

28. ANALYSIS OF SELECTED FRAGMENTS WITH
TARGETED cfDNA TESTING TECHNOLOGIES
Targeted Sequencing
Targeted SNP-counting
Digital analysis of selected regions (DANSR) MICROARRAY
NGS

29. Analysis of selected fragments performed by NGS in MULTIPLEXING setting
Different from MPSS: available reagents are utilized to analyse a narrowed subset of
chromosomes/sample
A high analysis depth is generally achieved
Patient 1 - chr13,18,21
Patient 2 - chr13,18,21
Patient 3 - chr13,18,21
Patient 4 - chr13,18,21
Patient 5 - chr13,18,21
Patient 6 - chr13,18,21
Patient 7 - chr13,18,21
Patient 8 - chr13,18,21
Patient 9 - chr13,18,21
Patient 10 - chr13,18,21
NGS Cartridge
TARGETED ANALYSIS BY NGS
Koumbaris et al, Clin Chem 62:6, 848-855, 2016; Zimmermann et al, Prenat Diagn. 2012
Dec;32(13):1233-41.
COUNTS
Z-score/NATUS  fetal trisomy risk score

30. Juneau et al., Fetal Diagn Ther. 2014;36(4):282-6; Stokowski et al, Prenatal Diagnosis 2015;
Chiu et al, BMJ 2011;342:c7401, doi:10.1136/bmj.c7401
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 96
Analysis support is microarray
SINGLEPLEX: no multiplexing - each sample is analyzed individually in each sub-array
Analytical conditions (depth and performances):
Independent on the # of sample input
Independent on reagent supply
Standardized in each experiment and any DANSR labs
TARGETED ANALYSIS BY MICROARRAY

31. Chromosome Counts
Fetal
Fraction
Maternal Age
Gestational
Age
Fetal trisomy
probability
score
FORTE Algorithm -
Fetal Fraction Optimized Risk of Trisomy Evaluation
FORTE™ incorporates different risk factors with the
outcome of cfDNA testing (counts) )()|(
)()|(
DPDxP
TPTxP
j
j
Ashoor G et al., Am J Obstet Gynecol. 2012 Apr;206(4):322.e1-5.
Bayesian analysis by odds ratio
comparing a model assuming a
disomic fetal chromosome and
a model assuming a trisomic
fetal chromosome.

32. FORTE Algorithm -
Fetal Fraction Optimized Risk of Trisomy Evaluation
Ashoor G et al., Am J Obstet Gynecol. 2012 Apr;206(4):322.e1-5.
FORTE trisomy risk score is independent on FF%
FORTE

33. OUTLINE
General principle of cfDNA tests
Description of genome-wide and targeted technologies
Analysis depth and breadth of chromosome targets
Fetal fraction measurement
level of validation, reliability, accuracy and consistency
Pros and cons of genome-wide and targeted technologies
CfDNA testing spreading process
Overview of different cfDNA tests: CE-IVD, home-brew, home-made, RUO, CLIA,
CAP, targeted, genome-wide

34. Chr 21, 18, 13, X & Y cfDNA
Other Chr cfDNA
Unmapped cfDNA
Origin of cfDNA in maternal bloodstream SNPs
If captured/analysed cfDNA fragments map SNPs variants  determination of the
FF% by SNP genotyping by quantification of the SNP variants differing from those of
the mother
C
G
T
A
FETAL FRACTION MESUREMENT WITH TARGETED METHODS
Koumbaris et al, Clin Chem 62:6, 848-855, 2016, Sparks AB et al., Am J Obstet Gynecol. 2012 Apr;206(4):319.e1-9;
Sparks AB et al., Prenat Diagn. 2012 Jan;32(1):3-9; Zimmermann et al, Prenat Diagn. 2012 Dec;32(13):1233-41.

35. FETAL FRACTION MEASUREMENT WITH MPSS
Decreased sequencing depth of only few bp of each circulating fragment, limits the
possibility to measure the FF% by SNP genotyping
Alternative methods:
Sex chromosome ratio - only in male pregnancies
Differences between maternal and fetal cfDNA characteristics (all prenatal samples regardless
fetal sex)
Methylation
Distribution of maternal and placental cfDNA fragment size
Indirect sequencing variables
DNA digestion of fetal and maternal cfDNA related to different nucleosome positioning
Preferred ending sites for maternal and fetal cfDNA
Fan et al, Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16266-71; Fan and Quake, PLoS One. 2010;5(5):e10439; Rava et al, Clin
Chem. 2014;60(1):243-50; Nygren et al, Clin Chem. 2010;56(10):1627-35; Yu et al, Proc Natl Acad Sci U S A. 2014;111(23):8583-8;
Kim et al Prenat Diagn 2015; 35: 810–815; Straver et al, Prenat Diagn 2016;36: 614-621; Chan et al, Proc Natl Acad Sci U S A
2016; 113: E8159–E8168

36. FETAL FRACTION MEASUREMENT
Yu et al. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8583-8;
DNA fragment size distributions6
Fetal main peak at 146bp; Maternal main peak at 166bp
Likely to be a rough estimation of FF% due to a substantial overlap of maternal and placental
distributions

37. LEVEL OF VALIDATION OF FF MEASUREMENTS METHODS
Low level of validation for the most of the methods
Reliability?
Unknown reproducibility
Unknown consistency between methods
Direct comparison of 6 methods in a series of 375
pregnancies with a male fetus
methods based on binned autosomal read counts and nucleosome
positioning did not perform reliably when compared to Y-based methods
van Beek et al, Prenat Diagn. 2017 May 31. doi: 10.1002/pd.5079; Kim et al, Prenat Diagn 2015; 35: 810–815;
Straver et al, Prenat Diagn 2016;36: 614-621

38. VALIDATION OF FF MEASUREMENT METHOD WITH SNPs IN DANSR
Direct comparison of SNP-genotyping with 576 SNPs across chr112 and Y-chr based FF
quantification with DANSR
Schmid et al, Ultrasound Obstet Gynecol. 2018 Feb 26. doi: 10.1002/uog.19036
RELIABILITY&CONSISTENCY
REPRODUCIBILITY

39. INFORMATIVITY IN DIFFERENT RATIAL POPULATIONS
VALIDATION OF FF MEASUREMENT METHOD WITH DANSR
Schmid et al, Ultrasound Obstet Gynecol. 2018 Feb 26. doi: 10.1002/uog.19036

40. OUTLINE
General principle of cfDNA tests
Description of genome-wide and targeted technologies
Analysis depth and breadth of chromosome targets
Fetal fraction measurement
level of validation, reliability, accuracy and consistency
Pros and cons of genome-wide and targeted technologies
CfDNA testing spreading process
Overview of different cfDNA tests: CE-IVD, home-brew, home-made, RUO,
CLIA, CAP, targeted, genome-wide

41. WHAT HAPPENDED FROM 2011 TO TODAY?
Jani et al, UOG 2015

42. RESULTS OF THE SPREADING

43. WHAT TYPES OF TESTS?
Home-brew/home-made/RUO/LTD/CLIA/CAP CE-IVD (software&reagents)
Locally
China/US
/Cyprus
MPSS/Genome-wide or
common aneuploidies
MPSS/common
aneuploidies onlyTargeted Targeted
Trident Study national
screening test
Based on VeriSeq
technology (Illumina)
Coming soon

44. IS AN INTERNALLY VALIDATED HOME-BREW/RUO cfDNA
TEST EQUIVALENT TO A CE-IVD TEST?
- YES, IF THE TEST IS A NEW INVENTION
- NO, IF THE TEST IS PERFORMED WITH RUO KIT
QUESTION
http://www.salute.gov.it/portale/temi/p2_6.jsp?id=10&area=dispositivi-
medici&menu=conformita

45. CE-IVD

46. CE-IVD
• ‘Il presente decreto non si applica ai dispositivi fabbricati ed utilizzati
unicamente nell'ambito della stessa struttura sanitaria e nel luogo di
fabbricazione o utilizzati in locali contigui, senza essere oggetto di
trasferimento ad altro soggetto.’
• Non fa distinzione tra enti pubblici ed enti privati ma usa genericamente il
termine di ‘operatori sanitari’