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.

1. Barbara MALVESTITI
TOMA Advanced Biomedical Assays, S.p.A.
bmalvestiti@tomalab.com
Validazione dei cfDNA Test e
Controlli di Qualità Esterni e Interni

2. 1980 2011
Screening Tests Evolution

3. Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015
VALIDATION and VERIFICATION Genetic Test
https://www.cdc.gov/genomics/gtesting/ACC
E/

4. ACCE Model Process for Evaluating Genetic
Tests
Analytic Validity
Clinical Validity
Clinical Utility
Ethical/ Legal/ Social
Implications
https://www.cdc.gov/genomics/gtesting/ACC
E/

5. ANALYTIC VALIDITY
https://www.cdc.gov/genomics/gtesting/ACC
E/
Diagnostic routine
New test blinded
for operators
Informed
consent for
the new test
Results
ANALYTIC SENSITIVITY
= ability of a test to identify a genetic change when it is
present.
How often is the test positive when an
aneuploidy is present?
ANALYTIC SPECIFICITY
= ability of a test to report a negative screen result when in
fact there is no aneuploidy
How often is the test negative when an
aneuploidy is not present?

6. cfDNA test – 22q11.2 results from ANALYTICAL
VALIDATION and CLINICAL VERIFICATION cohort
Schmid et al, Fetal Diagn Ther. 2017 Nov 8. doi: 10.1159/000484317
Multicentric worldwide prospective blinded clinical validation is ongoing

7. ANALYTICAL VERIFICATION 22q
ANALYTICAL VALIDATION
Total samples (N) 456
22q11.2 (n/N) 31/33
No evidence of a deletion (n/N) 422/423*
Sensitivity %, (95% CI) 93.94%(95%CI:80.39-98.32)
Specificity %, (95% CI) 99.76%(95%CI:98.67-99.96)
Deletions spanned through the A-D 22q11.2 LCRs, sizes ranged from 2.37 and 2.89Mb
Sensitivity verified on 2 maternal plasma samples and 31 simulated pregnancy samples
obtained combining genomic 22q11.2DS DNA with plasma from a related/unrelated non-
pregnant euploid females (23) or provided as internal control in the reagents kit (8)
*Specificity verified on presumed unaffected - commercial samples without a known
diagnosis of 22q11.2 deletion, presumed to be unaffected (may cause underestimation
of Specificity)
Performances overlap with those published by the source laboratory (Schmid et al 2017)
Simulated fetal fractions (FF) ranged from 7 to 39%
Schmid et al, Fetal Diagn Ther. 2017 Nov 8

8. CLINICAL VALIDITY
How well will it perform in the clinical setting?
https://www.cdc.gov/genomics/gtesting/ACC
E/
CLINICAL SENSITIVITY
CLINICAL SPECIFICITY
Maternal side
Maternal diseases / treatments
Maternal tumors
Abnormal maternal sex chromosomes
(constitutional or mosaic)
Transfusions and transplants
CNV and maternal autosomal
mosaicisms
Fetal side
Low FF
Fetal mosaicisms
Vanishing twins
PPV
NPV
If the test is positive, is my
baby really affected?
My baby is really normal if
the test says I am screen
negative?
PREVALENCE

9. CLINICAL VALIDITY: NEXT Study
Women (age 18-48, mean age of 30.7) with singleton pregnancies between 10
to 14 weeks gestation
First Trimester
Screening (FTS*)
cfDNA test&
Each pregnancy was followed.
Outcome data obtained by:
Genetic testing
Newborn Exam
n=15.841 (women with both FTS* & cfDNA & outcome data)
n=18.955 enrolled
Norton M, et al, N Engl J Med. 2015 Apr 23;372(17):1589-97.
Study Design

10. CLINICAL VALIDITY: NEXT Study
Norton M, et al, N Engl J Med. 2015 Apr 23;372(17):1589-97.
Sensitivity 100%
Positive Predictive
Value 81%
79%
3.4%

11. cfDNA testing performances: a Meta-Analysis
Type of aneuploidy Number of studies
N.
trisomic cases
N.
non-trisomic cases
Twin pregnancies
DR FPR
(95% CI) (95% CI)
T21 5 24 1111
100% 0%
(95.2-100%) (0-0.003%)
Type of aneuploidy Number of studies
N.
trisomic cases
N.
non-trisomic cases
Singleton pregnancies
DR FPR
(95% CI) (95% CI)
T21 30 1963 223932
99.7% 0.04%
(99,1-99,9%) (0,02-0,07%)
T18 25 563 222013
97.9% 0.04%
(94.9-99.1%) (0.03-0.07%)
T13 23 119 212883
99.0% 0.04%
(65.8-100%) (0,02-0,07%)
45,X 11 36 7677
95.8% 0.14%
(70.3-99.5%) (0.05-0.38%)
other SCA 8 17 5403
100% 0.003%
(83.6-100%) (0-0.07%)

12. SENSITIVITY and SPECIFICITY do not adequately
reflect the real-life performance of cfDNA testing
and the actual PPVs being far less than 100%
Conclusions - CLINICAL VALIDITY
CONFIRMATORY TESTING
is recommended in the presence of a positive report.

13. CLINICAL UTILITY
Lower AF and CV
due to a lower FPR

14. CLINICAL UTILITY

15. CLINICAL UTILITY
Improved pregnancies
management
Possibility to lower costs
Lower capacity of the:
Lower identification
of rare anomalies
Lower AF and CV
due to a lower FPR
New cytogeneticists
New gynecologist

16. Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015
VALIDATION and VERIFICATION Genetic Test

17. Testing Strategies
Targeted SNP-
Sequencing
Massive Parallel
Shotgun Sequencing
Digital ANalysis of
Selected Regions
NIPT - strategies

18. Pre-Installation
12-2015
Installation
01-2016
PerformancePerformance
Qualification
02-2016
Training
03/04 - 2016
Guide specification
site
Inspection site
Instrument installation
Installation qualification
Operational qualification
Performance qualification
Lab personnel training
- in San Jose Labs
- in TOMA Labs
Internal TOMA lab VERIFICATION from 05-2016
Internal VERIFICATION
Verify that the performance of
the test are similar to those of the
laboratory source.
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

19. Sample Collection Scheme
Routine Cytogenetic
Prenatal Diagnosis
Routine cfDNA Test
2 CfD Tubes
(2w5m at 20°C)
1 CfD Tubes
(2w5m at 20°C)cfDNA extraction
cfDNA test Blinded
for operators
Results
Comparison with previous results
Karyotype @ TOMA cfDNA test @ Ariosa
2 CfD Tubes
(max 1w at RT)
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

20. Analyzed Cohort
Description of the Analysed Cohort
SAMPLE TYPE n.
Euploid 380
Aneuploid 30
T21 14
T18 8
T13 3
MX 2
XXX 1
XXY 1
XYY 1
Total 410
VERIFICATION
171
196
43
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

21. Validation Results
SAMPLE TOTAL TN FP TP FN
Euploid 380 380 0
Aneuploid 30 29 1
T21 14 14
T18 8 8
T13 3 2 1
MX 2 2
XXX 1 1
XXY 1 1
XYY 1 1
RESULTS
NO fetal sex discordances
VERIFICATION
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

22. SAMPLE TOTAL TN FP TP FN SENSITIVITY % SPECIFICITY %
T21 14 14 0
Euploid 380 380 0 100% (76.84-100) 100% (99.03-100)
Total 394
T18 8 8 0
Euploid 380 380 0 100% (63.06-100) 100% (99.03-100)
Total 388
T13 3 2 1
Euploid 380 380 0 67% (9.43-99.16) 100% (99.03-100)
Total 383
MX 2 2 0
Euploid 380 380 0 100% (15.81-100) 100% (99.03-100)
Total 382
SCAs 3 3 0
Euploid 380 380 0 100% (29.24-100) 100%(99.03-100)
Total 383
PERFORMANCES
VERIFICATION
67%67%
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

23. 93,2%
2,6%
4,2%
-20% < FF Toma < 20%
FF Toma ≥ 20% FF Ariosa
FF Toma < -20% FF Ariosa
FETAL FRACTION MEASUREMENT
FF% ranges n. cases %
-20%<FF%<20% 354 93.2%
FF%>20% 10 2.6%
FF<-20% 16 4.2%
VERIFICATION

24. For all investigated chr aneuploidies, SENS and
SPEC are in agreement with source laboratory
performances (slightly lower performances for
T13 but not stat different; OR 0.1333, 95% CI
0.0082-2.1813)
PPV and NPV cannot be calculated because
the enrolled cohort does not represent a
clinical population enrolled with specific
clinical criteria
Fetal sex concordance was 100%
VERIFICATION
Conclusions – TEST PERFORMANCES
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

25. FF% fell in the arbitrary range of ±20%
measured FF% by source laboratory: 93,2% of
the cases
In about 2.6% and 4.2% of the cases, the FF%
was above or below of the ±20% of the
measured FF% by source laboratory,
respectively;
The described discrepancies can be explained by:
Different maintenance and handling of the two blood
aliquots (freezing and thowing and storage up to 6
months at -20°C in toma Lab – shipping to source
laboratory in USA max 1week at Room T)
Different cfDNA extraction method
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015
VERIFICATION
Conclusions – FETAL FRACTION

26. With the multiplication of available cfDNA test in
the market, trasparency around local validation and
verification of testing is becoming increasingly
important.
A standardized tech-transfer process
monitoring 172 IQC metrics
Is realiable;
Provides an assurance that the performances in the
decentralized laboratory maches that of the source
laboratory.
VERIFICATION
Conclusions – VERIFICATION
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

27. Description of the analysed Cohort
SAMPLE TYPE n.
Total sample analyzed 6701
NO Results 44
NO Results SCA 57
Euploid 6533
High Risk 67
T21 31
T18 5
T13 4
MX 3
XXX 13
XXY 9
XYY 2
ONGOING VALIDATION
Analyzed Cohort (June 2016– Dic 2017)
ONGOING
VALIDATION
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

28. NO RESULTS
Total sample
6701
No Results
291
Results
51%
No Results
Low FF <4%
22%
No Results
FF>4%
78%
Results
79%
No Results
21%
No Result
14%
Results
35%
1° tube
1° tube
1° draw
2° draw
2° tube
ONGOING
VALIDATION
1° draw
NRR:0.65%
Mattocks et al, European Journal of Human Genetics (2010) 18, 1276–1288; Norton et al, NEJM 2015

29. NO RESULTS - SCA
ONGOING
VALIDATION
Total sample
6701
No Results
57
No Results
SCA
57
Follow up
4
Follow up lost or
pregnances ongoing
53

30. High Risk
samples N. TP FP FN
Method Confirmation
NO follow
up
CVS AF TC
T21 31 28 0 10 17 1 3
T18 5 4 1 1 4
T13 4 3 0 1 2 1
MX 3 1 1 1 1 1
47,XXX 13 3 4 7 6
47,XXY 9 2 5 1 6 2
47,XYY 2 1 0 1 1
Total: 67
HIGH RISK
ONGOING
VALIDATION
24 6 9
15

31. SAMPLES ANALYSED No follow-up TN FP TP FN SENSITIVITY %
(95% CI)
SPECIFICITY %
(95% CI)
T21 31 3 28 0
100%
(87.66% to 100 %)
99.95%
(99.87% to 99.99%)
Euploid 6626 6629 3
Total 6657
T18 5 0 4 0
100%
(39.76% to 100.00%)
99.98%
(99.92% to 100.00%)
Euploid 6652 6655 1
Total 6657
T13 4 1 3 0
100%
(29.24% to 100.00%)
99.98%
(99.92% to 100.00%)
Euploid 6653 6656 1
Total 6657
MX 3 1 1 0
100%
(2.50% to 100.00%)
99.97%
(99.89% to 100.00%)
Euploid 6597 6600 2
Total 6600
SCAs 24 9 6 0
100%
(54.07% to 100.00%)
99.73%
(99.57% to 99.84%)
Euploid 6576 6579 18
Total 6600
HIGH RISK
ONGOING
VALIDATION
NO FOLLOW UP
>99.7%

32. Conclusions – Ongoing Validation
Sensitivity 100%
Specificity 99.95%
PPV 87%
NPV 100%

33. ONGOING
VALIDATION
External - QUALITY CONTROL