PrenaTest® – Clinical validation studies

Diagnostic value of the PrenaTest® for singleton and twin pregnancies as well as for gonosomal aneuploidies

The PrenaTest® has been successfully validated based on a total of 870 samples from single-fetus and multi-fetal pregnancies in multiple studies and provides a clear result in 99.8% of all completed analyses. Detailed data are presented below concerning the accuracy of the PrenaTest® based on various clinical studies.

Diagnostic value of the PrenaTest® for singleton pregnancies

Performance evaluation of the NGS-based PrenaTest® in studies of 2012/2013

The diagnostic accuracy of the PrenaTest® has been validated by clinical studies in which LifeCodexx AG was substantially involved. The European validation study (EVS) comprised the analysis and the reporting of 468 blood samples.2 Within the scope of a further study with samples from the partner company Sequenom Inc., USA, (Sequenom Collective Study, SCS) we analysed and reported an additional 340 samples. In summary, the performance qualification comprises the analysis of 808 samples including 75 trisomy 21 cases (EVS 41, SCS 34), 14 trisomy 18 cases (EVS 8, SCS 6) and 8 trisomy 13 cases (EVS 5, SCS 3). 806 out of 808 samples have been classified correctly (99.8%). Within the EVS, one result was false-negative for trisomy 21 and one result was false-positive for trisomy 18 (with an aberration of chromosome 10). This translates into a detection rate for trisomy 21 of 98.7% with a false-positive rate of 0%. The numbers of trisomy 13 and 18 cases tested are insufficient to draw conclusions on its sensitivity and specificity for these trisomies.

 

Table 1: PrenaTest® sensitivity & speciticity for the detection of fetal trisomy 21 for singleton pregnancies

 

EVS 2012SCS 2013
Overall
Sensitivity
(lower unilateral 95%
confidence interval)
97.6%
(88.9%)
100%
(91.6%)
98.7%
(93.8%)
Specificity
(lower unilateral 95%
confidence interval)
100%
(99.3%)
100%
(99.0%)
 100%
(99.6%)

Table 2: Number of cases and overall detection rates of fetal trisomies 13,18 and 21 within the study collectives

(mosaics as well as structural aberrations are not included).

 

EVS 2012SCS 2013
Overall
Correctly classified samples466/468 (99.6%)340/340 (100%)806/808 (99.8%)
Trisomy 135/5 (100%)3/3 (100%)8/8 (100%)
Trisomy 188/8 (100%)6/6 (100%)14/14 (100%)
Trisomy 2140/41 (97.6%)34/34 (100%)74/75 (98.7%)
Overall detection rate53/54 (98.1%)43/43 (100%)96/97 (99.0%)
False-positive rate1/414 (0.2%)0/297 (0%)1/711 (0.1%)

Performance evaluation of the new PrenaTest® Option 1 for the detection of trisomy 21 (study of December 2016)

The performance of the PrenaTest® Option 1 has been validated in a clinical study. The study results of the maternal plasma samples (n=966) demonstrated a positive percentage agreement (PPA; equates to sensitivity) of 100 % (lower 1-sided 95% confidence interval of 91.8 %; n=35/35) and a negative percentage agreement (NPA; equates to specificity; n=931/931) of 100% compared to NGS-based PrenaTest®. The negative predictive value (NPV) was 100 % (lower 1-sided 95 % confidence interval of 99.68 %). The average fetal fraction of all examined blood samples was 8.1%. In 54 blood samples with a fetal fraction below 4% and as low as 2.4% a correct test result was achieved.

 

Based on the 100% positive as well as negative percentage agreement of the results of the validated qNIPT concept (the new PrenaTest® Option 1) compared to the PrenaTest® based on next generation sequencing (NGS), the test quality for the PrenaTest® remains unchanged. Please note that the new PrenaTest® Option 1 cannot be used in the case of a twin pregnancy. Please also read the limitations.

 

Learn more about the method.

 

Tabelle 3: Performance evaluation of PrenaTest® Option 1 for the examination of chromosome 21 in the case of singleton pregnancy.

 

Study 2016
Correctly classified samples966/966 (100%)
Trisomy 21 positive35/35 (100%)
Trisomy 21 negative931/931 (100%)
Sensitivity
(lower 1-sided 95% condidence interval)
100% (91.8%)
Spezifität
(lower 1-sided 95% confidence interval)
100% (-)
NPV
(lower 1-sided 95% confidence interval)
100% (99.68%)
Diagnostic value of the PrenaTest® for twin pregnancies

Within the scope of the performance qualification for the NGS-based PrenaTest® for multiple pregnancies 60 twin as well as 2 triplet pregnancies have been investigated (comprising 16 samples from our partner company Sequenom Inc., USA). Among the twin pregnancies there were 6 trisomy 21 cases, which have been confirmed by karyotyping (1 monochorionic, concordant; 5 dichorionic, discordant). Using the PrenaTest® all 6 cases have been determined correctly. The remaining samples exhibited inconspicuous results. No fetal trisomy 13 or 18 occurred within the scope of the performance qualification, therefore no conclusions on the PrenaTest® accuracy for trisomies 13 and 18 in multiple pregnancies can be drawn. There were too few triplet samples for a performance qualification and a statement on test accuracy for triplets.

 

Table 4: Results of the performance review of the PrenaTest® for the determination of fetal trisomies 21, 18 und 13 for twin pregnancies

(all positive and one part of the negative results (SQNM study 2013) were tested using karyotyping)

 

LC study 2013SQNM study 2013
Overall
Correctly classified samples46/46*16/1662/62*
Trisomy 212/24/46/6
Trisomy 13/18000
Detection rate2/24/46/6
*including 2 triplet pregnancies
Diagnostic value of the PrenaTest® for gonosomal aneuploidies

The NGS-based PrenaTest® for gonosomal aneuploidy (Turner, Triple X, Klinefelter and XYY syndrome) was tested on a total of 434 specimens from single pregnancies. During this testing, 11 out of 12 affected fetuses, that is 92 %, were correctly determined. Moreover, five discordant, „false positive“ results were obtained. At present, based on the low number of cases examined, LifeCodexx AG will not separately report any sensitivities and specificities for gonosomal aneuploidy.

 

Table 5: Results of the performance review of the PrenaTest® for determination of gonosomal aneuploidies for singleton pregnancies

 

EVS 2012SQNM study 2013
Overall
Correctly classified samples377/383* (98.4%)51/51(100%)428/434* (98.6%)
Turner syndrome7/8 (87.5%)3/3 (100%)10/11 (90.9%)
Triple X syndrome**000
Klinefelter syndrome**000
XYY syndrome1/1 (100%)01/1 (100%)
Overall detection rate8/9 (88.9%)3/3 (100%)11/12 (91.7%)
False-positive rate5/374 (1.3%)0/48 (0%)5/422 (1.2%)
* Three samples with normal, male karyotype were classified as false-positive for Klinefelter syndrome. In two of the samples the cffDNA content was below 5%. The third sample showed an abnormal chromosome X value, which could be a maternal triple X and which was not determined by conventional karyotyping in the study. The reason for the false-positive results of the two other samples classified for Turner syndrome could be a maternal mosaic or a low cffDNA content.
** The Triple X and Klinefelter syndrome were examined independently of this performance qualification in the context of research projects andsuccessfully determined.
Validity of the PrenaTest® to exclude or detect a 22q11.2 microdeletion, associated with DiGeorge syndrome and velo-cardio-facial syndrome (Shprintzen syndrome)

Validation of the test method:
Data from synthetic pooled DNA specimens as well as several specimens from pregnant women whose unborn child had a 22q11.2 microdeletion were investigated using next generation sequencing. In all cases, a 22q11.2 microdeletion was correctly detected. The validation process was performed in three phases:

 

Phase 1
Four synthesized specimens, each of which had a different cffDNA level (16%, 8%, 4% and 2%) and which had a 22q11.2 microdeletion, were analyzed several times. The 22q11.2 microdeletion of the two specimens with a cffDNA level of 8% and 16% was correctly determined, while the results of the specimens with a lower cffDNA level were not significant.

 

Phase 2
Four specimens from pregnant women whose unborn children had a 22q11.2 microdeletion were retrospectively tested and the 22q11.2 microdeletion was correctly detected in each case. In this phase, the 22q11.2 microdeletion was previously confirmed in three of the specimens using invasive diagnostic procedures. In the case of the fourth specimen, a cardiac defect (DORV; double outlet right ventricle) was confirmed by ultrasound. The DiGeorge syndrome was confirmed after birth.

 

Phase 3
In a final internal blinded study, specimens from phase 2 with a 22q11.2 microdeletion as well as euploidic specimens were investigated. All specimens were correctly classified. Due to the low number of cases, a concrete test sensitivity and specificity cannot currently be derived.

 

Correctly classified specimens       16/16
Total detection rate                          2/2
False-positive rate                          0/14

 

 

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