THE USE OF THE EAC PRIMERS IN DIGITAL PCR FOR MONITORING MINIMAL RESIDUAL DISEASE IN CML RESULTS IN A SHIFT BETWEEN MOLECULAR RESPONSE GROUPS.
(Abstract release date: 05/19/16)
EHA Library. Niederwieser D. 06/09/16; 132634; E1085
Dr. Dietger Niederwieser
Contributions
Contributions
Abstract
Abstract: E1085
Type: Eposter Presentation
Background
Digital PCR (dPCR) generates an absolute read out that is largely robust to variations in PCR efficiency and should reduce the requirement for standardisation by laboratory-specific conversion factors.
Aims
The aim of this study was to compare the results of dPCR to qPCR (quantitative PCR) in blinded samples from two independent laboratories with respect to the observed rates of molecular response (MR) in CML patients (pts) having undergone 18 months of nilotinib treatment in the ENEST1st trial (ClinicalTrials.gov:NCT01061177).
Methods
A total of 230 cDNA samples from CML pts with e13 or e14/a2 BCR-ABL fusion genes who were treated within the ENEST1st trial between 2012 and 2013 were analysed in Leipzig (L, n=75) or Mannheim (M, n=155) with qPCR. BCR-ABL levels were determined relative to those of ABL and standardization was achieved using plasmid DNA. Both labs are accredited by the European Treatment and Outcome Study (EUTOS) collaboration. The cDNA samples were blinded for the qPCR results and re-analysed in L with a duplex dPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer’s recommendations, duplicate samples yielding a minimum of 3 positive droplets from the 12-19.000 routinely analysed droplets were scored as positive. Depth of MR was scored using the EUTOS definitions used in the ENEST1st trial.
Results
A previous comparison between dPCR and qPCR showed significant differences in the distribution of the molecular response (MR) categories (p = 0.02) with more residual disease (RD) measured by dPCR. In detail, significantly fewer patients achieved deep molecular responses (MR4 to MR5) by dPCR (p = 0.035). Of the 230 samples analysed by the two methods, 58% (n=134) were classified in the same MR category, while 11% (n=25) skipped to classes with less RD and 31% (n=71) moved to classes with more RD by dPCR, mostly over a range of 1-2 classes. ABL copies in non-concordant samples were median 1.1 fold higher by dPCR than by qPCR, while BCR-ABL transcripts were 3 fold higher by dPCR in 56/71 samples analysed, resulting in significantly more patients having more RD by dPCR. Experimental tests of the sensitivity between 0.25 and 25 BCR-ABL copies showed a LOQ (limit of quantitation) of 3 positive droplets in duplicates (cut-off 3). Interestingly, application of the laboratory-specific qPCR conversion factor to the dPCR data (cut-off 3) independently reduced the difference in distribution categories between the qPCR and dPCR to below significance (Table 1).
Table 1: Distribution of MR classes
Conclusion
The use of M-BCR-ABL primers according to the EAC (Europe Against Cancer) protocol in dPCR with a cut-off of 3 droplets results in a shift of molecular response categories towards more minimal RD by dPCR than by qPCR. This difference is reduced to below significance by application of the laboratory specific qPCR conversion factor to the dPCR data.
Session topic: E-poster
Keyword(s): BCR-ABL, PCR
Type: Eposter Presentation
Background
Digital PCR (dPCR) generates an absolute read out that is largely robust to variations in PCR efficiency and should reduce the requirement for standardisation by laboratory-specific conversion factors.
Aims
The aim of this study was to compare the results of dPCR to qPCR (quantitative PCR) in blinded samples from two independent laboratories with respect to the observed rates of molecular response (MR) in CML patients (pts) having undergone 18 months of nilotinib treatment in the ENEST1st trial (ClinicalTrials.gov:NCT01061177).
Methods
A total of 230 cDNA samples from CML pts with e13 or e14/a2 BCR-ABL fusion genes who were treated within the ENEST1st trial between 2012 and 2013 were analysed in Leipzig (L, n=75) or Mannheim (M, n=155) with qPCR. BCR-ABL levels were determined relative to those of ABL and standardization was achieved using plasmid DNA. Both labs are accredited by the European Treatment and Outcome Study (EUTOS) collaboration. The cDNA samples were blinded for the qPCR results and re-analysed in L with a duplex dPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer’s recommendations, duplicate samples yielding a minimum of 3 positive droplets from the 12-19.000 routinely analysed droplets were scored as positive. Depth of MR was scored using the EUTOS definitions used in the ENEST1st trial.
Results
A previous comparison between dPCR and qPCR showed significant differences in the distribution of the molecular response (MR) categories (p = 0.02) with more residual disease (RD) measured by dPCR. In detail, significantly fewer patients achieved deep molecular responses (MR4 to MR5) by dPCR (p = 0.035). Of the 230 samples analysed by the two methods, 58% (n=134) were classified in the same MR category, while 11% (n=25) skipped to classes with less RD and 31% (n=71) moved to classes with more RD by dPCR, mostly over a range of 1-2 classes. ABL copies in non-concordant samples were median 1.1 fold higher by dPCR than by qPCR, while BCR-ABL transcripts were 3 fold higher by dPCR in 56/71 samples analysed, resulting in significantly more patients having more RD by dPCR. Experimental tests of the sensitivity between 0.25 and 25 BCR-ABL copies showed a LOQ (limit of quantitation) of 3 positive droplets in duplicates (cut-off 3). Interestingly, application of the laboratory-specific qPCR conversion factor to the dPCR data (cut-off 3) independently reduced the difference in distribution categories between the qPCR and dPCR to below significance (Table 1).
| MR class dPCR versus qPCR | dPCR withCut-off 3 for positivity | dPCR multiplied byCF qPCR |
| Concordance [%] | 58 | 61 |
| Less RD [%] | 11 | 16 |
| More RD [%] | 31 | 23 |
| X-fold more RD/less RD | 2.8 | 1.4 |
Conclusion
The use of M-BCR-ABL primers according to the EAC (Europe Against Cancer) protocol in dPCR with a cut-off of 3 droplets results in a shift of molecular response categories towards more minimal RD by dPCR than by qPCR. This difference is reduced to below significance by application of the laboratory specific qPCR conversion factor to the dPCR data.
Session topic: E-poster
Keyword(s): BCR-ABL, PCR
Abstract: E1085
Type: Eposter Presentation
Background
Digital PCR (dPCR) generates an absolute read out that is largely robust to variations in PCR efficiency and should reduce the requirement for standardisation by laboratory-specific conversion factors.
Aims
The aim of this study was to compare the results of dPCR to qPCR (quantitative PCR) in blinded samples from two independent laboratories with respect to the observed rates of molecular response (MR) in CML patients (pts) having undergone 18 months of nilotinib treatment in the ENEST1st trial (ClinicalTrials.gov:NCT01061177).
Methods
A total of 230 cDNA samples from CML pts with e13 or e14/a2 BCR-ABL fusion genes who were treated within the ENEST1st trial between 2012 and 2013 were analysed in Leipzig (L, n=75) or Mannheim (M, n=155) with qPCR. BCR-ABL levels were determined relative to those of ABL and standardization was achieved using plasmid DNA. Both labs are accredited by the European Treatment and Outcome Study (EUTOS) collaboration. The cDNA samples were blinded for the qPCR results and re-analysed in L with a duplex dPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer’s recommendations, duplicate samples yielding a minimum of 3 positive droplets from the 12-19.000 routinely analysed droplets were scored as positive. Depth of MR was scored using the EUTOS definitions used in the ENEST1st trial.
Results
A previous comparison between dPCR and qPCR showed significant differences in the distribution of the molecular response (MR) categories (p = 0.02) with more residual disease (RD) measured by dPCR. In detail, significantly fewer patients achieved deep molecular responses (MR4 to MR5) by dPCR (p = 0.035). Of the 230 samples analysed by the two methods, 58% (n=134) were classified in the same MR category, while 11% (n=25) skipped to classes with less RD and 31% (n=71) moved to classes with more RD by dPCR, mostly over a range of 1-2 classes. ABL copies in non-concordant samples were median 1.1 fold higher by dPCR than by qPCR, while BCR-ABL transcripts were 3 fold higher by dPCR in 56/71 samples analysed, resulting in significantly more patients having more RD by dPCR. Experimental tests of the sensitivity between 0.25 and 25 BCR-ABL copies showed a LOQ (limit of quantitation) of 3 positive droplets in duplicates (cut-off 3). Interestingly, application of the laboratory-specific qPCR conversion factor to the dPCR data (cut-off 3) independently reduced the difference in distribution categories between the qPCR and dPCR to below significance (Table 1).
Table 1: Distribution of MR classes
Conclusion
The use of M-BCR-ABL primers according to the EAC (Europe Against Cancer) protocol in dPCR with a cut-off of 3 droplets results in a shift of molecular response categories towards more minimal RD by dPCR than by qPCR. This difference is reduced to below significance by application of the laboratory specific qPCR conversion factor to the dPCR data.
Session topic: E-poster
Keyword(s): BCR-ABL, PCR
Type: Eposter Presentation
Background
Digital PCR (dPCR) generates an absolute read out that is largely robust to variations in PCR efficiency and should reduce the requirement for standardisation by laboratory-specific conversion factors.
Aims
The aim of this study was to compare the results of dPCR to qPCR (quantitative PCR) in blinded samples from two independent laboratories with respect to the observed rates of molecular response (MR) in CML patients (pts) having undergone 18 months of nilotinib treatment in the ENEST1st trial (ClinicalTrials.gov:NCT01061177).
Methods
A total of 230 cDNA samples from CML pts with e13 or e14/a2 BCR-ABL fusion genes who were treated within the ENEST1st trial between 2012 and 2013 were analysed in Leipzig (L, n=75) or Mannheim (M, n=155) with qPCR. BCR-ABL levels were determined relative to those of ABL and standardization was achieved using plasmid DNA. Both labs are accredited by the European Treatment and Outcome Study (EUTOS) collaboration. The cDNA samples were blinded for the qPCR results and re-analysed in L with a duplex dPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer’s recommendations, duplicate samples yielding a minimum of 3 positive droplets from the 12-19.000 routinely analysed droplets were scored as positive. Depth of MR was scored using the EUTOS definitions used in the ENEST1st trial.
Results
A previous comparison between dPCR and qPCR showed significant differences in the distribution of the molecular response (MR) categories (p = 0.02) with more residual disease (RD) measured by dPCR. In detail, significantly fewer patients achieved deep molecular responses (MR4 to MR5) by dPCR (p = 0.035). Of the 230 samples analysed by the two methods, 58% (n=134) were classified in the same MR category, while 11% (n=25) skipped to classes with less RD and 31% (n=71) moved to classes with more RD by dPCR, mostly over a range of 1-2 classes. ABL copies in non-concordant samples were median 1.1 fold higher by dPCR than by qPCR, while BCR-ABL transcripts were 3 fold higher by dPCR in 56/71 samples analysed, resulting in significantly more patients having more RD by dPCR. Experimental tests of the sensitivity between 0.25 and 25 BCR-ABL copies showed a LOQ (limit of quantitation) of 3 positive droplets in duplicates (cut-off 3). Interestingly, application of the laboratory-specific qPCR conversion factor to the dPCR data (cut-off 3) independently reduced the difference in distribution categories between the qPCR and dPCR to below significance (Table 1).
| MR class dPCR versus qPCR | dPCR withCut-off 3 for positivity | dPCR multiplied byCF qPCR |
| Concordance [%] | 58 | 61 |
| Less RD [%] | 11 | 16 |
| More RD [%] | 31 | 23 |
| X-fold more RD/less RD | 2.8 | 1.4 |
Conclusion
The use of M-BCR-ABL primers according to the EAC (Europe Against Cancer) protocol in dPCR with a cut-off of 3 droplets results in a shift of molecular response categories towards more minimal RD by dPCR than by qPCR. This difference is reduced to below significance by application of the laboratory specific qPCR conversion factor to the dPCR data.
Session topic: E-poster
Keyword(s): BCR-ABL, PCR
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