2.3 RESULTS
2.3.5 GF EGFR Assay Version 5
Version 5 was the final version of the EGFR assay made available by GeneFirst for this project. 30 samples were tested, 11 wild-types and 19 mutants. Unfortunately all the samples panel either contained very low remaining sample volume (after routine testing by the Pathology department) or were of low DNA concentration. 12 samples (11 wild-type and 1 mutant) contained DNA concentrations that were below the level which could be accurately quantified by the Qubit instrument. The results for the individual samples tested with this final version of the assay can be found in table A2.1.5 (chapter two appendix). Some samples are indicated with “Unknown (low)” in the DNA loading field of the table.
Overall concordance with the original Therascreen results for this version of the assay was 26.67%. This is a reduction of 20% compared to version 4. A breakdown of the results are shown in table 2.3.5.1. Despite this being the most recent version of the assay, the performance of the assay was poorer than the previous two versions. Again, the persistent issue of false positive/ discrepant results were a feature of the version 5 dataset. Only 2 of
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17 (10.5%) mutant samples and 6 of 11 (55%) wild-type samples matched the original Therascreen results.
Table 2.3.5.1: Summary table showing the concordance % and discrepancy % of the GeneFirst EGFR PCR assay version 5. WT = Wild-type.
To accurately assess the performance of version 5 of the EGFR assay, the data was reanalysed to examine the results given by each of the assays four separate reaction master mixes. The EGFR assay consisted of four master mixes, with each mix using two reporter fluorophores (VIC and FAM, linked to mutant target probes) therefore it was possible to generate eight signals per sample. From our dataset of 30 samples, it is therefore possible to have up to generate 240 PCR signals across the eight reactions. The results from all four master mixes are shown in table 2.3.5.2. In total there were 125 signals detected, of which 41 (32.8%) were either false positives or false negatives. Of the individual master mixes, the data suggests that master mix C performed the best, with only 5 false results, whereas master mix D performed the worst, with 19 false results.
Match Discrepant Totals Mutant
Samples WT samples Mutant 2 17 19 10.5 WT 6 5 11 54.5 Totals 8 22
30
Concordance % 26.7 Discrepancy % 73.3 To ta lGeneFirst EGFR v5 Sample Concordance %
Th er as cr ee n
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Table 2.3.5.2: Complete analysis of PCR results from all GF EGFRv5 master mixes. Designations ‘True Positive’, ‘True Negative’, ‘False Positive’ and ‘False Negative’ are assigned based on the original Therascreen results.
The data from table 2.2.5.2 was used to calculate the sensitivity and specificity of the version 5 assay. The results of these calculations are shown in table 3.2.5.3. The GF EGFR version 5 assay achieved a sensitivity and specificity of 68.2% and 67.0% respectively.
A B C D True Pos True Neg False Pos False Neg
EGFRv5 09 Exon 19 Del FALSE POS TRUE NEG TRUE NEG TRUE POS 1 2 1 0 EGFRv5 01 Exon 20 Ins TRUE NEG TRUE NEG TRUE POS FALSE POS 1 2 1 0 EGFRv5 02 L861Q and G719X FALSE NEG TRUE POS x2 TRUE NEG FALSE POS 2 1 1 1 EGFRv5 03 Exon 19 Del TRUE NEG TRUE NEG TRUE NEG TRUE POS 1 3 0 0 EGFRv5 04 T790M FALSE NEG FALSE POS TRUE NEG FALSE POS 0 1 2 1 EGFRv5 05 L861Q TRUE NEG TRUE POS, FALSE
POS TRUE NEG FALSE POS 1 2 2 0
EGFRv5 06 L858R TRUE NEG FALSE POS FALSE POS FALSE NEG 0 1 2 1 EGFRv5 07 T790M FALSE NEG FALSE POS TRUE NEG FALSE POS 0 1 2 1 EGFRv5 08 S768I TRUE NEG TRUE NEG TRUE POS FALSE POS 1 2 1 0 EGFRv5 18 Exon 19 Del TRUE NEG TRUE NEG TRUE NEG TRUE POS 1 3 0 0 EGFRv5 10 Exon 20 Ins TRUE NEG TRUE NEG TRUE POS FALSE POS x2 1 2 2 0 EGFRv5 11 L861Q and G719X FALSE POS,
FALSE NEG TRUE POS x2 TRUE NEG FALSE POS 2 1 2 1 EGFRv5 12 Exon 19 Del TRUE NEG FALSE POS TRUE NEG TRUE POS 1 2 1 0 EGFRv5 13 T790M FALSE NEG TRUE NEG TRUE NEG FALSE POS 0 2 1 1 EGFRv5 14 L861Q TRUE NEG TRUE POS TRUE NEG FALSE POS 1 2 1 0 EGFRv5 15 L858R FALSE POS FALSE POS TRUE NEG FALSE POS 0 1 3 0 EGFRv5 16 T790M TRUE POS TRUE NEG TRUE NEG FALSE POS 1 2 1 0 EGFRv5 17 S768I TRUE NEG TRUE NEG TRUE POS FALSE POS 1 2 1 0 EGFRv5 21 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0 EGFRv5 22 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0 EGFRv5 23 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0 EGFRv5 19 WT FALSE POS TRUE NEG TRUE NEG FALSE POS 0 2 2 0 EGFRv5 20 Del TRUE NEG TRUE NEG TRUE NEG FALSE NEG 0 3 0 1 EGFRv5 24 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0 EGFRv5 29 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0 EGFRv5 25 WT TRUE NEG FALSE POS FALSE POS TRUE NEG 0 2 2 0 EGFRv5 26 WT TRUE NEG TRUE NEG FALSE POS FALSE POS 0 2 2 0 EGFRv5 27 WT TRUE NEG FALSE POS FALSE POS FALSE POS 0 1 3 0 EGFRv5 28 WT TRUE NEG TRUE NEG FALSE POS TRUE NEG 0 3 1 0 EGFRv5 30 WT TRUE NEG TRUE NEG TRUE NEG TRUE NEG 0 4 0 0
TRUE 22 25 23 12
FALSE 9 7 5 19
Result GF EGFRv5 PCR Master Mix
Study ID Mutation
type
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Table 2.3.5.3: Summary of GF EGFR version 5 data.
GeneFirst EGFR Assay version 5
Test Mutant Present Mutant absent
Positive True Positive 15 False Positive 34
Negative False Negative 7 True Negative 69
Statistic Value
Sensitivity Specificity
Positive Predictive Value Negative Predictive Value
68.2%
67.0%
30.6%
90.8%
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Table 2.3.5.4 Endogenous control results from samples tested using the EGFR version 5 assay. Samples with DNA concentrations below the lower quantifiable range were designated ‘unknown’. ROX A = ROX reporter signal in master mix A, ROX B = ROX reporter signal in master mix B, ROX C = ROX reporter signal in master mix C, ROX D = ROX reporter signal in master mix D, NTC = No template control, WT = Wild-type.
Table 2.3.5.4 shows the results for the endogenous control assay for all samples tested using the GF EGFR assay version 5. In total 9/ 25 samples failed the endogenous control in at least one master mix, resulting in a failure rate of 26.5%. Of the NTCs, only 1/4 failed which showed an improvement on the results from version 4 of the assay.
By the end of the investigation, 142 FFPE tissue samples had been tested using the five versions of the GF EGFR assays. A summary of the testing is shown in table 2.3.5.5. Overall
Study ID Mutation type DNA (ng, total) Ct Mean
Control Pos/ Neg Ct Mean
Control Pos/ Neg Ct Mean
Control Pos/ Neg Ct Mean
Control Pos/ Neg
Assay Pass/ Fail
EGFRv5 01 Exon 19 Del 30 23.134 POS 28.003 POS 25.352 POS 24.045 POS PASS
EGFRv5 02 Exon 20 Ins 30 21.175 POS 25.900 POS 22.624 POS 23.324 POS PASS
EGFRv5 03 L861Q and G719X 30 NEG 31.618 POS 26.254 POS 28.079 POS FAIL EGFRv5 04 Exon 19 Del 30 21.406 POS 26.556 POS 23.608 POS 25.294 POS PASS
EGFRv5 05 T790M 30 22.427 POS 27.174 POS 22.891 POS 23.724 POS PASS
EGFRv5 06 L861Q 30 22.427 POS 27.211 POS 23.094 POS 25.043 POS PASS
EGFRv5 07 L858R 30 NEG NEG 35.664 POS NEG FAIL EGFRv5 08 T790M 30 22.169 POS 27.137 POS 23.467 POS 24.119 POS PASS
EGFRv5 09 S768I 30 23.633 POS 29.029 POS 25.153 POS 25.799 POS PASS
NTC 0 NEG NEG NEG NEG PASS
EGFRv5 10 Exon 19 Del 15 22.997 POS 27.510 POS 24.121 POS 24.274 POS PASS
EGFRv5 11 Exon 20 Ins 15 21.364 POS 25.800 POS 22.607 POS 23.523 POS PASS
EGFRv5 12 L861Q and G719X 15 21.782 POS 27.274 POS 23.397 POS 24.312 POS PASS
EGFRv5 13 Exon 19 Del 15 22.673 POS 27.365 POS 24.074 POS 24.663 POS PASS
EGFRv5 14 T790M 15 20.775 POS 25.400 POS 22.101 POS 23.096 POS PASS
EGFRv5 15 L861Q 15 22.766 POS 27.961 POS 23.937 POS 24.853 POS PASS
EGFRv5 16 L858R 15 26.743 POS 39.233 POS 27.878 POS 36.766 POS PASS
EGFRv5 17 T790M 15 20.693 POS 25.101 POS 21.914 POS 22.526 POS PASS
EGFRv5 18 S768I 15 22.614 POS 27.341 POS 23.800 POS 24.778 POS PASS
NTC 0 NEG NEG NEG NEG PASS
EGFRv5 19 WT Unknown 23.298 POS 29.563 POS NEG NEG FAIL EGFRv5 20 WT Unknown 24.694 POS NEG NEG NEG FAIL EGFRv5 21 WT Unknown 27.948 POS NEG 17.898 POS 20.483 POS FAIL EGFRv5 22 WT Unknown 16.599 POS 22.002 POS 18.245 POS 18.875 POS PASS
EGFRv5 23 Del Unknown 16.897 POS 22.018 POS NEG NEG FAIL EGFRv5 24 WT Unknown 25.133 POS NEG NEG NEG FAIL
NTC 0 NEG NEG 39.173 POS NEG FAIL
EGFRv5 25 WT Unknown 17.268 POS 23.712 POS 18.924 POS 22.147 POS PASS
EGFRv5 26 WT Unknown 22.769 POS 34.479 POS 28.061 POS NEG PASS
EGFRv5 27 WT Unknown 20.868 POS 28.288 POS 22.299 POS 26.968 POS PASS
EGFRv5 28 WT Unknown 22.383 POS 33.795 POS 28.589 POS 33.378 POS PASS
EGFRv5 29 WT Unknown 23.426 POS NEG 30.235 POS 38.301 POS PASS
EGFRv5 30 WT Unknown 27.161 POS NEG NEG NEG FAIL
NTC 0 NEG NEG NEG NEG PASS
PCR Pass: 25
PCR Fail: 9
Failure rate: 26.5%
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49.3% of results matched the original Therascreen results. The assays generally worked better with wild-type samples, which were concordant with the original results in 67.3% of cases, whereas only 38.9% of mutant samples matched the original results. The relatively low sensitivity and specificity of the GF EGFR assays is reflected in the relatively low level of concordance seen in this phase of the project.
Table 2.3.5.5: Summary table showing the concordance % and discrepancy % of all the samples tested with the GeneFirst EGFR PCR assay versions 1- 5. WT = Wild-type.