Mutation Detection by SEQ
Initial amplification PCR of exons 18-21 was standardized by performing a series of gradient PCRs to better accommodate assays for all four exons in a single thermal cycler setting. We found that at a lower annealing temperature than that described in previous studies, samples with low yield were better amplified, due to controlled reduction in specificity of the primers (see details in the discussion section). The standardization of the annealing temperature was performed using FFPE tissue-derived wildtype DNA (Fig 2). At 58℃, we observed satisfactory amplification for all four exons, following which all SEQ initial amplification PCR was conducted at this temperature.
With SEQ, 275 FFPE tumor samples received at MODL were screened for sequence variations in EGFR TKD (exons 18, 19, 20, and 21). About 27% of the samples (75/275) contained at least one pathogenic variant in any of the four exons. However, samples with well-differentiated adenocarcinoma of lung showed a 37% mutation occurrence rate. On the other hand, in poorly differentiated adenocarcinoma, mutation was detected in only 11% of the samples. Among the different exons of EGFR TKD which had mutation, exon 19 variations constituted about 69% (52/75) of the cases. Mutation in exon 21 was found in about 21% of the cases (16/75), and exons 18 and 20 variations were detected in one and eight samples, respectively.
Limit of detection (LOD) of mutation fraction by SEQ
The DNA concentration for a typical SEQ was 2.5ng/μl. In order to determine the lower limit of detection (LOD) of MAF, we used two commercially available standards of EGFR TKD mutants (mutDNA), 1) isolated DNA containing the exon 19 deletion, p.E746_A750del (Ex19_std) with 50% MAF (# HD251, Horizon Discovery Ltd, Ireland, UK), each vial contains 1μg DNA in Tris-EDTA buffer (pH: 8.18, concentration: 50ng/μl) with 50% mutant allele fraction of EGFR ΔE746-A750 (SNP ID: rs121913421) validated by digital droplet PCR, and 2) FFPE tissue containing the exon 21 mutation, L858R (Ex21_std) with 50% MAF (#HD130, Horizon Discovery Ltd, Ireland, UK) each vial contains one section FFPE cell pellet of human cell lines, 15-20μm thick, with an approximate cell density of 3.5 x 105 cells/section, containing roughly 400ng total DNA with 50% mutant allele fraction of EGFR L858R (SNP ID: rs121434568) validated by digital droplet PCR.
The LOD of MAF of Ex19_std and Ex21_std was separately determined by performing a series of assays with different total DNA concentrations ranging from 0.25ng/μl (5ng/assay of mutDNA + wtDNA) to 2.5ng/μl (50ng/assay of mutDNA + wtDNA). Fig 3a and 3b show the chromatograms of Ex19_std at two different total DNA concentrations (a: 2.5ng/μl, b: 0.25 ng/μl), and Fig 3c and 3d show the chromatograms of Ex21_std at two different total DNA concentrations (c: 2.5ng/μl, d: 0.25ng/μl). As seen from Fig 3b and 3d, there was a sudden absence of the mutant peak at 5% MAF for Ex19_std and 2.5% MAF for Ex21_std at 0.25ng/μl assay. However, in the 2.5ng/μl, the mutant peak was not detected at 0.5% MAF for Ex19_std and 0.125% for Ex21_std. This shows that the LOD of MAF for a 0.25ng/μl assay was 10% for Ex19_std, and 5% for Ex21_std for a 0.25ng/μl assay. On the other hand, in a 2.5ng/μl assay, lowest LOD for Ex19_std and Ex21_std were 1% and 0.25% respectively (Fig 3a & 3c).
Since the origins of wtDNA and mutDNA were different, there can be a difference in amplification due to the quality of DNA. In order to address this, we performed SEQ with both wtDNA and mutDNA separately as well as in 1:1 mix. At 25% MAF, the wtDNA: mutDNA ratio was 1:1, for a total DNA concentration of 2.5 ng/μl. The peak heights of both wtDNA and mutDNA species were found to be equal, irrespective of cell-derived or tissue derived DNA (See Fig 3b, 3d and supplementary Fig S1c). Moreover, the amplification peak heights of both tissue-derived wtDNA and cell-derived mutDNA, when sequenced separately were comparable as that of 1:1 mix (Fig S1a and S1b). This shows that there was minimal difference between the quality of DNA obtained from both sources.
Validation of SEQ LOD by Real time PCR
With the intention of assessing both mutant and wildtype DNA amplification in mixed assays used in SEQ, multiplex real time PCR with allele-specific primer-probe for the detection of exon 19 deletion was performed. MAFs of 10%, 5%, 1% and 0.1% were assessed in triplicates in mixed DNA assays. Similarly, Ex19_std and wtDNA were also separately assessed. The multiplex allele-specific primer-probe is incorporated with two fluorescent dyes, FAM and VIC, detecting mutant and wildtype alleles, respectively. Accordingly, the amplifications of both the alleles were determined by the Ct values obtained for both dyes, respectively, in each assay. According to the manufacturer’s instruction, ΔCt (The difference between Ct of mutant and Ct of wildtype) ≤ 12, indicates positive for the deletion of exon 19. The FAM Ct values showed wide variation across all MAFs, the lowest being detected in 50% MAF, while the highest in 0.1% MAF (Fig 4 and Table 1). On the other hand, VIC Ct was comparable in all assays between all the MAFs (Fig 4c). ΔCt of all the MAFs, except 0.1% MAF, were detected as positive for exon 19 deletion (Table 1). Thus, the wildtype DNA as well as the mutant standard DNA were amplified in the mixed assays, thereby validating the lowest MAF detected in the identical assay using SEQ.
Standardization of HRM assay
In order to assess the HRM detectability, a previous study by Do et al. included 5ng of DNA per 20μl assay (0.25ng/μl final concentration) with 50% MAF [18]. We examined the LOD of initial DNA concentration by performing a dilution curve of mutDNA standard mixed with wtDNA in a real-time PCR assay using the same HRM conditions. Different mutDNA standard concentrations from 0.5pg/μl to 0.25ng/μl at 50% MAF were employed and assessed against Ct of real-time PCR assay. Ct value increases with decreasing concentration (Fig 5). We found that the relative change in Ct values (ΔCt) was proportional to the natural logarithm of DNA concentration/assay as per the equation given below.
See formula 1 in the supplementary files.
By applying the above equation, the saturation concentration of DNA for no change in ΔCt value was ~0.249ng/μl. Hence, the total DNA concentration/assay was fixed as 0.25ng/μl for heteroduplex HRM analysis. This concentration is 10 times lower than the amount of DNA required per assay for SEQ.
LOD of MAF by HRM
In order to examine whether the LOD of MAF decreases with increasing concentration of total DNA (mutDNA+ wtDNA) per assay, we titrated mutDNA with wtDNA in a series of separate HRM assays containing varying total DNA concentrations. HRM MAF titration was conducted by incorporating decreasing MAF in a mixed DNA sample from 10% to 0.005% of both Ex19_std & Ex21_std, in an increasing total DNA concentration/assay from 0.25ng/μl to 2.5ng/μl (Fig 6 and Fig 7). For both the mutDNA standards, lowest LOD observed was 0.25% in a 2.5ng/μl HRM assay, while in a 0.25ng/μl assay, 5% MAF was the lowest LOD detected with the Ex19_std, and 10% with the Ex21_std.
Comparative performance analysis
Of the 116 samples that were included in the study, 67 samples were mutation positive using the SEQ method (Table 2). 47 of the 67 positive samples had deletion mutation in exon 19, and substitution mutations were detected in 15 and five samples in exon 21 (Table 3), and exon 20 respectively. None of the samples in this group were found to have a mutation in exon 18. Hence, the performance characteristics for exons 18 and 20 were not separately included in the comparative performance analysis. HRM positive samples were distributed as 72% positive and 28% negative in SEQ samples with the exon 19 mutation (Table 3). Furthermore, HRM positivity was distributed as 60% positive and 40% negative in SEQ samples with the exon 21 mutation (Table 3). The sensitivity of detection was 100% for both exon 19 and exon 21 mutations, while specificity was higher with the exon 21 variants compared to exon 19 variants (90% vs. 74%). McNemar’s comparison of these two methods was significant (p value < 0.01) for both the groups, and overall performance differences between these two methods. Overall specificity of HRM over SEQ was about 67% with 86% accuracy (Table 2). The positive predictive value (PPV) was 60% for exon 21 variants, about 72% for exon 19 variants, with an overall 80% PPV. However, the negative predictive value (NPV) was 100% in individual variant groups and overall values as well.