Sample size
A prospective series study in which consecutive FFPE tumor samples, sent to Molecular Oncology Diagnostics Laboratory (MODL) for SEQ of the EGFR gene TKD region and mutation analysis were subjected to HRM. Samples irrespective of the tissue of origin, stage of the disease and demographic characteristics were included in the study. Following the screening of 275 FFPE samples, a total of 116 samples were taken for the comparative study. Pathogenic variations in EGFR TKD were identified in 75 out of 275 samples by SEQ. HRM was performed in 67 of the 75 positive samples, and first 49 of the 200 negative samples, in chronological order. Eight positive mutant samples were not included for comparative analysis due to low quantity of DNA.
Sample preparation
Tumor tissue samples obtained from patients were subjected to grossing and dissection, fixed in neutral buffered formalin solution, embedded in paraffin, sectioned, and stained. The tissues in paraffin blocks (FFPE) were sectioned and subjected to histopathological examination by the pathologists in the Department of Pathology. Following the determination of the histological as well as immunohistochemical characteristics of the tumor, and the subsequent assessment of the adequacy of the cellular content in the sections, region of interest from each tumor tissue was selected to enrich tumor cell content to about 50% for molecular genetic analysis. Approximately 10 sections of the selected regions of each tumor tissue, with a thickness of 10μm, were obtained in a 1.5ml tube and transferred to MODL at room temperature for mutation analysis.
The DNA from FFPE sections was extracted using QIAmp® FFPE DNA extraction kit (Qiagen, USA), according to the procedure described by the manufacturer. DNA quantification was performed by checking the absorbance at 260nm and 280nm by spectrophotometric analyzer (Thermo Fisher Scientific, USA), and fluorometric method using Qubit3.0® fluorometer (Thermo Fisher Scientific, USA). Downstream processing of the extracted DNA was performed only when the ratio of absorbance at 260/280 was ≥ 1.8 with the concentration of DNA ≥ 5ng/μl.
Initial Amplification of DNA
In a 20μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5μM). Approximately 50ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18]. Thermal cycler settings included an initial denaturation of 95°C for 15 minutes, followed by 45 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds and a final extension at 72°C for 10 minutes. The amplicons were assessed using 2% Agarose gel (SeaKem® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure® PCR product purification kit (Roche Molecular Diagnostics, Switzerland).
PCR for High Resolution Melting Analysis
In a 20μl assay, 1X of High-Resolution Master mix (Roche Molecular Diagnostics, Switzerland), 300nΜ each of forward and reverse primers, and 2.5mM of MgCl2 were added. As described in the previous study [18], 5ng of template DNA was added, and the assay was made up with PCR- grade distilled water. The assay strip-tubes were loaded onto the LightCycler480®. The assay was optimized based on previously described method [18] with minor modifications. The standardized thermal cycler settings include - an initial denaturation at 95°C for 15 minutes followed by 50 cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 10 seconds and extension at 72°C for 30 seconds, with initial 10 cycles of touchdown (1°C /cycle). This is followed by final denaturation at 95°C for 1 minute and cooling at 4°C for 2 minutes. The high-resolution melting was performed from 65°C to 95°C at a ramp rate of 0.02°C/s with 25 fluorescence data acquisition points, followed by cooling to 4°C for 30 seconds.
Sanger Sequencing
DNA sequencing using Sanger’s dideoxy method was performed using BigDye® Terminator cycle sequencing kit v3.1compatible with ABI 3500® Genetic analyzer. The original reaction setup, recommended by the manufacture, was optimized with the following modifications. In a total assay volume of 10μl of separate forward and reverse reactions, BigDye® Terminator, 1X sequencing buffer, 0.8μM of m13 forward or reverse primer were added to the respective reaction assays. Post-PCR purified DNA amplicon was added, and the volume was made up with distilled water. Assay conditions were setup by the manufacturer recommendations with the following modifications. Initial denaturation at 96°C for 1 minute, 15 cycles of denaturation (at 96°C for 10 seconds), annealing (at 50°C for 5 seconds) and extension (at 60°C for 75 seconds), followed by final extension (2 set of 5 cycles with expanding the extension phase 60°C to 90 seconds and 2 minutes subsequently). The amplified product was stored at 4°C. Post-sequencing PCR clean-up was done using Sephadex® G-50 medium (molecular weight cut-off of 30,000 Mr) (GE Lifesciences), and Whatman UNIFILTER® filtration plates (Sigma, St. Louis MO, USA). The products were then subjected to clean up by Sephadex gel column filtration using Sephadex® G-50 medium. Capillary electrophoresis was performed using ABI 3500® Genetic analyzer (Thermo Fisher Scientific Inc. USA). The electropherogram obtained from the ABI 3500 Genetic Analyzer is exported to the sequence analysis software, Codoncode® aligner program version 7.0. Comparison of sequences in the contig automatically identifies the variation in the sample sequence when aligned to a reference sequence, and located in the genome by Basic Local Alignment Search Tool (BLAST) [19] from National Center for Biotechnology Information (NCBI). Pathogenicity of variants was identified from different public databases such as dbSNP (NCBI, NIH USA), ClinVar (NCBI, NIH USA), and/or COSMIC database (Sanger Institute, UK). The pathogenicity of variants that were not reported in public databases or previous publications, were tested using computer-aided public accessible prediction tools such as Mutation Taster [20], Polyphen [21] or Sorting Intolerant from Tolerant – SIFT- algorithm [22].
HRM Analysis
HRM was performed using the LightCycler® 480 real-time PCR and data was analyzed in LightCycler® 480 Gene scanning software 1.5, Windows version. In a typical HRM, during the melting phase, with increasing temperature, fluorescence decreases as the saturated dye detaches from the denatured amplicon DNA. The change in fluorescence per unit change in temperature (𝒹F/𝒹T) is plotted. Since the fluorescence intensity changes (HRM signal curve) for different samples have different end points, the data is normalized using the pre-melt and post-melt temperature ranges. Hence, each species of DNA is delineated according to the melting temperature. In the difference plot, the pre-assigned wild-type DNA is set as the baseline curve and the DNA from tumor tissues are plotted with different colors from that of the wild-type DNA. Significant deviation from the baseline curve is indicative of and assigned as mutant species by the software. The standard sensitivity is kept as 0.3 and as the sensitivity is increased, smaller deviations can be identified as mutant. The difference plot of heteroduplexes (where one strand is mutant and other strand is wild-type) shows maximum deviation from the wild-type species, while that of homoduplex mutant DNA shows intermediate deviation. Samples with an aberrant HRM curve are identified as mutant species. HRM results were compared with the results from SEQ for validating HRM analysis in the detection of EGFR mutation (Fig 1).
Statistical Analysis
Statistical analysis was performed using IBM SPSS Windows version 20.0 software. Categorical variables are expressed using frequency and percentage. Diagnostic measures such as sensitivity, specificity and accuracy were calculated. McNemar’s test was used to test the statistical significance of the difference between HRM compared to standard test of SEQ.