Next Generation Sequencing: Are there any variants that you would eliminate from

Question

Next Generation Sequencing:

Are there any variants that you would eliminate from further consideration based on the NGS sequencing parameters and why?

Which of the variants are clinically significant for lung adenocarcinoma and what is the effect of these variants?

For the KRAS variants listed what would the position be in HG38? Why are the positions different between the 2 builds?

Sample Chromosome Position(hg19) ReferenceBase AlternateBase DepthOfCoverage PercentVariant AA change Gene Name # of times detected HC 1 115256530 G T 12771 0.129590478 p.Q61K NRAS 1655 HC 12 25398281 C T 16321 0.15654678 p.G13D KRAS 2555 HC 12 25398284 C T 16313 0.087966652 p.G12D KRAS 1435 HC 15 90631830 G A 8312 0.013715111 IDH2 114 HC 3 178936091 G A 34625 0.085545126 p.E545K PIK3CA 2962 HC 3 178952085 A G 40806 0.173185316 p.H1047R PIK3CA 7067 HC 4 1807894 G A 4241 0.996227305 FGFR3 4225 HC 4 55141055 A G 31553 0.997718125 p.P567P PDGFRA 31481 HC 4 55599321 A T 11789 0.106200696 p.D816V KIT 1252 HC 7 55241707 G A 12291 0.27361484 p.G719S EGFR 3363 HC 7 55249001 G T 36209 0.025325195 EGFR 917 HC 7 55249063 G A 36786 0.1548687 p.Q787Q EGFR 5697 HC 7 55249071 C T 36786 0.01130865 p.T790M EGFR 416 HC 7 55259515 T G 44515 0.028305066 p.L858R EGFR 1260 HC 7 140453136 A T 17520 0.101997717 p.V600E BRAF 1787 HC 9 133738370 A G 39258 0.10451373 p.Y257C ABL1 4103

Explanation / Answer

The data is from a lung biopsy from a patient recently diagnosed with a lung adenocarcinoma being considered for targeted therapy with tyrosine kinase inhibitors.
We first sought to profile the mutational landscape of lung adenocarcinomas, focussing on copy number aberrations (amplifications and deletions), single nucleotide variations (SNVs: non-synonymous, missense, and nonsense mutations), and frameshift mutations (truncating and in frame) across a panel of key driver genes. We identified a panel of 14 oncogenes previously reported to be of key importance in lung adenocarcinoma. Alterations in these genes are present in 82% of our test population of 230 patients. The landscape here is dominated by missense mutations in KRAS (present in >30% cases) and copy number gains primarily in EGFR, DDR2, BRAF, MET and PIK3CA. Importantly, targeted therapies have been developed for each of these driver genes. Aside from KRAS, SNVs are relatively evenly distributed across these driver oncogenes. Samples were sorted by overall mutational burden. Despite covering the vast majority of patients, our oncogene panel did not cover the bulk of a patients’ mutational burden, likely due to a high proportion of low-frequency ‘passenger’ mutations within the clinical population.

Most techniques aimed at detecting mutational events within a gene, such as digital droplet PCR or SNV array technologies, detect specific base-pair substitutions or frameshift mutations at a defined genomic locus rather than across the entire gene length. Despite the fact that over 30% of patients harbour a missense mutation in KRAS, many of these mutations could be missed without proper direction. Thus, it is important to identify specific hotspot loci within driver genes to create targeted panel.

We identified several such hotspot regions in recurrently mutated oncogenes (representative examples KRAS and EGFR. For example, 73 of the 75 SNVs in KRAS resulted in an amino acid substitution at position 12 in the Ras domain. Notably, 17% of KRAS mutant lung adenocarcinomas harbour the G12D substitution (glycine to aspartic acid at position 12) which confers a more invasive tumour phenotype and a reduced response to anti-EGFR targeted therapies. EGFR, the second most recurrently mutated oncogene in lung adenocarcinoma, showed a much more dispersed pattern of mutational events across its protein-coding domains. Missense mutations were preferentially localised to the phosphor-tyrosine kinase and eight resulted in an amino acid substitution from leucine to arginine at position 858 (L858R).

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