Transl Res: Major advances in concomitant diagnostics with targeted drugs for lung cancer: a systematic assessment of the sensitivity of 74,389 rare EGFR mutations to 5 first-line targeted inhibitors

The incidence of epidermal growth factor receptor (EGFR) mutations in the non-small cell lung cancer (NSCLC) population is 28.
6%, and the incidence is much higher in East and Southeast Asian populations than in European populations
.
Clinical trials have shown that EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are superior to chemotherapy in terms of progression-free and adverse effects in patients with NSCLC, and several EGFR-TKIs (eg afatinib, erlotinib, gefitinib, icotinib, osimertinib) have been approved for first-line treatment in patients with advanced NSCLC with activated EGFR mutations
。 Clinically, doctors usually perform EGFR mutation testing to check the patient's mutation pattern to determine treatment options
.
However, at present, the companion diagnosis of EGFR is mainly aimed at the mutation hotspot area, and there is nothing that can be done for rare mutations
.

Saturation mutation scanning (DMS) is a new high-throughput, systematic study of gene mutants, enabling rapid functional verification
of a large number of VUS mutants.
The technology was established around 2010, developed slowly in the following 6 years, and entered a period of rapid development after 2016
.
An Lei's team from Henan University and Xu Hongen's team from Precision Medicine Center of Zhengzhou University are the first teams in China to pay attention to and develop DMS technology, and the team used DMS technology to systematically study 74389 EGFRs for the first time The sensitivity of rare variant mutations to five clinical first-line EGFR-TKI targeted drugs, the study method is universal, and can be used to study the sensitive mutations and drug-resistant mutations
of other targeted inhibitors.

Recently, the research results were published in Translational Research under the title "Defining the Sensitivity Landscape of EGFR Variants to Tyrosine Kinase Inhibitors
。

The article was published in Translational Research

Clinical significance

1.
The study of the sensitivity of EGFR rare variants to targeted drugs can promote the solution of clinical precision drug use problems of a large number of rare variants and promote the in-depth development
of targeted drug companion diagnosis.

2.
The functional interpretation of unknown meaning mutations (VUS mutants) is the bottleneck problem faced by the gene sequencing industry, and deep mutational scanning technology is expected to help the rapid solution
of the bottleneck problem in the industry.

Main research content

To cover all major mutation types, the research team constructed two EGFR libraries using the MITE method, all of which were located in EGFR mutation hotspots (exons 18-21).
Parallel synthesized oligonucleotides were used to construct saturated EGFR Sub-Del (Substitution and Deletion mutants) libraries, and multiple degenerate primers were used to construct saturated EGFR Ins (Exon-20 insertion mutant) library, EGFR replacement deletion (Sub-Del) and exon 20 constructed in the construct A total of 3,914 and 70,475 variants were detected in the insertion (Ins) library (Figure 1).

Figure 1 Experimental design and data analysis

To systematically assess the sensitivity of EGFR mutants to different TKIs, the research team used the endogenous EGFR-depleted cell line, the human lung adenocarcinoma cell line PC9 For the model, Sub-Del and Ins mutants were introduced into PC9-Cas9 cells and 5 TKIs were used Process
it.
The results showed that in erlotinib, gefitinib, icotinib, afatinib (50 nM) and osimertinib (200 nM).
In the trial, the number of cells containing the Sub-Del variant decreased significantly on days 5 and 7, reached its lowest on day 9, and then began to increase
。 In the afatinib (600 nM) and osimertinib (600 nM) tests, the number of cells was 11th, respectively Day and day 15 reached the lowest point
.
For cells containing the Ins variant, afatinib (600 nM) and osimertinib (600 nM).
The number of cells detected is minimized on days 9 and 13, respectively, while the number of cells detected by other TKIs is at TKI There was no reduction
during the two-week period of processing.

To investigate the enrichment of variants after TKIs incubation, the research team collected cells from each TKI experiment for gDNA extraction, mutation region amplification, and high-throughput sequencing
through massively parallel cytotoxicity assays.
Compared to reversible TKIs (erlotinib, gefitinib and icotinib), irreversible TKIs (afatinib50 nM and osimertinib200 nM) showed stronger inhibition against the Sub-Del variant
.
For the Ins variant, osimertinib (200 nM) has a greater inhibitory effect
than other TKIs.
The above results suggest that the Ins library variant is more resistant to TKI treatment than the Sub-Del library variant, whereas osimertinib ( 200 nM) showed stronger inhibition of the variants in both libraries (Figure 2), and cytotoxicity screening results were consistent
with known clinical notes.

Figure 2.
EGFR-TKIs have different inhibitory effects on Sub-Del and Ins library variants

The research team then presented the variant enrichment fraction of each variant in each TKI trial in the form of a heat map (Figure 3).

In the systematic cytotoxicity screening, 813 and 51 surrogate variants were identified, respectively, that may mediate resistance to reversible and irreversible TKIs of EGFR
.
Cell proliferation experiments found that 221 of the 3,914 Sub-Del variants proliferated rapidly in controlled trials and were sensitive to EGFR-TKI, which is potential EGFR-TKIs sensitive variants
.
For 70,475 Ins variants, insertions of amino acids at positions 770-774 were highly enriched
in all five TKI cytotoxicity assays.

In addition, the first 5% of enriched insert variants include high-frequency glycine or serine insertions
.

Figure 3.
Enrichment of EGFR Sub-Del library mutants and EGFR Ins-1 library variants for the cytotoxicity of five TKIs

In summary, the sensitivity of relevant mutations to five first-line EGFR-TKI-targeted drugs was evaluated by systematic drug sensitivity screening experiments for 7,4389 rare variants of EGFR gene in lung cancer.
NSCLC patients with rare EGFR mutations were found to be most likely to benefit
from osimertinib treatment.
This research method is versatile and can be used to study sensitive mutations and drug-resistant mutations of other targeted inhibitors, and the development of small molecule targeted inhibitors
.

In addition, related technologies are also used to study the relationship between other gene mutations and the sensitivity of targeted inhibitors, and promote the rapid development
of tumor precision medicine.

References:

Lei An, Yueqiang Wang, Guangyao Wu.
et al.
Defining the Sensitivity Landscape of EGFR Variants to Tyrosine Kinase Inhibitors.
Transl Res.
2022 Nov 5; S1931-5244(22)00244-4.
doi: 10.
1016/j.
trsl.
2022.
11.
002.