[It is expected that the ending will be overhauled before New Year's Day and will be completed. The new book should be opened in early January]

The activation mutation of the epidermal growth factor receptor (EGFR) gene occurs in 10-20% of white people and at least 50% of Asian non-small cell lung cancer (NSCLC) patients. Two mutations, the deletion of exon 19 and the replacement of single amino acids of exon 21, are commonly referred to as the "classic" epidermal growth factor receptor mutation, accounting for approximately 85% of the observed epidermal growth factor receptor mutations in non-small cell lung cancer,

It also confers increased sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor (EGFRI). Rare mutations account for about 15% of non-small cell lung cancer gene mutations, including point mutations, deletions and insertions of exons 18-25 of the EGFR gene. Although it is a low-frequency mutation, given the high overall incidence of lung cancer, it is estimated that more than 30,000 new diagnoses in NSCLC patients will contain rare EGFR mutations each year.

Therefore, it is crucial to understand the biology of rare EGFR mutations and evaluate the effectiveness of current treatment options. One obstacle to understanding the biological differences between rare EGFR mutations and classic EGFR mutations is the lack of patient-derived NSCLC cell line models containing endogenous rare EGFR mutations. Therefore, many preclinical studies rely on exogenous expression of rare EGFR mutants in model cell lines, such as mouse pro-b cell line BA/F3 and mouse fibroblast cell line NIH-3T3. Despite these limitations, structural and preclinical data have been used to predict the efficacy of different EGFR for specific rare EGFR mutations.

However, few clinical trials can systematically and vigorously evaluate the efficacy of EGFRI in patients with non-small cell lung cancer carrying rare EGFR mutations. Due to the lack of clinical data, the field relies heavily on post-summarized analysis of clinical trials and case series to evaluate EGFRI responses in this group of different types of patients. For example, in 2018, FDA approved the second generation of EGFRI      Afatinib for the treatment of SC768I,

L861q and g719x rare EGFR point mutations are evidence based on pooled analysis of three clinical trials Lux-long   2, Lux-long  3 and Lux-long   6. In the following chapters, we will describe the structural characteristics of the rare EGFR mutation and elaborate on preclinical and clinical evidence of EGFR response in patients carrying this rare EGFR mutation.

picture

【Classic egfr mutation】

picture

Picture [Rare egfr     18 exon mutation]

【Deletion of e709x and 18 exons】

picture

【Preclinical data】

To study the E709x mutation, Kobayashi et al. expressed E709k and Dele709-t710insd in ba/f3 and nih-3t3 cells. Ba/f3 cells expressing E709x mutation grew in the absence of il-3, while Nih-3t3 cells expressing the mutation structure formed lesions in the lesion formation experiment, indicating that these mutations are oncogenic drivers. To detect the EGFRI sensitivity of these mutations, the authors conducted dose-response experiments on ba/f3 cells using 7 different EGFRIs; the first generation inhibitor G

Efitinib and erlotinib, second-generation inhibitors Afatinib, itinib and neratinib, and third-generation inhibitors Osimertinib and rociletinib. They found that E709k and Dele709-T710insd were significantly less sensitive to gefitinib, erlotinib and osimertinib than BA/F3 cells expressing ex19del, with Dele709-T710insd being the most resistant to these inhibitors.

However, E709k and Dele709-T710insd were sensitive to the second-generation inhibitors Afatinib and Neratinib as that of Ex19del. At the same time, westernblot analysis of Hek293 cells expressing E709k, Dele709-T710insd and Ex19del showed that after afatinib and neratinib treatment, the tyrosine phosphorylation of EGFR in each cell line was inhibited. In contrast, only Ex19del showed EGFR inhibition after the same dose of gefitinib, erlotinib or Osimertinib, which indicated that the second-generation EGFR had the greatest affinity for the rare E709x and exon 18 deletion mutations compared with the first-generation or third-generation inhibitors.

【Clinical Data】

Table 1 summarizes the results of trials evaluating the prognosis of non-small cell lung cancer patients with rare EGFR exon 18 mutations. Due to the low incidence of e709x replacement and exon 18 deletion mutations, little is known about the clinical response of these mutations to EGFRI. Kobayashi et al. reported that 15 patients with E709x compound mutations had rr of 53% and 4 patients with Dele709_t710insd who received first-generation EGFRI treatment had rr of 25%.

This data supports preclinical evidence that the replacement mutation of E709 is generally more sensitive to first-generation EGFRI than the deletion of exon 18. A report evaluating the use of afatinib found that in four patients with E709x mutations, drug activity was associated with the time to treatment failure (ttf) and 12 months, however, all patients had complex mutations with additional l858r or g719x EGFR mutations. Further clinical studies will be needed to validate preclinical data that second-generation EGFRI is more effective for E709x mutations, which in turn may depend on the absorption of diagnostic tests capable of detecting these rare EGFR variants.

Picture [g719x]

Among the rare EGFR mutations of NSCLC, g719x substitutions (including g719s, g719a, g719c and g719d substitutions) are one of the more common mutations second only to exon 20 insertion, accounting for about 1.5-3% of all EGFR mutations in NSCLC. G719x mutations can appear as independent EGFR mutations, or as complex EGFR mutations combined with other point mutations (such as s768i or l819q).

【Preclinical data】

Picture [Clinical Data]

In one of the largest clinical studies, Chiu et al. focused on the unusual EGFR mutations. The efficacy of gefitinib and erlotinib in 78 patients with single g719x mutations was evaluated and compared with the common ex19del and l858r mutations. G719x single mutation patients were sensitive to EGFRI treatment (total effective (orr) = 36.8%, disease control rate (dcr) = 72.4%, n=78), but compared with the l858r mutation (orr=67.5%, dcr=95.6%, n=256) or ex19del (orr=65.3%, dcr=94.5%, n=94.5%, n=1858r mutation (orr=67.5%, dcr=95.6%, n=256) or ex19del (orr=65.3%, dcr=94.5%, n=1858r mutation (orr=67.5%, dcr=95.6%, n=256) or ex19del (orr=65.3%, dcr=94.5%, n=1858r mutation (orr=67.5%, dcr=94.5%, n=1858r mutation (orr=67.5%, dcr=95.6%, n=256) or ex19del (orr=65.3%, dcr=94.5%, n=1858r mutation (orr=67.5%, dcr=94.5%, n=1858r mutation (orr=67.5%, dcr=95.6%, n=256) or ex19del (orr=65.3%, dcr=94.5%, n=1858r mutation (orr=65.3%, dcr=94.5%, n=1858r mutation (

=222) had significantly reduced sensitivity. These findings were accompanied by poor patient outcomes, with median PFS of 6.3 months in patients with g719x, while median PFS of 10-13 months in patients with l858r and ex19del. In line with preclinical studies, second-generation EGFRI has been shown to be more effective than first-generation inhibitors in patients with g719x. In an early phase II clinical trial of Neratinib (nct00266877), although the overall results for EGFR mutant lung cancer were disappointing, 3 of the four patients with rare EGFR mutant G719x responded partially to Neratinib, while

The fourth patient was stable within 40 weeks. However, severe dose-limiting toxicity of nelatinib was observed in this trial, which hindered further studies of non-small cell lung cancer with EGFR mutations. The g719x mutation was also proven to respond to second-generation EGFRI    Afatinib. Post hoc analysis of 32 patients from Lux-long    2, Lux-long   3 and Lux-long    6 trials showed that Afatinib was clinically active against rare EGFR mutations (g719x, s786i, l861q) lung cancer. In 8 people carrying a single g7

Among the 19x mutations and six patients carrying complex g719x mutations, afatinib treatment resulted in 77.8% RR and 13.8 months of PFS, which led to FDA expanding the indication of afatinib in January 2018 to include non-small cell lung cancer patients carrying g719x mutations. A recent phase II trial showed that the third-generation inhibitor Osimertinib may also have clinical activity in this patient population, with 52.6% RR reported in 19 patients carrying g179x mutations. However, further trials are needed to determine whether osimertinib provides significant survival benefits compared to afatinib treatment.

【Rare egfr    19 exon mutations】

It is reported that in non-small cell lung cancer, the low incidence of exon 19 insertion accounts for about 2% of exon 19 abnormalities and 1% of all egfr mutations. The exon 19 insertion is characterized by the insertion of 18 base pairs, resulting in an increase of 6 amino acid sequences, which in most cases begins with the 744 or 745 codons of the egfr gene. Although the inserted amino acid sequences are different, the 4 amino acid sequences of pvai are identical to all reported exon 19 insertion sequences.

【Preclinical data】

Although there are slight differences in the exact sequence of the insertion of exon 19 mutation, there are two conservative effects on the 3D structure: the addition of 6 residues to the loop connecting the β3-strand and the αc-helix (Figure 3), and the alteration of the last 2 residues of e746 and l747 of the β3-strand in WT    EGFR. Since this loop is flexible in the structure of WT    EGFR, insertion of 6-residue sequences may be well tolerated.

Afterwards, the movement of amino acids changes the identity of the E746 residue, and may not have any structural effect because it is exposed to the surface of the kinase. In contrast, the l747 residue is involved in an important hydrophobic core, stabilizing the inactive form of the kinase domain. He et al. reported 11 patients with exon 19 insertion, in each patient, the common effect of the insertion is that the identity of the leucine residue at position 747 in egfr is changed to proline at this position

Residue (l747p). It is predicted that l747p is not conducive to the formation of this hydrophobic core, resulting in structural activation of EGFR; this mechanism is similar to the mechanism of l858r replacement, which is located in the hydrophobic core immediately adjacent to l747. To evaluate the sensitivity of exon 19 insertion mutations to EGFR, they expressed two different exon 19 insertion fragments in ba/f3 cells (i744_k745inskipv

Ai and K745_e746instpvaik). Dosage response experiments showed that the insertion of these exons 19 was similar to that of gefitinib and afatinib compared with ba/f3s expressing ex19del. Westernblot analysis of nih-3t3 cells expressing exon 19 showed that egfr phosphorylation was depleted after gefitinib or afatinib treatment, similar to what was observed in ex19del expressed cells.

This data led the authors to conclude that exon 19 insertion mutations are egfri sensitive and suggest that NSCLC patients carrying these mutations should receive egfri treatment.

【Clinical Data】

picture

【Rare egfr    20 exon mutation】Pictures

Table 3 summarizes clinical trials of EGFRI in patients with non-small cell lung cancer who evaluated rare EGFR mutations in exon 20. Clinical studies have shown that among the different types of mutations that support preclinical observations, the EGFRI sensitivity of different types of exon 20 inserts differed significantly. Most patients with NSCLC carrying EGFR exon 20 inserts are resistant to first and second generation EGFRI, with reported response rates ranging from 0 to 27%. However, as predicted by structural data and kinetic analysis of in vitro drug binding, the insertion of the EGFR gene portion encoding the αc-helix, rather than the insertion within the loop immediately following it, is

Exceptions to the rules. Multiple reports show that patients carrying A763_y764insfqea inserts have partial responses to erlotinib, which has a sensitivity to the first generation of EGFRI similar to the classic EGFR mutation. While currently licensed EGFRI has not shown any significant benefits for other EGFR exon 20 inserts, emerging EGFRIs include Poziotinib, Tak-788 and Tarloxotinib are in clinical development and have good activity for NSCLC patients in this subset. More details of these drugs have been reviewed recently by laboratories elsewhere in our area.

Although the s768i mutation can occur alone, it is more common that s768i coexist with other point mutations in egfr to form complex mutations, which may confuse the interpretation of clinical data. For example, in multiple case studies of patients with s768i mutation, huge differences in response to first-generation egfri were reported, ranging from non-responsive and progressive disease to good partial response and moderate sensitivity. Leventakos et al.

Heterogeneity of response durability was emphasized. Asymptomatic survival time of 3-30 months in 9 patients with s768i alone or s768i/g719x or s768i/l858r complex mutations were reported. The low frequency of these mutations and the variation of co-occurring EGFR point mutations is a huge challenge for the analysis of these inconsistencies in clinical data. Interestingly, Chiu et al. observed a single s768i point mutation with

Compared with the s768i/g719x complex mutation is more sensitive to the treatment of the first generation EGFR. The rr of the s768i complex mutation is 50% (n=10), and the rr of the single s768i mutation is 33% (n=7). Preclinical data show that the second generation inhibitor Afatinib may be more effective against the s768i mutation EGFR than the first generation inhibitor. In lux-long    2, lux-long

In the later analysis of the 3 and lux-long trials, the second-generation EGFR     Afatinib obtained 100% orr and 14.7 months of median PFS in 8 patients with s768i mutations, resulting in FDA approval of the drug for NSCLC patients with egfr                                                                                                                                                                                                                  

Picture [Preclinical data]

Molecular dynamic simulations show that l861q stabilizes the active αC-in conformation by forming new hydrogen bonds near the C-terminus of the αC-helix. Research on l861q in the ba/f3 model system shows that similar to the s768i mutation, l861q is resistant to the first-generation EGFRI compared with l858r, but unlike s768i, l861q is sensitive to afatinib and osestinib treatment. This is in line with western blot analysis that expressing l861q

After cells were treated with erlotinib, egfr phosphorylation was retained, but after treatment with 10                                                                                                                                                                                                                                                 

【Clinical Data】

picture

【Other rare egfr mutations】

Image [egfr kinase domain replication (egfr-kdd)]

【Preclinical data】

When EGFR-KDD is expressed in mouse fibroblast cell lines NR6 and BA/F3 cells, EGFR-KDD shows high levels of constitutive receptor phosphorylation compared with WT     EGFR[71]. Galant et al. also found that A1235 cells, a human glioblastoma cell line containing endogenous EGFR, show high levels of constitutive EGFR phosphorylation in the presence and absence of serum. Calculation model shows that EGFR-KDD can form an asymmetric dimer from N-leaf to C-leaf within the molecule, resulting in

Structural activation of receptors. To evaluate the sensitivity of EGFR-KDD to currently available EGFR, Galant et al. treated EGFR-KDD with erlotinib, afatinib and ossitinib. They found EGFR-KDD to be most sensitive to afatinib. They also showed that after EGFR treatment, phosphorylation of downstream signal node Erk was reduced, and similar results were observed in NR6-EGFR-KDD and A1235 cells, indicating that ERK signaling is important for the survival of EGFR-KDD-dependent cells.
Chapter completed!