Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions

In PDX model LG1423 harboring V769_D770InsASV, daily 25 mg/kg osimertinib induced superior tumor growth inhibition to 20 mg/kg afatinib (60% vs 18% at day 22).

“in a PDX model (LG1423) harboring the same prevalent EGFR Ex20ins V769_D770InsASV variant as used in H2073 studies, daily dosing of 25 mg/kg osimertinib induced a superior tumor growth inhibition than the higher 20mg/kg dose of afatinib (60%, p<0.001 & 18%, p: non-significant respectively at day 22) when compared to the control group (Fig. 5A).”

Ex20Ins SVD and ASV confer primary resistance to afatinib at 7.5 mg/kg in H2073 xenografts.

“the Ex20Ins SVD and ASV confer primary resistance to 7.5 mg/kg, a dose that represents the 40mg clinical starting dose of afatinib (Fig. 3A–B).”

• AssumptionThe authors state this as an assumption: A 7.5 mg/kg dose of afatinib in mice represents the 40 mg clinical starting dose.

The efficacy of osimertinib was associated with robust and sustained inhibition of phospho-EGFR and downstream signalling markers after a single 25 mg/kg dose.

“The efficacy of osimertinib was associated with robust and sustained inhibition of phospho-EGFR and downstream signalling markers (Fig. 5C and Fig. S8) following a single 25mg/kg dose.”

Gefitinib and afatinib showed high potency only against H2073-FQEA, similar to their activity against wild-type EGFR.

“both gefitinib and afatinib also showed high potency only against the clinically sensitive H2073-FQEA, which was also similar to their wtEGFR activity (Fig. 2D–E).”

Osimertinib and AZ5104 have preclinical activity against the two most clinically prevalent Ex20Ins variants.

“here we report pre-clinical activity of osimertinib and AZ5104 (a circulating metabolite of osimertinib) (13), against these two most clinically prevalent variants of Ex20Ins mutations.”

• cites [13] Cross, 2014, AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer, Cancer Discov sourceNot checked yet

  Not checked in the free run.

Gefitinib, erlotinib and afatinib showed high potency against phosphorylation of the Ex20InsFQEA variant in the Cos-7 engineered cell line model.

“the first- and second-generation approved TKIs (gefitinib, erlotinib and afatinib) showed high levels of potency against phosphorylation of the Ex20InsFQEA variant using the Cos-7 engineered cell line model (Fig. 2A)”

Without ligand, wild-type EGFR localized mainly to the plasma membrane in H2073-WT cells.

“As expected, wt-EGFR was mostly located at the plasma membrane in the H2073-WT cells in the absence of ligand stimulation.”

In a separate experiment, osimertinib induced superior tumor growth inhibition to erlotinib (93% vs 15% at day 20).

“osimertinib induced superior tumor growth inhibition than erlotinib (93%, p<0.001 & 15%, p: non-significant respectively at day 20) when compared to the control group (Fig. 5B).”

Knocking down EGFR with siRNA reduced survival of the SVD and ASV mutant H2073 lines.

“Similarly, siRNA knockdown of EGFR inhibited the survival of both H2073-SVD and -ASV cell lines (Fig. S2A).”

• cites Fig. S2ANot checked yet

  Not checked in the free run.

In proliferation assays, osimertinib did not show the same margin of selectivity between Ex20Ins and wild-type EGFR that was seen in pEGFR assays.

“in contrast to the pEGFR activity, osimertinib did not exhibit the same margin of selectivity between Ex20Ins and wtEGFR”

Osimertinib and AZ5104 produced similar potent inhibition of proliferation in H2073-FQEA, H2073-SVD and H2073-ASV cell lines.

“osimertinib (Fig. 2B and Fig. S3A) and AZ5104 (Fig. 2C and Fig. S3B) demonstrated similar potent levels of proliferation inhibition against H2073-FQEA, H2073-SVD and H2073-ASV cell lines.”

Minimal body weight loss of less than 10% of starting body weight was observed in the PDX efficacy experiments.

“Minimal body weight loss (less than 10% of starting body weight) was observed in these efficacy experiments (Fig. S7A–B).”

In a separate study, AZ5104 at 25 mg/kg daily induced 7% regression at day 15.

“In a separate study, AZ5104 dosed at 25mg/kg daily induced 7% regression (p<0.001) at day 15 when compared to the control group (Fig. S7C).”

Compared with afatinib, osimertinib displayed greater activity towards Ex20Ins relative to wild-type EGFR.

“compared to afatinib, osimertinib consistently displayed a greater level of activity towards Ex20Ins compared to wtEGFR”

The growth behavior caused by these insertions tracks with continued EGFR pathway activity under low-serum conditions.

“This Exon20 insert-driven growth phenotype correlates with sustained EGFR pathway activation in low serum, shown in Fig. 1B.”

• cites Fig. 1BNot checked yet

  Not checked in the free run.

The stronger efficacy of AZ5104 compared with osimertinib or afatinib correlated with more profound and less transient inhibition of EGFR and downstream signalling pathways.

“The stronger efficacy observed with AZ5104 when compared to osimertinib or afatinib correlated with a more profound and less transient inhibition of EGFR and downstream signalling pathways.”

Osimertinib and AZ5104 showed greater relative potency than erlotinib across each Ex20Ins variant tested.

“consistently exhibited greater relative potency compared to the reversible TKI erlotinib across each variant (Fig. 2A).”

Osimertinib and AZ5104 potently inhibited phosphorylation of the Ex20Ins variants NPG, SVD and ASV.

“Osimertinib and AZ5104 also potently inhibited phosphorylation of other EGFR Ex20Ins variants NPG, SVD and ASV”

Afatinib showed high activity against all Ex20Ins variants tested.

“the second-generation irreversible TKI afatinib similarly showed high activity against all the Ex20Ins variants (Fig. 2A).”

Earlier generation TKIs had significantly lower activity against H2073-SVD and H2073-ASV cell lines than against wild-type EGFR.

“the earlier generation TKIs demonstrated significantly lower levels of activity against H2073-SVD and H2073-ASV cell lines compared to wtEGFR (Fig. 2D–E and Fig. S3C–D).”

The level of efficacy achieved by osimertinib was significantly greater than that of clinically relevant doses of afatinib.

“Moreover, the level of efficacy achieved was significantly greater than clinically relevant doses of afatinib.”

The mutant-selective profile of osimertinib appeared to remain for Ex20Ins variants, in contrast to other TKIs.

“the mutant-selective profile of osimertinib appeared to remain for Ex20Ins variants, in contrast to the other TKIs.”

In vivo, osimertinib delivered high anti-tumor activity across both Ex20Ins models at 25 mg/kg/day, a dose modelled to be approximately consistent with the 80 mg clinically approved dose for T790M tumors.

“In vivo, osimertinib delivered high levels of anti-tumor activity across both Ex20Ins models at 25mg/kg/day, a dose modelled to be approximately consistent with the 80 mg clinically-approved dose for targeting T790M tumors.”

• AssumptionThe authors state this as an assumption: A 25 mg/kg dose of osimertinib in mice approximates the clinically approved 80 mg dose.

In two other PDX models harboring less prevalent Ex20ins, osimertinib delivered high anti-tumor activity compared with afatinib.

“in two other PDX models harboring less prevalent Ex20ins, osimertinib similarly delivered high levels of anti-tumor activity compared to afatinib (M766_A767insASV; H773_V774insNPH).”

The margin of selectivity between Ex20Ins mutants and wild-type EGFR seen in pEGFR assays is not apparent for osimertinib in proliferation assays.

“the margin of selectivity between Ex20Ins mutants and wtEGFR observed in pEGFR assays is not apparent for osimertinib in the more chronic proliferation assays”

Osimertinib and AZ5104 effectively inhibited Ex20Ins mutant forms of EGFR at potencies comparable to those previously published for common activating and T790M mutants.

“Using in vitro phospho-EGFR studies, osimertinib and its active metabolite AZ5104 effectively inhibited Ex20Ins mutant forms of EGFR, at potencies comparable to the common activating and T790M mutants previously published (13).”

• cites [13] Cross, 2014, AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer, Cancer Discov sourceNot checked yet

  Not checked in the free run.

Osimertinib retains a more comparable balance of potency between wild-type EGFR and all Ex20Ins mutations.

“osimertinib retains a more comparable balance of potency between wtEGFR and all Ex20Ins mutations.”

Afatinib and gefitinib have very similar potencies between wild-type EGFR and the FQEA variant, which is clinically sensitive to these TKIs.

“both afatinib and gefitinib harbor very similar potencies between wtEGFR and the FQEA variant which has been demonstrated to be sensitive to these TKIs clinically.”

Proliferation activity is much lower against the other prevalent Ex20Ins variants compared with wild-type EGFR for afatinib and gefitinib.

“proliferation activity is much lower against the other prevalent Ex20Ins variants compared to wtEGFR for these TKIs.”

Osimertinib at 25 mg/kg did not achieve the same levels of tumor shrinkage observed in in vivo models representing canonical EGFR activating and T790M mutants.

“osimertinib at the maximally tolerated pre-clinical dose of 25mg/kg is not able to achieve the same levels of shrinkage observed across in vivo models representing canonical EGFR activating and T790M mutants (14).”

• cites [14] Hirano, 2015, In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer, Oncotarget sourceNot checked yet

  Not checked in the free run.

The AZ5104 metabolite was similarly able to achieve high levels of tumor inhibition.

“Consistent with in vitro pharmacology, the AZ5104 metabolite was similarly able to achieve high levels of tumor inhibition.”

Most exon 20 insertion mutations, except the rare A763_Y764insFQEA, make tumors resistant to the standard first-line EGFR inhibitors erlotinib, gefitinib, and afatinib.

“EGFR Ex20Ins tumors are generally unresponsive to 1st and 2nd generation EGFR inhibitors”

• Also stated in: § Introduction · plain

  “Unlike Ex19del and L858R, most of the Ex20Ins mutations have been associated in pre-clinical and clinical studies with de novo resistance to the currently approved first-line EGFR-tyrosine kinase inhibitors (TKI), erlotinib, gefitinib and afatinib (2,4–8).”

• cites [2] Arcila, 2013, EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics, Mol Cancer Ther sourceNot checked yet

  Not checked in the free run.

• cites [4] Yasuda, 2012, EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications, Lancet Oncol sourceNot checked yet

  Not checked in the free run.

• cites [5] Chen, 2016, Clinical efficacy of first-generation EGFR-TKIs in patients with advanced non-small-cell lung cancer harboring EGFR exon 20 mutations, Onco Targets Ther sourceNot checked yet

  Not checked in the free run.

• cites [6] Naidoo, 2015, Epidermal growth factor receptor exon 20 insertions in advanced lung adenocarcinomas: Clinical outcomes and response to erlotinib, Cancer sourceNot checked yet

  Not checked in the free run.

• cites [7] Yasuda, 2013, Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer, Sci Transl Med sourceNot checked yet

  Not checked in the free run.

• cites [8] Yang, 2015, Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6, Lancet Oncol sourceNot checked yet

  Not checked in the free run.

Survival of H2073-WT cells depends on EGFR.

“As demonstrated previously using EGFR TKI (13) or using siRNA against EGFR (Fig. S2A), the survival of H2073-WT cell line is dependent on EGFR.”

• cites [13] Cross, 2014, AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer, Cancer Discov sourceNot checked yet

  Not checked in the free run.

• cites Fig. S2ANot checked yet

  Not checked in the free run.

Osimertinib and AZ5104 achieve robust Ex20Ins-mediated pathway inhibition in vivo that is associated with tumor growth inhibition.

“These data demonstrate that both osimertinib and AZ5104 can achieve robust Ex20Ins-mediated pathway inhibition in vivo that is associated with tumor growth inhibition.”

The findings justify testing osimertinib clinically in NSCLC patients whose tumors have EGFR exon 20 insertions.

“Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC.”

• AssumptionRests on an assumption: Conventional cytotoxic chemotherapy remains the usual treatment for NSCLC patients with EGFR exon 20 insertions.

Osimertinib consistently showed more meaningful efficacy than earlier generation TKIs at clinically relevant doses.

“Despite experimental variability, osimertinib therefore consistently showed more meaningful efficacy compared to the earlier generation TKIs, at clinically relevant doses.”

In mice, the pharmacokinetic half-life of osimertinib and AZ5104 is approximately 3 hours.

“in mice the pharmacokinetic half-life of osimertinib and AZ5104 is only approximately 3 hours (13)”

• cites [13] Cross, 2014, AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer, Cancer Discov sourceNot checked yet

  Not checked in the free run.

The two most prevalent Ex20Ins forms, D770_N771InsSVD and V769_D770InsASV, account for approximately 40% of patients with Ex20Ins mutations.

“the two most prevalent forms of Ex20Ins (D770_N771InsSVD and V769_D770InsASV), which account for approximately 40% of such patients (2,4,18).”

• cites [2] Arcila, 2013, EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics, Mol Cancer Ther sourceNot checked yet

  Not checked in the free run.

• cites [4] Yasuda, 2012, EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications, Lancet Oncol sourceNot checked yet

  Not checked in the free run.

• cites [18] Planchard, 2016, Osimertinib Western and Asian clinical pharmacokinetics in patients and healthy volunteers: implications for formulation, dose, and dosing frequency in pivotal clinical studies, Cancer Chemother Pharmacol sourceNot checked yet

  Not checked in the free run.

Maximal proliferation of Ex20Ins mutant cells occurs in low serum.

“indicating that maximal proliferation occurs in this low serum context.”

The two most clinically prevalent Ex20Ins variants, SVD and ASV, are constitutively active in a manner analogous to canonical activating EGFR mutations.

“Our findings provide evidence that the two most clinically prevalent variants of Ex20Ins mutations, SVD (D770_N771InsSVD) and Ex20Ins ASV (V769_D770InsASV) (2,4,18), are constitutively active in an analogous manner to canonical activating-mutations.”

• cites [2] Arcila, 2013, EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics, Mol Cancer Ther sourceNot checked yet

  Not checked in the free run.

• cites [4] Yasuda, 2012, EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications, Lancet Oncol sourceNot checked yet

  Not checked in the free run.

• cites [18] Planchard, 2016, Osimertinib Western and Asian clinical pharmacokinetics in patients and healthy volunteers: implications for formulation, dose, and dosing frequency in pivotal clinical studies, Cancer Chemother Pharmacol sourceNot checked yet

  Not checked in the free run.

The resistance to afatinib is due to the Ex20Ins mutation rather than the H2073 cell line background.

“The H2073 parental cell line is sensitive to a similar dose of afatinib (Fig. S4), indicating resistance is due to the Ex20Ins rather than the cell line background.”

Ex20Ins activating mutants likely signal in a mechanistically analogous way to Ex19del and L858R mutants despite structural differences.

“more generally Ex20Ins activating mutants likely signal in a mechanistically analogous manner to Ex19del and L858R mutants despite structural differences.”

Altered EGFR localisation suggests that endosomal signalling of EGFR Ex20ins may be an important mechanism regulating EGFR-dependent tumorigenesis.

“Altered EGFR localisation suggests that endosomal signalling of EGFR Ex20ins may be an important mechanism regulating EGFR-dependent tumorigenesis”

Ex20Ins mutants lack T790M-associated structural differences proximate to the ATP-binding site.

“Although Ex20Ins mutants lack T790M-associated structural differences proximate to the ATP-binding site (7)”

• cites [7] Yasuda, 2013, Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer, Sci Transl Med sourceNot checked yet

  Not checked in the free run.

Ex20Ins mutations share many signalling and cell biology characteristics with common activating EGFR mutants despite their disparate mutational changes.

“Taken together, these studies support the notion that Ex20Ins mutations have many similar signalling and cell biology characteristics as common activating mutants, despite their disparate mutational changes.”

The balance of activity between mutant EGFR and wild-type EGFR is important for driving clinical benefit.

“This data provides a useful benchmark and further supports the notion that it is the balance of activity between mutant EGFR and wtEGFR that is important for driving clinical benefit”

The lower in vitro potency and reduced tumor growth inhibition of osimertinib against Ex20Ins mutant models compared with common activating mutants may suggest that increased dose levels, such as 160 mg osimertinib, may be required clinically.

“the lower in vitro potency and reduced tumor growth inhibition of osimertinib against Ex20Ins mutant models compared to common activating mutants may suggest that increased dose levels (e.g. 160 mg osimertinib (20)) may be required clinically.”

• cites [20]Not checked yet

  Not checked in the free run.

TKIs that effectively target the prevalent Ex20Ins mutations are likely to provide at least significant clinical benefit to this EGFR-mutant subset.

“we propose that TKIs that can effectively target these prevalent mutations are likely to at least provide significant clinical benefit to this EGFR-mutant subset.”

Clinical benefit is seen only in FQEA, where wild-type EGFR pharmacology is not the primary limiting activity.

“i.e. benefit is only seen in FQEA in which the wtEGFR pharmacology is not the primary limiting activity.”

These data are the first to demonstrate that osimertinib can achieve superior growth inhibition across diverse Ex20Ins PDX models at clinically relevant doses in vivo compared with afatinib.

“These data are the first to demonstrate that osimertinib can achieve superior growth inhibition across diverse Ex20Ins PDX models at clinically relevant doses in vivo compared to afatinib.”