Timing of Osimertinib Impacts RT-Induced Tumor Immunogenicity

Abstract

In a phase II randomized clinical trial, oligometastatic NSCLC patients (12% with EGFR mt) who did not progress after 1st-line therapy and completed multi-site SBRT displayed not only an improved PFS but also better OS compared to those who received maintenance chemotherapy or observation (Gomez et al JCO 2019). The ability of RT to increase immunogenicity of tumor cells is well-characterized and could have been a contributor to these clinical observations. Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) that is currently approved as a first-line treatment for patients with non-small cell lung cancer displaying an exon 19 deletion or an exon L858R mutation. We hypothesized that the timing of RT relative to the administration of an EGFR-TKI may impact tumor immunogenicity (RT-induced tumor cell senescence as a cancer vaccine substrate [Meng et al Molecular Therapy 2012] and RT-induced type 1 interferon release as a viral mimicry signal for DC recruitment and activation [Vanpouille-Box et al Nature Communications 2017]). We report the use of a syngeneic murine Lewis lung model (LLC1) to evaluate the impact of osimertinib treatment on RT-mediated immunogenicity in vitro. LLC1 cells were seeded in 6-well plates and were treated with 6 Gy RT or osimertinib (1 mM for 24 hrs) or both. Combination treatment was tested in three sequencing schedules where osimertinib treatment was done (1) one day before RT, (2) concurrent with RT or (3) one day after RT. Type 1 interferon in the supernatant was measured using ELISA and normalized to baseline production in untreated cells. Identification of senescent cells relied on enhanced expression of pH-dependent beta-galactosidase activity on fixed cells. Baseline production of interferon beta in LLC1 lung cancer cells was intrinsically low and not improved with either RT or osimertinib as single agents. Interestingly, we observed differential induction related to the sequence of combination treatment. Only marginal IFNb levels were observed when osimertinib was given prior to or concurrent with radiotherapy but was increased 1.5-fold when cells were treated with osimertinib 1 day after RT. Furthermore, osimertinib treatment after RT was accompanied by the highest number of senescent LLC1 cells (7-fold increase). Whereas single 6Gy dose was sufficient to induce a 5-fold increase of senescent cells; osimertinib alone had no effect. No further induction of senescent cells was seen when osimertinib treatment was done prior to or concurrent with RT. Overall, these data support testing the combination of osimertinib and tumor-targeted radiotherapy. Ongoing pre-clinical work will identify optimal timing of EGFR-TKI treatment to potentiate RT-induced immunogenicity in vivo and to inform the subsequent development of a prospective clinical trial.