Longitudinal monitoring for the emergence of epidermal growth factor C797S resistance mutations in non-small cell lung cancer using blood-based droplet digital PCR

Introduction

The third-generation epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib is approved for front-line use as well as for use as a second-line treatment when EGFR-mutant, non-small cell lung cancer (NSCLC) becomes resistant to erlotinib, afatinib or gefitinib. This resistance is most frequently mediated by the T790M point mutation^[1]. Unfortunately, acquired osimertinib resistance, considered to be associated with the C797S point mutation in many cases, can emerge within about 10 months in a high proportion of patients^[2]. While no formal clinical study has yet validated the utility of the C797S mutation as a biomarker for osimertinib resistance, such information could guide treatment decision-making for multidrug resistant NSCLC.

Case report

In October 2014, a never-smoker female presented with new-onset seizures and was diagnosed with an extensive brain and bony metastatic TTF-1 positive lung adenocarcinoma. Tissue EGFR exon 19 deletion (del19) was identified. After an excellent initial response to whole-brain radiation therapy and ongoing erlotinib, she was switched to osimertinib when T790M resistance emerged in July 2016. Molecular testing was conducted using the validated GeneStrat ddPCR platform (Biodesix, Inc., Boulder, Colorado USA) for EGFR L858R, del19 (E746-A750), and T790M^[3]. By January 2018 (osimertinib day 540) the patient had discontinued therapy due to resistance and disease progression. She died in hospice care at age 46.

We used a newly developed ddPCR assay to retrospectively evaluate the emergence of the C797S mutation in six remnant plasma samples from this patient. All Biodesix personnel were blinded to patient case information. The C797S mutation was undetectable on treatment days 0 and 42 but was detectable by day 287 [Figure 1]. Orthogonal testing with next-generation sequencing showed that only the C797S variant (G>C) was present in circulation. The EGFR del19 and T790M mutations both declined after initial osimertinib treatment but re-emerged by day 287 [Figures 2 and 3].

Figure 1. Kinetics of emergence of the C797S (G>C) somatic mutant variant during osimertinib treatment cycle. Results are shown as %MVF. For timepoints 0 and 42 C797S was ND. Although results were obtained for del19 and T790M for the day-164 specimen, the remaining specimen QNS for C797S testing at this time point. ND: not detected; QNS: quantity was not sufficient; %MVF: percent minor variant frequency

Figure 2. Evolution of EGFR del19, T790M, and C797S during the 540-day osimertinib treatment cycle. Data are displayed as relative fluorescence units, and the calculated %MVF values are shown for each test. A and B: Del19 on days 0 and 540, respectively; C and D: T790M on days 0 and 540, respectively; E and F: C797S on days 0 and 540, respectively. EGFR L858R was consistently found to be negative by both ddPCR and NGS tests (data not shown). EGFR: epidermal growth factor; %MVF: percent minor variant frequency; ddPCR: droplet digital PCR; NGS: next-generation sequencing

Figure 3. Detection of del19 driver and T790M TKI resistance mutations compared to C797S osimertinib resistance mutation (%MVF) over 540-day osimertinib treatment cycle. Note the day-164 specimen was unavailable for testing for C797S because the sample was exhausted. Insets: patient PET-CT scan on days 385 and 540 (showing disease progression). EGFR: epidermal growth factor; %MVF: percent minor variant frequency; TKI: tyrosine kinase inhibitor

Discussion

As hypothesized, the detected emergence of C797S mutations correlated with clinical signs of treatment resistance. Interestingly, while signals for the del19 driver mutation and T790M resistance mutations were detected on initiation of osimertinib therapy (July 2016), and subsequently declined (August through December 2016), both re-emerged and with greater detectable signal than C797S [Figure 3]. However, while the detection of the del19 variant reached a steady state and T790M decreased as disease progressed, the levels of the C797S variant in circulation continued to rise. We hypothesize that these results indicate the emergence, over time, of a clonal C797S variant sub-population. It is noteworthy that while the C797S mutation allele frequency increased at later timepoints, the T790M frequency declined across those same timepoints. There could be several reasons for this observation. One possibility is that osimertinib treatment results in specific killing of T790M only cells, which contributes to the decline in detectable T790M signal, but not for C797S. Additionally, the kinetics of death and active nucleic acid shedding by cells are complex and are mostly for monitoring purposes at this time. In any event, given this patient’s young age, had these data been available to aid treatment decision in real-time and based on the cis/trans nature of the T790M and C797S mutation, there might have been an opportunity to implement a first-generation TKI, with hope for recaptured disease response and control, instead of treatment cessation^[5].

The results of this study, using blinded case specimens, show that highly sensitive ddPCR methods can be used for the monitoring of emergent EGFR somatic variant mutations in circulation. In this case study, we have demonstrated that this method has potential clinical utility for detecting and monitoring osimertinib treatment resistance via the C797S escape mechanism.