br Methods An ongoing prospective study at the Princess Marg
Methods An ongoing prospective study at the Princess Margaret Cancer Centre is enrolling consenting patients with stage IV EGFRm NSCLC for longitudinal blood collection. Patients may enrol at any point in their treatment. In this study, we performed a cross-sectional analysis of 72 patient liquid biopsies using an NGS panel assessing regions of 11 doripenem receptor (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, TP53) and >150 hotspots with a limit of detection (LOD) down to 0.1% (Oncomine™ Lung cfDNA Assay; Thermofisher). Conduct of this study was approved by the University Health Network Research Ethics Board.
Discussion As the treatment landscape in EGFRm NSCLC continues to evolve, resistance mechanisms are becoming better understood. More comprehensive analysis of ctDNA not only overcomes limitations of single site tissue biopsies but may also have an important role in longitudinal analyses, both in monitoring for clearance of primary EGFR mutations but also for the emergence of potential subclones and acquired mutations. While the main focus of research to date has been on the detection of T790M in ctDNA, our analyses show that when stable on treatment with an EGFR-TKI, the presence not only of T790M but other EGFR mutations, including co-occurring mutations, predicts a shorter progression free survival. This finding is supported by recent data from AURA3, suggesting that the persistence of an EGFRm results in an inferior response to osimertinib . Also, in the small group of patients receiving a TKI in our study the percentage variant allele fraction of the EGFRm also predicted time to confirmed progression. Others studies have shown that the % allelic fraction can impact treatment outcomes and survival  although this requires validation in well conducted large trials. Furthermore, it remains unknown as to whether the detection of resistance mechanisms prior to confirmatory RECIST progression or the rise in mutation allelic fraction should prompt a change in systemic treatment. The APPLE trial (NCT02856893) will evaluate sequencing of gefitinib and osimertinib and provides an opportunity to assess the impact of switching treatment based on the detection of T790M in ctDNA. Multi-gene NGS panels (ThermoFisher Oncomine™ Lung ctDNA Research Assay in this study) provide a practical method to characterise resistance mechanisms and to detect variants in multiple genes relevant in NSCLC including KRAS, PIK3CA, TP53 and ALK. A PIK3CA mutation was detected in the presence of T790M in a patient receiving a 3rd generation TKI. Chabon et al using CAPP-seq identified this mutation as a resistance mutation and this has been supported by more recent reports [3,9]. A de novo KRAS mutation was discovered in one patient who had a PFS of 14 months on gefitinib. While some studies suggest EGFR and KRAS mutations are mutually exclusive, others have shown that their co-occurrence does not impact treatment response . The major limitation in this study is the analysis of ctDNA at a single time point in different patients with EGFRm NSCLC. However given our findings, we do show the potential utility in ctDNA analysis at any time point in EGFRm NSCLC.
Conclusion The presence of any EGFR mutation including co-occurring mutations in ctDNA may predict a shorter progression free survival interval. The evaluation of changes in percentage variant allele fraction may be an important biomarker of disease progression and warrants further evaluation.
Conflict of interest
Acknowledgements This project was supported by the Princess Margaret Cancer Foundation, including through the OSI Pharmaceuticals Foundation Chair (NBL) and the Alan B. Brown Chair (GL).
Introduction Lung adenocarcinoma is the most prevalent type of pulmonary malignancy, accounting for approximately half of lung cancer cases [, , , ]. A high percentage of adenocarcinomas harbour known driver molecular aberrations, some of which are therapeutically targetable. This is the case of EGFR activating mutations, accounting for up to 20% of lung adenocarcinoma cases in Caucasian cohorts and in up to 30–50% of patients of Asian origin [5,6]. The development of small molecule inhibitors targeting the EGFR receptor has improved treatment outcomes in patients with tumors possessing these mutations [, , , ]. However, the use of such inhibitors is currently restricted to adenocarcinomas with known EGFR activating mutations. Thus, tumors exhibiting high constitutive EGFR activation mediated by either unknown EGFR mutations or by other molecular mechanisms, which may also benefit from these therapies, are not candidates for treatment with EGFR inhibitors. Furthermore, not all tumors harbouring known EGFR-activating mutations respond to EGFR inhibitors [3,, , ]. Therefore, the discovery of novel biomarkers predicting EGFR therapy efficacy, as well as the identification of therapies achieving higher efficacy against EGFR signalling-dependent tumors is an unmet clinical need.