Background: The treatment of patients with advanced NSCLC has become reliant on tissue specimens and biomarkers to help guide appropriate treatment options. Currently, there are numerous lung cancer biomarker-defined patient subgroups, with evidence showing that fixation conditions may alter their expression on FFPE specimens, but little evidence on the ensemble of targetable biomarkers and CKI targets. Methods: We assessed 30 adult patients with primary NSCLC tumors. 25 specimens were properly fixed with neutral 10% formalin using gentle agitation for 24-48 hours, whereas 5 specimens were over-fixed (52-108 hours). We examined expression of actionable and exploratory biomarkers ALK, ROS1, BRAF, EGFR, c-Met, panTRK, HER2 and MEK1. We also examined CKI targets such as PDL1, CD3/CD8, CD3/CD8/FoxP3, PD1, CD3/CD8/PD1. We performed IHC on all slides which were scored by a thoracic pathologist per standard clinical practice. Descriptive statistics were applied to further explore the impact of overfixation on IHC results. Results: Results are shown in the table below. Patients had an average age of 64, were mostly male (22/30) with adenocarcinoma histology (21/30). Specimens were collected in compliance with local rules and regulations and tumor staging was from IB-IV. For the 5 overfixed specimens, multiplex PD-L1 was less expressed when compared to the normally fixed specimens. We also surprisingly observed that cMet and HER2 had stronger expression with overfixed tissue. All other targets had no differences observed in this small patient population. Conclusions: Overfixation of NSCLC FFPE had little to no detrimental impact on most targetable biomarkers, except multiplex PD-L1. We also observed that cMet and HER2 had a slightly stronger expression level with overfixed tissue. As we do not have a reference of the overfixed samples fixed from 24-48 hrs, we cannot be assured that the differences in expression levels are not inherent to the sample itself and independent of fixation time. Additional tests of the same sample fixed over different periods will help to identify the true cause. We propose a consistent approach on tissue fixation time and process as the ensemble of preanalytical conditions is critical in ensuring proper biomarker characterization and validation with IHC.