PD-L1 expression in cell-blocks of non-small cell lung cancer: The impact of prolonged fixation

Introduction: In the selection of non-small cell lung cancer (NSCLC) patients for immunotherapy, specimen processed as cell blocks (CBs) may be the only available material to assess PD-L1 expression. Therefore, optimal CB preparation becomes paramount. In this context, here we assessed whether inadequate fixation time might be one of the pre-analytical factors affecting PD-L1 expression. Methods: Ex vivo CBs from placental (n = 3) and NSCLC (n = 8) resection specimens were obtained. PD-L1 staining was performed on CBs prepared at increasing fixation times (12 hours, 48 hours, 72 hours, 96 hours, 168 hours and 504 hours) using the companion diagnostic SP263 Assay and a validated 22C3 laboratory developed test (LDT). Staining intensity and percentage of positive cells were evaluated. Results: All placental CBs showed moderate to strong PD-L1 positivity in most cells, regardless of the fixation time. Likewise, the percentage of SP263-stained NSCLC cells was similar at all fixation times except for one case, which showed less intense SP263 staining at 168 hours. Conversely, in 5/8 cases, the 22C3 LDT percentage of positive cells and staining intensity decreased at 168 hours and 504 hours. Conclusions: Our results show that fixation time influences the performance of 22C3 LDT on CBs. Thus, we recommend that the fixation time of cytological materials be carefully checked, especially when PD-L1 testing is delayed until the oncology request. Indeed, delays in tissue processing and paraffin embedding may lead to sub-optimal performance of PD-L1 staining on CBs.