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

Abstract

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.

Evaluation of the Molecular Landscape in PD-L1 Positive Metastatic NSCLC: Data from Campania, Italy

“…In this study, we retrospectively analyzed a total of 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our referral clinic for the molecular evaluation of at least five driver genes, namely, EGFR , KRAS , BRAF , ALK and ROS1 . In our experience, both histological ( n = 110, 65.9%) and cytological ( n = 57, 34.1%) samples were analyzed, strongly supporting the evidence that evaluation of PD-L1 expression levels and molecular profiling of advanced stage NSCLC patients is feasible by using both types of specimens [ 21 , 22 , 29 , 30 ]. In this context, studies have shown that NGS (both DNA- and RNA-based approaches) represents a valid solution to analyze clinically relevant biomarkers simultaneously in small tissue samples [ 32 , 33 ].…”

“…PD-L1 IHC/ICC evaluation was performed with a validated laboratory developed test (LDT), consisting of the use of Dako’s concentrate 22C3 anti-PD-L1 primary antibody with a Ventana’s detection systems on the BenchMark XT platform, or by using the companion diagnostic kit SP263 assay (Ventana Medical Systems, Tucson, AZ, USA) [ 29 , 30 ]. The level of PD-L1 expression was determined by using tumor proportion score (TPS).…”

“…Indeed, evaluating PD-L1 expression levels and the genomic assessment of clinically relevant oncogenic targetable drivers would be crucial to broaden the treatment options for NSCLC patients. In our referral laboratory experience at the Molecular Predictive Pathology Laboratory at the Department of Public Health of the University of Naples Federico II, we routinely perform immunohistochemistry/immunocytochemistry (IHC/ICC) to evaluate PD-L1 expression [ 29 , 30 ]. In addition, we perform both DNA-based next generation sequencing (NGS) and fully automated real-time polymerase chain reaction (RT-qPCR), namely, Idylla™ (Biocartis, Mechelen, Belgium) to evaluate point mutations, deletions and insertions [ 31 , 32 , 33 ] and IHC/ICC and RNA-based NGS analysis to identify gene fusions [ 34 ].…”

Tissue Microarray from Cell Block Material (cbTMA)—An Additional Shot for Cytology in the Predictive Pathology Era: The PD-L1 Experience

“…CBs were prepared using the Shandon Cytoblock Cell Block Preparation System (Thermo Scientific, Waltham, MA) according to the manufacturer's instructions, as previously described [15]. The original hematoxylin and eosin (H&E)-stained slides were examined by two different pathologists to outline the area of interest with respect to representative cellularity.…”

PD‐L1 and beyond: Immuno‐oncology in cytopathology

“…Indeed, CB preparatory techniques may vary significantly depending on several factors, that is, the choice of the fluid medium used for the FNA needle rinse (formalin, saline or alcohol‐based fixatives followed by formalin post‐fixation), the fixation time, and the method of concentration. 19 , 21 , 22 , 23 , 24 Despite the lack of standardized preparation protocols, several lines of evidence have demonstrated that the type of fixative does not affect PD‐L1 staining. In fact, Wang et al 21 observed that fixation with formalin only, methanol/alcohol only, or both did not affect PD‐L1 expression.…”