Department of Pathology, University Medical Centre Utrecht, Utrecht University, Utrecht, the Netherlands.
Department of Pathology, Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands.
Histopathology. 2021 Oct;79(4):480-490. doi: 10.1111/his.14373. Epub 2021 Jun 8.
Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation.
FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results.
Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.
程序性死亡配体 1（PD-L1）免疫组化用于预测哪些非小细胞肺癌（NSCLC）患者对程序性死亡蛋白 1/PD-L1 抑制剂的治疗反应最佳。PD-L1 免疫组化有时在酒精固定的细胞学标本上进行，而不是在福尔马林固定石蜡包埋（FFPE）活检或切除标本上进行。我们研究了与福尔马林固定相比，经支气管内超声引导经支气管针吸活检（EBUS-TBNA）的血凝块用乙醇预处理是否会导致 PD-L1 免疫组化减弱。
鉴定了 54 例 NSCLC 患者的 EBUS-TBNA 标本的 FFPE 细胞块。对于每个病例，都有配对的样本，包括直接浸入福尔马林的血凝块和在 Fixcyt（50%乙醇）中预固定的血凝块。使用标准化 SP263 检测法和 22C3 抗体（实验室开发的测试（LDT））对 PD-L1 进行免疫组织化学染色。使用两个截断值（1%和 50%）确定 PD-L1 阳性。评估福尔马林固定（金标准）和乙醇预固定材料之间 PD-L1 阳性的一致性。当使用 22C3 LDT 时，分别有 30%和 36%的乙醇预固定标本在 1%和 50%截断值时显示假阴性结果（kappa 0.64 和 0.68）。当使用 SP263 时，22%的乙醇预固定标本在 1%截断值时显示假阴性结果（kappa 0.67）。在 50%截断值时，一致性更高（kappa 0.91），有 12%的乙醇预固定标本显示假阴性结果。
在使用 SP263 或 22C3 LDT 进行免疫组化染色时，使用 1%截断值时，EBUS-TBNA 标本在福尔马林固定之前用乙醇固定可导致相当数量的 PD-L1 免疫组化假阴性结果。当使用 22C3 LDT 进行免疫组化染色时，使用 50%截断值也是如此。
