Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.
Department of Pathology, Isala Hospitals, Zwolle, the Netherlands.
Cancer Cytopathol. 2021 Apr;129(4):304-317. doi: 10.1002/cncy.22383. Epub 2020 Oct 27.
Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied.
Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration.
Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay.
Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.
程序性死亡配体 1（PD-L1）的免疫组化染色用于确定哪些非小细胞肺癌（NSCLC）患者最有可能从免疫治疗中获益。许多 NSCLC 患者的治疗管理基于细胞学而不是组织学。在这项研究中，分析了细胞学细胞块与组织学对应物之间 PD-L1 免疫染色的一致性。此外，还研究了各种固定剂和固定时间对 PD-L1 免疫反应性的影响。
通过对肺切除术/肺叶切除术标本进行细针穿刺，收集了 67 例 NSCLC 患者的配对组织学和细胞学样本。使用福尔马林固定、琼脂基或 CytoLyt/PreservCyt 固定的 Cellient 细胞块制备。从细胞块和组织块的切片用 SP263（标准化检测）和 22C3（实验室开发的检测）抗体染色。比较组织学和细胞学之间的 PD-L1 评分。此外，还比较了在不同固定剂中固定时间逐渐增加的情况下，表达 PD-L1 的人细胞系的免疫染色。
琼脂细胞块和组织块显示出实质性的一致性（κ=0.70 和 κ=0.67），而 Cellient 细胞块与组织学之间则存在适度至良好的一致性（κ=0.28 和 κ=0.49）。与福尔马林相比，用各种醇基固定剂固定的细胞系显示出较低的 PD-L1 免疫反应性。与 SP263 不同，在用醇固定后再用福尔马林固定，使用 22C3 实验室开发的检测和 22C3 pharmDx 检测可保留更高和更与组织学一致的染色强度。
在醇基固定剂固定的细胞学标本上进行 PD-L1 染色可能导致免疫染色结果为假阴性，而福尔马林固定则导致更高和更与组织学一致的 PD-L1 免疫染色。通过在福尔马林后再次固定，可以在一定程度上逆转醇固定的有害影响。
