Immunocytochemistry for predictive biomarker testing in lung cancer cytology

Abstract

With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers. In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.

Keywords: cell blocks; immunocytochemistry; lung cancer; predictive; smears.

Figure 1.

ALK immunocytochemistry images of ALK re-rearranged pulmonary adenocarcinomas on previously Papanicolaou-stained non-CB cytology. (A, B) Homogeneously positive tumor cells with admixed ALK-negative benign cells. A laboratory-developed test using 5A4 antibody (Novocastra) was performed on an automated immunostainer (Leica Bond), and AEC (3-amino-9-ethylcarbazole) was used as a chromogen (A, magnification ×200; B, magnification ×400). (C, D) CB specimen stained with the Ventana ALK (D5F3) CDx Assay (C, magnification ×400) and ALK 5A4 antibody (D, magnification ×400) on the Ventana BenchMark system using 3,3’-diaminobenzidine (DAB) as a chromogen.

Figure 2.

Two immunocytochemistry images of ROS1 re-rearranged pulmonary adenocarcinomas using D4D6 antibody (Cell Signaling). (A) Cell block specimen on the Ventana BenchMark system using 3,3’-diaminobenzidine (DAB) as a chromogen (magnification ×400). (B) Cell block specimen on previously Papnicolaou-stained non-CB cytology using Leica Bond system and AEC (3-amino-9-ethylcarbazole) as chromogen (magnification ×400).

Figure 3.

PD-L1 immunocytochemistry images of non-CB cytology specimens. (A, B, E, F) NSCLC with almost all tumor cells positive for PD-L1. Both membranous as well as diffuse staining is evident. (C, D) PD-L1–negative tumor cells with macrophages serving as an internal positive staining control. (A-D) Laboratory-developed test using concentrated SP142 antibody on Leica Bond. (E, F) SP263 IHC assay on Ventana Benchmark XT (E, magnification ×400; F, magnification ×200).

Figure 4.

PD-L1 immunocytochemistry images of CB specimens. (A, B) NSCLC with all tumor cells being positive for PD-L1 (hematoxylin and eosin and PD-L1, magnification ×200). (C) PD-L1–negative aggregate of adenocarcinoma cells and adjacent histiocytes, some of which are weakly PD-L1–positive (magnification ×400). (D) NSCLC with most tumor cells being PD-L1–positive (magnification ×200). (B-D) Laboratory-developed tests using DAKO 22C3 on Ventana BenchMark. (E, F) PD-L1–positive NSCLC by Ventana PharmDx Assay on BenchMark (magnification ×400).