Comparison between Immunocytochemistry, FISH and NGS for ALK and ROS1 Rearrangement Detection in Cytological Samples

Abstract

The detection of ROS1 and ALK rearrangements is performed for advanced-stage non-small cell lung cancer. Several techniques can be used on cytological samples, such as immunocytochemistry (ICC), fluorescence in situ hybridization (FISH) and, more recently, next-generation sequencing (NGS), which is gradually becoming the gold standard. We performed a retrospective study to compare ALK and ROS1 rearrangement results from immunocytochemistry, FISH and NGS methods from 131 cytological samples. Compared to NGS, the sensitivity and specificity of ICC were 0.79 and 0.91, respectively, for ALK, and 1 and 0.87 for ROS1. Regarding FISH, the sensitivity and specificity were both at 1 for ALK and ROS1 probes. False-positive cases obtained by ICC were systematically corrected by FISH. When using ICC and FISH techniques, results are very close to NGS. The false-positive cases obtained by ICC are corrected by FISH, and the true-positive cases are confirmed. NGS has the potential to improve the detection of ALK and ROS1 rearrangements in cytological samples; however, the cost of this technique is still much higher than the sequential use of ICC and FISH.

Keywords: ALK; FISH; NGS; ROS1; adenocarcinoma; cytology; immunocytochemistry; lung cancer.

Figure 1

Example of malignant cells with or without ALK and ROS1 rearrangements. (A–D) Represent the cytology of pleural effusion (P1) containing ALK rearranged malignant cells. (A) Papanicolaou stain, (B) May–Grünwald–Giemsa (MGG) stain, (C) immunocytochemistry (ICC) (peroxidase staining) using antibody against ALK (clone 5A4), (D) ALK FISH probe. (E–H) Represent the cytology of pleural effusion (P2) containing ROS1 rearranged malignant cells. (E) Papanicolaou stain, (F) May–Grünwald–Giemsa stain, (G) immunocytochemistry (peroxidase staining) using antibody against ROS1 (clone D4D6), (D) ROS1 FISH probe. (I–L) Represent the cytology of a lymph node (P3) containing ALK rearranged malignant cells. (I) Papanicolaou stain, (J) May–Grünwald–Giemsa stain, (K) immunocytochemistry (peroxidase staining) using antibody against ALK (clone 5A4), (L) ALK FISH probe. Of note, the false-negative result was obtained with ICC, whereas the rearrangement is observed with FISH. (M–R) Represent the cytology of pleural effusion (P4) containing malignant cells without ALK and ROS1 rearrangements. (M) Papanicolaou stain, (N) May–Grünwald–Giemsa stain, (O) immunocytochemistry (peroxidase staining) using antibody against ALK (clone 5A4), (P) ALK FISH probe. (Q) Immunocytochemistry using antibody against ROS1 (clone D4D6), (R) ROS1 FISH probe. Of note, the false-positive results were obtained with ICC using antibodies against ALK and ROS1, whereas the FISH shows more than two signals without rearrangement using ALK and ROS1 probes. (S) H2228 cell line showing positive staining against anti-ALK antibody (positive control, C+). (T) HCC78 cell line showing a positive staining against anti-ROS1 antibody (positive control, C+). Black scale bar represents 20 µm. White scale bar represents 10 µm.

Figure 2

A Venn diagram of ALK-positive (A) and ROS1-positive (B) cases detected by NGS, ICC and FISH in cytological samples. Four patients and one patient were excluded for ALK or ROS1 results, respectively, because of non-interpretable results in one of the methods.