Comparison of Different ROS1 Immunohistochemistry Clones and Consistency with Fluorescence In Situ Hybridization Results in Non-Small Cell Lung Carcinoma

Background: The study of ROS1 rearrangement in non-small cell lung carcinoma (NSCLC) has gained importance as it enables personalized treatment of NSCLC with tyrosine kinase inhibitors. Therefore, it is important that the ROS1 assessment tests become more standardized. In this study, we compared the two immunohistochemistry (IHC) antibodies (D4D6 and SP384 clones) and consistency with the fluorescence in situ hybridization (FISH) results in NSCLC. Aims: To investigate the effectiveness of the commonly used two IHC antibodies (SP384 and D4D6 clones) to detect ROS1 rearrangement in NSCLC. Study Design: A retrospective cohort study. Methods: The study included 103 samples diagnosed with NSCLC, confirmed using IHC and FISH ROS1 results (14 positives, four discordant, and 85 consecutive negatives), with sufficient tissue samples (≥ 50 tumor cells). All samples were initially tested with ROS1-IHC antibodies (D4D6 and SP384 clones); their ROS1 status was then analyzed using the FISH method. Finally, samples with discordant IHC and FISH results were confirmed using the reverse transcription polymerase chain reaction method. Results: The sensitivity of SP384 and D4D6 clones of ROS1 antibody was 100% with a ≥ 1 + cut-off. When the ≥ 2 + cut-off was used, the sensitivity rate for the SP384 clone was 100%, whereas the sensitivity for the D4D6 clone was 42.86%. ROS1 FISH rearranged samples were positive for both clones, but SP384 had generally higher intensity than D4D6. The mean IHC score was + 2 for SP384 and + 1.17 for D4D6. SP384 mostly tended to have a higher IHC score intensity, which made the evaluation easier than D4D6. SP384 has a higher sensitivity than D4D6. However, false positives were found in both clones. There was no significant correlation between ROS1 FISH-positivity percentage with SP384 (p = 0.713, p = 0.108) and D4D6 (p = 0.26, p = -0.323) IHC staining intensity. The staining patterns of both clones were similar (homogeneity/heterogeneity). Conclusion: Our findings show that the SP384 clone is more sensitive than D4D6. However, SP384 can also cause false positive results like D4D6. Knowing the variable diagnostic performance of different ROS1 antibodies before using them in clinical applications is necessary. IHC-positive results should be confirmed using FISH.

Concordance of PD-L1 expression and CD8+ TIL intensity between NSCLC and synchronous brain metastases.

Programmed death-ligand 1 (PD-L1) is suggested to be a predictive biomarker in non-small-cell lung carcinoma (NSCLC). However, the differential expression of PD-L1 in primary lung tumor vs. synchronous metastases, especially brain metastasis (BM), remains unclear. This study assessed the concordance of PD-L1 expression on tumor cells and tumor-infiltrating lymphocytes (TILs) and CD8+ TIL intensity between primary lung tumors and synchronous BMs from 24 NSCLC patients. PD-L1, CD3, and CD8 positivity was determined by immunohistochemistry (IHC). PD-L1 scoring was based on the proportion of tumor cells with membranous expression of PD-L1 and the cutoff values <1%, 1-49%, and ≥50%. CD3 and CD8 positivity in TILs was evaluated semi-quantitatively and the proportion of CD3+/CD8+ TILs was determined. PD-L1 expression on tumor cells and TILs was evaluated in relation to CD3+/CD8+ TIL proportions and the intensity of CD8+ TILs between the paired primary lung and BM tissues. In the primary lung tumors, PD-L1 positivity was observed in 25%, 37.5%, and 37.5% cases for the cutoff values <1%, 1-49%, and ≥50%, respectively. PD-L1 expression on tumor cells was strongly correlated between the paired primary lung and BM tissues, in all cutoff groups. However, PD-L1 expression on TILs and the proportion of CD3+/CD8+ TILs were not strongly correlated in all three groups between the paired primary lung tumors and BMs. The intensity of CD8+ TILs was concordant in only 54.16% of the paired primary lung tumors and BMs. This study showed a high concordance of PD-L1 expression in neoplastic cells between primary NSCLC and synchronous BMs.