Frequencies of ALK and ROS in NSCLC FFPE tumor samples utilizing a highly specific immunohistochemistry-based assay and FISH analysis.

Abstract

10536 Background: Receptor tyrosine kinases ALK and ROS are expressed as oncogenic fusion proteins in approximately 2%-5% of all non-small cell lung cancer (NSCLC) patients. Most recently, ALK targeted therapies have been shown to be effective in ALK positive patients. This may also be true for ROS but is less validated. The ability to identify patients that harbor these fusion proteins is desirable. Currently, molecular assays such as fluorescence in situ hybridization (FISH) and RT-PCR are used to identify these patients. We reasoned that an immunohistochemistry (IHC)-based assay could provide a fast, inexpensive alternative for diagnosing patients with ALK and ROS abnormalities. We have developed an IHC-based assay for the detection of low level ALK and ROS in NSCLC patients with high specificity and sensitivity. Methods: We generated highly specific rabbit monoclonal antibodies against ALK and ROS. ALK and ROS antibodies were evaluated in a panel of 656 NSCLC FFPE tumor tissues. The majority of tissues (n = 409) were analyzed in tissue microarray format while the remaining (n = 247) were whole tissue sections. IHC positive tissues were also analyzed by FISH to compare protein expression to the presence of ALK and ROS rearrangements. Results: Western blot, IHC and FISH experiments confirm that our monoclonal antibodies can specifically detect low levels of both ROS and ALK in NSCLC tumor samples. Approximately, 4.1% (27 cases) of NSCLC cases analyzed were ALK positive by IHC. We were able to confirm these results by FISH in 25/27 cases. The remaining 2 cases we were unable to score due to poor tissue quality. We identified 2.6% (17/656) of the NSCLC tissues to be ROS positive by IHC. We confirmed these results in 16/17 tissues by FISH analysis. Due to technical issues, we were unable to score the remaining tissue. Both the ALK and ROS antibodies are highly specific. We are currently performing additional studies to determine sensitivity. ALK and ROS were never observed in the same tumor sample. Conclusions: We conclude that IHC staining with ALK and ROS monoclonal antibodies is a highly specific assay that reliably identifies aberrant ALK and ROS protein expression in NSCLC patients. No significant financial relationships to disclose.