Assessment of the ALK Antibody, 5A4 in Detecting ALK Rearrangments in Non-Small Cell Lung Cancer Specimens
John Glassco, Ainura Kyshtoobayeva, Kenneth J Bloom. Clarient, A GE Healthcare Company, Aliso Viejo, CA
Background: Translocations involving the kinase domain of the anaplastic lymphoma kinase (ALK) gene have been identified in a number of different malignancies including non-small cell lung cancer (NSCLC). Although ALK has been demonstrated to fuse with a number of different genes, the echinoderm microtubule-associated protein-like 4 (EML4) gene is commonly associated with ALK in NSCLC. Crizotinib was recently approved for the treatment of ALK positive NSCLC patients as determined by the presence of an ALK gene rearrangement based on a companion diagnostic FISH assay. We sought to determine if a commercially available ALK antibody could identify tumors assessed as ALK positive by the approved FISH assay.
Design: 100 formalin fixed embedded tissue sections, 50 from tumors assessed as ALK positive by FISH and 50 tumors assessed as ALK negative by FISH were pulled from our archives. 4 micron sections were stained with a monoclonal antibody, 5A4 (Leica, Buffalo Grove, Il) 1:50, following heat-induced epitope retrieval, 100o C, pH9, 20 minutes. FISH was performed using the Abbott LSI ALK break apart probe. 50 tumor cells were enumerated for the presence of a break apart signal which was considered as present when at least one set of orange and green signals were 2 or more signal diameters apart or when a single orange signal without a corresponding green signal was observed in more than 15% of tumor cells.
Results: 41 (82%) of the 50 tumors assessed as ALK positive by FISH showed expression of ALK protein with the 5A4 clone ranging from 1+ to 3+ intensity. The remaining 9 tumors showed no expression with the ALK antibody. All 50 tumors assessed as ALK negative by FISH showed no expression of ALK protein.
Conclusions: When ALK expression was noted with the 5A4 clone, the tumor was assessed as ALK positive by FISH; however, 9 (18%) of the tumors assessed as ALK positive by FISH lacked expression of the ALK protein. The 5A4 clone lacks the sensitivity to replace the FISH assay in clinical practice based on this series.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 306, Tuesday Morning