Detection of antibodies to ALK proteins in patients with ALK-positive ALCL.

(A) Immunoperoxidase labeling of transfected COS-1 cells. Left panel: Plasma from a patient with ALK-positive ALCL (diluted 1:250) strongly labeled COS-1 cells expressing NPM-ALK (arrow). This pattern of labeling is comparable to that obtained with monoclonal anti-ALK (inset). Central panel: Strong staining by this plasma is also seen in COS-1 cells expressing full-length ALK protein. In contrast, no labeling is seen in COS-1 cells that have been transfected with the “empty” pcDNA3 vector (inset). Right panel: No staining is observed when plasma from a control subject is tested on COS-1 cells expressing either NPM-ALK or ALK. (B) Detection of antibodies to NPM-ALK by biochemical analysis. Left panel: Cell protein extracts were immunoprecipitated with plasma from 3 ALK-positive ALCL patients (numbers 1-3) and subjected to an in vitro kinase assay. A strongly autophosphorylated 80-kd protein is observed in immunoprecipitates of the t(2;5)–positive cell line SU-DHL-1 identical in size to that seen in immunoprecipitates prepared with the control monoclonal anti-ALK antibody. No corresponding phosphorylated proteins are present in the tonsil preparations. Comparable results were obtained with the use of plasma from all the other ALK-positive ALCL patients (not shown). Right panel: The control subjects comprise 5 normal patients (12-16), 5 patients with carcinoma (17-21), and 3 patients with ALK-negative ALCL (22-24). A weak 80-kd band (arrowed) is precipitated from SU-DHL-1 lysates by plasma from 3 control subjects (13, 17, and 24), but the other controls are negative.