Analysis of ALK protein expression in ALCL and IMT. (A) (Upper Left) Giemsa stain of ALCL cell line showing large neoplastic cells with large nuclei and abundant pale cytoplasm. (Lower Left) Immunocytochemistry for ALK in ALCL showing nuclear and cytoplasmic distribution of ALK protein. (Upper Right) Hematoxylin/eosin (H&E) of IMT tissue biopsy showing a spindle cell neoplasm. (Lower Right) Exclusive cytoplasmic reactivity for ALK in the neoplastic cells of the IMT tissue sample. (B) Immunoblot analysis of SUDHL-1 cell lysate and IMT tissue lysate using the anti-ALK antibody. The reported NPM–ALK protein was seen in the SUDHL-1 control cell line at 80 kDa, whereas an unknown ALK fusion protein was seen in the IMT tissue lysate at 104 kDa. (C) Immunoprecipitation of SUDHL-1 cell lysate and IMT tissue lysate using anti-ALK antibody. Lysates were incubated with ALK antibody, and the antibody selected proteins were resolved by SDS/PAGE. After silver staining, the ALK fusion protein was seen in the IMT lysate pull-down lane at 104 kDa. The IgG control lane and the SUDHL-1 lane containing the 80-kDa NPM–ALK are shown for comparison.

Mass spectra of tryptic peptide K.CGSGPVHISGQHLV-VYRRK identifying the NPM–ALK fusion.

Fusion mapping of the NPM–ALK protein by MS/MS. The strategy of combining peptide sequences generated by multiple enzyme digests yields enhanced coverage of the partner proteins involved in the chimeric fusion (82.6%). Blue, trypsin; green, lysine-C; orange, glutamic-C; pink, proteinase K.