Representative cases of IHC staining, FISH and qRT-PCR analysis in lung adenocarcinoma. (A-C) ALK IHC staining using CST’s D5F3 antibody. (A) Cytoplasmic reactivity of strong intensity in tumor cells (original magnification, x40). (B) Weak to moderate cytoplasmic reactivity in tumor cells (original magnification, x100). (C) No staining in tumor cells (original magnification, x200). (D-F) FISH analysis using Vysis ALK Break-Apart probes. (D) The ALK+ case in which the majority of cells contained more than one copy of a single green signal without a corresponding orange signal in addition to fused signals using FISH analysis. Green arrow represents more than one copy of a single green signal, red arrow represents single red or split red-green signals indicative of ALK-rearrangement and yellow arrow represents touching red-green signals not indicative of ALK-rearrangement. (E) ALK+ case with split red-green signals. (F) NSCLC case without ALK rearrangement. (G-I) VENTANA ALK (D5F3) IHC assay revealed no expression in ALK- patients and strong expression in ALK+ patients. (G) Strong ALK expression (original magnification, x20). (H) Unspecific staining (original magnification, x40). (I) No ALK expression (original magnification, x20). (G-L) Graphs from qRT–PCR showing change in the normalized reporter signal (delta Rn) against PCR cycle number. (J) ALK fusion was detected at around 14 cycles of qRT-PCR analysis in a case with strong ALK expression. (K) ALK fusion was detected at around 28 cycles in a case with weak ALK expression. (L) No ALK fusion was detected with endogenous control gene, beta-actin, expressed normally.