Metaphase FISH with LSI ALK probe (A) and subsequent R-banding analysis (B) shows insertion of genomic material into theALK locus indicated by spatial separation of the red signal for 3′ALK (telomeric) and the green signal for 5′ALK(centromeric) on the short arms of both der(2) chromosomes (arrows) as compared with the intact chromosome 2 (arrowhead). (C) In interphase nuclei of tumor cells, insertion into the ALK locus on both derivative chromosome 2 leads to separation of 3′ and 5′ALKsignals in addition to each 1 colocalized, indicating the intactALK locus. (D) In contrast to the larger nucleus of the tumor cells, the smaller nuclei of nonneoplastic cells show 2 colocalized signals for 3′ and 5′ALK, which is the regular signal constellation for 2 intact chromosomes 2. (B, small picture) FISH with a chromosome 1 painting probe on R-banded chromosomes shows a major part of the short arm of the der(2) chromosomes to derive from chromosome 1. (E) RT-PCR amplifies a PCR product of approximately 300 bp specific for TPM3-ALK fusion (Lane 2). Lane 1, 100-bp ladder; lane 3: H₂O control.