1. Volume 204, Issue 1, Pages 45-52 (January 2011)
Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer 
Heather R. Sanders, Hai-Rong Li, Jean-Marie Bruey, Jay A. Scheerle, Aurelia M. Meloni-Ehrig, JoAnn C. Kelly, Constance Novick, Maher Albitar 
Cancer Genetics 
Volume 204, Issue 1, Pages (January 2011)
DOI: /j.cancergencyto
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
Fragment analysis and FISH staining in the specimen positive for the novel EML4–ALK fusion variants 8a and 8b. (A) RNA from FFPE tissue was amplified by RT-PCR using unlabeled EML4 exon 17 forward primer and FAM-labeled ALK exon 20 reverse primer. Two peaks were observed, at positions 171 bp and 238 bp, indicating the presence of at least two fusion transcript variants (the actual nucleotide length of variant 8b is 236 bp, slightly shifted from the observed peak position due to mobility shift differences of some primer/PCR products labeled with FAM). (B) FISH using break-apart probes for ALK revealed abnormalities. The normal allele shows cohybridization of 5′ (green) and 3′ (red) ALK probes, as demonstrated by overlapping signal (yellow) in the pseudo-colored FISH image (arrowhead). The second allele shows split signal from 5′ and 3′ ALK probes (arrows), demonstrating presence of an ALK rearrangement.
Cancer Genetics  , 45-52DOI: ( /j.cancergencyto )
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 2
Sequencing of transcript fusion sites in novel EML4–ALK fusion variants 8a and 8b with electropherograms from reverse sequencing reactions (A,B) and with representation of fusion transcripts (C). (A) The 171-bp peak consisted of a complete EML4 exon 17 and ALK exon 20 separated by 30 bp of adjacent ALK intron 19–20 sequence. (B) The larger product, 238 bp, also contained a complete EML4 exon 17, ALK exon 20, and adjacent ALK intron 19–20 with an added 4 bp (boxed) of that intron. It also contained 60 bp of nonadjacent EML4 intron 17–18 sequence separating the EML4 exon 17 and ALK intron sequence. (C) Fusion transcripts resulted from an EML4 (green) and ALK (red) genetic rearrangement. The precise gene breakpoints were not identified in the present study; dotted borders indicate intronic and exonic sequences that appear to be carried over to the fusion transcripts.
Cancer Genetics  , 45-52DOI: ( /j.cancergencyto )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 3
Putative proteins formed as a result of EML4–ALK variant 8a and 8b fusions. (A) Amino acid sequence of the putative truncated EML4 protein resulting from EML4–ALK fusion transcript variant 8a. EML4 residues are shown in black, intron-derived residues in blue. (B) Amino acid sequence of variant 8b putative EML4–ALK fusion protein. EML4 residues are shown in black, intron-derived residues in blue, and ALK residues in red. (C) Putative EML4 truncation containing the N-terminal region of EML4 consisting of HELP and partial WD repeat domains (variant 8a) and fusion of the N-terminal region of EML4 to the C-terminal region of ALK containing the protein tyrosine kinase domain (variant 8b). Inferred chimeric protein fusion sites are indicated with a dotted line. IDS, intron-derived sequence; TM, transmembrane domain.
Cancer Genetics  , 45-52DOI: ( /j.cancergencyto )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 4
Immunohistochemistry staining confirms expression of ALK1 protein. Formalin-fixed, paraffin-embedded tissue sections were stained by IHC using ALK1 antibodies (A,B) or nonimmune control antibodies (C,D). Malignant cells stain positive for ALK1. Images were obtained with a 20× objective (ScanScope; Aperio Technologies, Vista, CA) and were digitally magnified an additional 10× (A,C) or 20× (B,D) to achieve 200× and 400× magnification, respectively. All four images are from the case positive for the novel EML4–ALK fusion variants 8a and 8b.
Cancer Genetics  , 45-52DOI: ( /j.cancergencyto )
Copyright © 2011 Elsevier Inc. Terms and Conditions