IASLC Sept 2013 | Page 42

41 Chapter 5: Reverse-Transcriptase Polymerase Chain Reaction and Multiple Gene Assays EML4 72F; 5'-GTCAGCTCTTGAGTCACGAGTT-3' (EML4 exon 2, forward) !"#$(2-H,(3 %DBC&DCBCBBD&DBC&CD&DBB%+ ()!"#$(;EF>(-0(KFGLMG:/( Fusion-RT-S; 5'-GTGCAGTGTTTAGCATTCTTGGGG-3' (EML4 exon 13, forward), HI?=F>%AB%J,(3 %DBDC&DBDBBB&DC&BBCBBDDDD%+ ()!"#$(;EF>(*+0(KFGLMG:/0(( &#'(+.26AA0(3 %&BCC&DBBCDBCCBDBBC&D&DC%+ 20, reverse) ALK 3078RR, 5'-ATCCAGTTCGTCCTGTTCAGAGC-3' (ALK exon ()&#'(;EF>(-.0(G;1;G?;/( !"#$%&#'()!*+,&-./0(1* EML4-ALK (E13;A20), v1 EML4-ALK (E20;A20), v2 !"#$%&#'()!-.,&-./0(1- EML4 !"#$(2-HN( 72F/ &#'(+.26AA( ALK 3078RR ()*02+345/ (1,735bp) Fusion HI?=F>(AB%JN( RT-S/ &#'(+.26AA( ALK 3078RR (($+-45(432bp (2,488bp) ()-0$6645/ (1,849bp) ()*06$845/ 454bp ($3$45( (1,804bp) ()*06.$45/ ()*062+45/ (1,873bp) 1,185bp (*0*6345( 546bp (3$745( 501bp (3.*45( (32.45( 570bp (32845( 579bp (88845( 999bp 'OH3P-3++HN( ALK 3078RR &#'(+.26AA( EML4-ALK (E6;A20), v3 !"#$%&#'()!7,&-./0(1+ EML4-ALK (E14;del49A20), v4 !"#$%&#'()!*$,:;<$8&-./0(1$ EML4-ALK (E2;A20), v5 !"#$%&#'()!-,&-./0(13 EML4-ALK (E13;ins69A20), v6 !"#$%&#'()!*+,=>?78&-./0(17 !"#$%&#'()!*$,:;<*$&-./0(12 EML4-ALK (E14;del14A20), v7 913bp*, 946bp* (8*+4590(8$7459( !"#$%&#'()!*3:;<*8,:;<-.&-./0(1$@( v4' (3,732bp) EML4-ALK (E15del19;del20A20), ()+02+-45/ !"#$%&#'()!*6,&-./0(13@ EML4-ALK (E18;A20), v5' ()-0+.-45/ (2,302bp) KIF5B867F 5'-ATTAGGTGGCAACTGTAGAACC-3' (exon 10) 'OH3P672H(3@%&BB&DDBDDC&&CBDB&D&&CC%+@(();EF>(*./( KIF5B2533F'OH3P-3++H((3@%DBDC&C&&&C&DBBDDB&CDBD%+@()!EF>(-+%-$(4FI>:MGQ/( 5'-GTGCACAAACAGTTGGTACGTG-3' (Exon 23-24 boundary) 'OH3P%&#'()'-$,&-./ KIF5B-ALK (K24;A20) 'OH3P%&#'()'*3,&-./ KIF5B-ALK (K15;A20) 'OH3P%&#'()'*2,&-./ KIF5B-ALK (K17;A20) KIF5B867F/ 'OH3P672HN( ALK 3078RR &#'(+.26AA( (3$745546bp ( ( KIF5B2533F/ ()+07.+45/(( (3,603bp) (*0*2745(( 1,176bp (*0$6+45( 1,483bp 9(BR;(M?S;G=?T(:;>FS;?((5GF:IUS?(FK(1MG=M>S(+M(M>:(40(G;?5;UV1;9* 6 =* 4 <* 2 ;* 0 :* 9* ALK ALK ALK Kinase Kinase domain domain 3’ region region 3' region 5' region 5’ region 5' region 10 8 6 4 2 0 >9* =* <* ;* :* 9* 5' region region 3' region region 5' region region 3' region region 5' region 3' region 5' region 3' region Figure 3. In normal lung cells, ALK is expressed at a very low or undetectable level of mRNA, whereas ALK-positive tumor cells produce abundant chimeric ALK transcript. When the 3' and 5' regions of ALK are separately analyzed, a high transcript of ALK 5' region is detected exclusively in ALK-positive cells. This method is not influenced by mixed normal cells and is independent of the fusion partners.