Introduction

Anaplastic large cell lymphoma (ALCL) expressing anaplastic lymphoma kinase (ALK; also referred to as ALK-positive ALCL or ALKoma) is a distinct clinicopathological and molecular entity characterized by a proliferation of T/ null lymphoid cells that show a diverse immunostaining pattern for ALK protein (1-3). This aberrant ALK expression is associated with chromosomal translocations involving the ALK gene at 2p23 (4). These translocations lead to the synthesis of novel chimeric proteins that retain the C-terminal portion of ALK, where the tyrosine kinase domain is located. In most of these tumors, the t(2;5)(p23;q35) translocation causes fusion of the ALK gene to the 5' region of the nucleophosmin (NPM) gene (5). Several cytogenetic and molecular studies also have demonstrated that chromosomal aberrations other than the t(2;5)(p23;q35) may give rise to novel ALK fusion genes in ALCL. So far, five different genes, nonmuscle tropomyosin (TPM3), TRK-fused gene (TFG), 5' aminoimidazole-4-carboxamide ribonucleotide formyltranferase/IMP cyclohydrolase (ATIC), clathrin heavy chain gene (CLTC), and moesin (MSN), have been cloned as alternative partners to NPM in ALCL (see Fig. 1) (6-13).

The characterization of these ALK partners has been performed using different molecular methods, including the 5' Rapid Amplification of complementary deoxyribonucleic acid (cDNA) Ends (5' RACE) technique. This approach allows the potential amplification and identification of either 5' or 3' messenger ribonucleic acids (mRNA) ends from an internal known sequence. In the present work, the goal of the polymerase chain reaction (PCR)-based 5' RACE technique is to identify the 5' gene involved in new ALK translocations using primers designed within the known 3' catalytic domain of the ALK. Although the precise protocol varies among different users, the general strategy remains consistent (see Fig. 2) (14). First-strand cDNA synthesis is primed using a gene-specific antisense primer (GSP1), performing the cDNA conversion of specific mRNAs and maximizing the potential for complete extension to the 5'-end of the message. For 5'RACE of ALK chimeric variants, a common GSP1 primer (ALK1 in Fig. 2) is used and this primer anneals in a sequence near the ALK 5' region that is juxtaposed with the NPM gene in the classic rearrangement of NPM-ALK. After the synthesis of cDNA, the first-strand product is

Alk Translocation Alcl
Fig. 1. ALK translocation variants in ALCL. ALK portion is showed in solid gray (retaining the marked kinase domain).

purified from unincorporated dNTPs and GSP1. Terminal deoxynucleotidyl transferase (TdT) is used to add homopolymeric tails to the 3' ends of the cDNA. Tailed cDNA is then amplified by PCR using a nested gene-specific primer (GSP2; ALK 2 in the Fig. 2), which anneals 3' to GSP1, and a complementary homopolymer containing an anchor primer (AAP), which permits amplification from the homopolymeric tail. This allows amplification of unknown sequences between the GSP2 and the 5'-end of the mRNA. Further, nested PCRs usually are required to confer an adequate level of specificity to the process to permit the characterization of RACE products. The reamplification is achieved using a nested gene-specific primer (GSP3; ALK 3 in the Fig. 2), which anneals 3' to GSP2, and a universal amplification primer (UAP), which anneals to the 5' sequence previously introduced by the APP primer.

The result of the 5' amplification yields a product corresponding to a fragment of the fusion gene, including a partial fragment of the unknown ALK partner. Hybridization with an internal ALK primer is needed to confirm the specificity of the obtained PCR fragments because unspecific bands frequently are generated in these amplifications (see Fig. 3). The confirmed specific PCR

Fig. 2. 5'RACE ALK strategy showing the localization of the primers used.

product subsequently is purified and sequenced. The obtained sequence can now be compared with the GenBank sequences to identify the corresponding ALK partner. If the new sequence were not known, further rounds of 5'RACE using 3' primers of the obtained new sequence eventually would lead to the characterization of the whole 5' sequence of the chimeric gene. In any case, once this gene is identified, terminal primers could be designed to amplify the entire coding region of the fusion gene to be cloned for a complete sequencing analysis, thus allowing further functional studies of the new chimeric ALK gene.

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