Molecular Screening in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma: Anaplastic Lymphoma Kinase Analysis, Next-Generation Sequencing Fusion Gene Detection, and T-Cell Receptor Immunoprofiling

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. Over ten fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10-20% of ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the TCR repertoire. We devised a quantitative RT-qPCR to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using Rapid Amplification of 5´cDNA (RACE) or NGS. T-cell Receptor (TCR) immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus two previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%), we discerned at least one (ranging from 1-4) clonal TCR rearrangement. In 59% of patients, clonal TCRB junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. RT-qPCR detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both the malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.