Identification of CARS-ALK Fusion in Primary and Metastatic Lesions of an Inflammatory Myofibroblastic Tumor

Abstract

Interphase fluorescence in situ hybridization demonstrated ALK rearrangements in both primary and metastatic lesions. Rapid amplification of cDNA ends (5'RACE) identified cysteinyl-tRNA synthetase (CARS) gene fused to ALK, which predicts an in-frame chimeric protein with the preserved functional catalytic domain of ALK at the C terminus. Amplification and sequencing of tumor DNA confirmed the breakpoint at the genomic level. Restriction analysis of DNA from primary soft tissue and recurrent lung tumors showed identical patterns, indicating the same clonal origin of both lesions. Western blot analysis with C-terminus ALK antibody showed expression of an aberrantly sized chimeric protein of approximately 130 kd in tumor tissue. This is the second case of IMT demonstrating CARS as the ALK fusion partner, which confirms the recurring involvement of ALK in IMT by a common genetic mechanism. Moreover, identical clonality of separate lesions involving different sites supports metastasis in IMT. (Lab Invest 2003, 83:1255-1265.

Renal cell carcinoma with novel VCL–ALK fusion: new representative of ALK-associated tumor spectrum

“…11 Subsequently, recurrent ALK chimeric oncogenes involving diverse partner genes (RanBP2, CLTC, CARS, and others) were identified in inflammatory myofibroblastic tumors. [15][16][17][18] More recently, the ALK gene has been shown to be involved in small subsets of epithelial malignancies, including carcinomas of pulmonary, esophageal, mammary gland, and gastrointestinal origin. [19][20][21][22] To the best of our knowledge, this is the first report of a recurrent ALK locus rearrangement in a primary kidney tumor.…”

“…Appropriate positive and negative controls were employed. The two anti-ALK antibodies used were a rabbit polyclonal ALK-11 developed and characterized in detail for clinical utility in anaplastic large cell lymphoma by Steven Morris's laboratory 28 and a mouse monoclonal (clone 5A4) ALK/ p80 (Thermo Fisher Scientific, Fremont, CA, USA) 18 An anaplastic large cell lymphoma with NPM-ALK fusion was used as a positive control for both anti-ALK antibodies utilized in the study. The immunohistochemistry results were scored by two pathologists and recorded by consensus as either positive, focally positive, focally weakly positive, or negative (Table 3).…”

“…18 The 5 0 RACE products were analyzed on 1.2% agarose gels and cloned onto PGEM-T Easy Vector Systems (Promega, Madison, WI, USA) according to the manufacturer's instructions. The cloned DNA was analyzed for inserts by restriction digestion with EcoR1 (Promega); three clones were selected and sequenced using an automated sequencer (Biosystems, Foster City, CA, USA) at the sequencing facility of Hartwell Center for Bioinformatics and Biotechnology (St Jude).…”

“…18 Positive (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) and negative (no reverse transcriptase) controls were concurrently run.…”

“…11 Subsequently, ALK rearrangements were found in several phenotypically different hematologic neoplasms [12][13][14] and in histogenetically unrelated inflammatory myofibroblastic tumor. [15][16][17][18] Recently, ALK rearrangements and EML4-ALK and KIF5B-ALK fusions were discovered in a subset of non-small cell lung carcinomas; 19,20 proteomics reports suggested a possible TPM4-ALK fusion in esophageal carcinoma; 21,22 and an exon array profiling study demonstrated the EML4-ALK fusion in 2.5% of breast and colon cancers. 23 ALK is also activated in B5 to 35% of neuroblastomas and neuroblastoma cell lines by a different mechanism involving point mutations and/or gene locus amplifications.…”