Connective Tissue Oncology Society

2001 CTOS Annual Meeting Posters— Biology

FUSION OF THE ALK GENE TO THE CLATHRIN HEAVY CHAIN GENE, CLTC, IN INFLAMMATORY MYOFIBROBLASTIC TUMOR
Julia A Bridge¹, Masahiko Kanamori¹, Zhigui Ma², D Ashley Hill², William Lydiatt¹, Man Yee Lui³, Gisele WB Colleoni³, Cristina R Antonescu³, Marc Ladanyi³, Stephan W Morris²
¹Departments of Pathology/Microbiology and/or Pediatrics and/or Orthopaedic Surgery and/or Otoloaryngology, University of Nebraksa Medical Center, ²Departments of Pathology and/or Hematology/Oncology, St. Jude Children's Research Hospital, ³Department of Pathology, Memorial Sloan-Kettering Cancer Center

OBJECTIVE: Recently, clonal mutations [fusion of the tropomyosin (TPM) N-terminal coiled-coil domains to the ALK C-terminal-kinase domain] have been detected in a subset of inflammatory myofibroblastic tumors (IMTs) supporting a neoplastic pathogenesis for this biologically controversial entity (Am J Pathol 157:377, 2000). The ALK gene is localized to chromosomal band 2p23 and the TPM3 and TPM4 genes are localized to 1q22-23 and 19p13.1 respectively. In the current study, cytogenetic analysis of an IMT revealed a 2;17 translocation [t(2;17)(p23;q23)]. Because 17q23 is not the chromosomal locus of either TPM3 or TPM4, our objective was to determine the ALK fusion gene partner in this case and in an additional IMT not exhibiting a TPM3-ALK or TPM4-ALK fusion gene transcript.

METHODS: Case 1 was obtained from an IMT of the neck of a 3-year-old female and Case 2, an IMT of the pelvis of a 37-year-old male. Conventional cytogenetic analysis was performed on a sterile, representative tissue sample from Case 1. Material suitable for cytogenetic analysis was not available for Case 2. Fluorescence in situ hybridization (FISH) studies employing 2p23 (ALK) breakpoint spanning and flanking probes (Vysis, Downers Grove, IL) were executed on metaphase preparations of Case 1 and on cytologic touch preparations of both cases. Rapid amplification of cDNA ends (RACE) studies were performed on Case 1 using the 5' RACE system from Gibco BRL (Rockville, MD). The products were analyzed on agarose gels. Sharp product bands were subjected to direct sequencing, whereas non-discrete products were cloned and multiple independent clones were sequenced. Following RACE analyses, heminested reverse transcriptase - polymerase chain reaction (RT-PCR) studies were performed to confirm the presence of a CLTC-ALK fusion gene in both cases. The identified product bands were directly sequenced.

RESULTS: Case 1 exhibited the following: 46,XX,t(2;17)(p23;q23),add(16)(q24)[5]/ 92,idem x2[1]/46,XX[4]. Case 1 metaphase cell FISH confirmed the presence of a 2;17 translocation involving the ALK locus. Cases 1 and 2 interphase cell FISH revealed a split of one set of the two-color probe signals, indicating a disruption of the 2p23 breakpoint (ALK) on one chromosome 2 homologue in 52% and 46% of the cells, respectively. 5' RACE of Case 1 revealed an ALK fusion with the clathrin heavy chain gene (CLTC) localized to 17q23. This CLTC-ALK fusion incorporates 1,634 residues of the 1,675-aa clathrin heavy chain. Heminested RT-PCR confirmed the presence of a CLTC-ALK fusion gene product in Case 1 and demonstrated the identical fusion product in Case 2. The ALK and CLTC breakpoints in these IMTs were identical to those recently reported in CLTC-ALK fusions in anaplastic large cell lymphoma (ALCL), (Blood 95:3204, 2000).

CONCLUSION: The CLTC-ALK fusion oncogene represents a novel mechanism of ALK activation in IMT and demonstrates that, similar to ALCL, the fusion partners of the ALK gene in IMT are diverse. ALK protein expression is an independent predictor of survival and serves as a useful biological marker of a specific disease entity within the spectrum of ALCL. Additional studies are warranted to determine whether ALK protein expression is likewise associated with specific clinicopathological traits in IMT.