KIF5B-ALK, a Novel Fusion Oncokinase Identified by an Immunohistochemistry-based Diagnostic System for ALK-positive Lung Cancer

Abstract

Purpose: EML4-ALK is a transforming fusion tyrosine kinase, several isoforms of which have been identified in lung cancer. Immunohistochemical detection of EML4-ALK has proved difficult, however, likely as a result of low transcriptional activity conferred by the promoter-enhancer region of EML4. The sensitivity of EML4-ALK detection by immunohistochemistry should be increased adequately. Experimental Design: We developed an intercalated antibody-enhanced polymer (iAEP) method that incorporates an intercalating antibody between the primary antibody to ALK and the dextran polymer-based detection reagents. Results: Our iAEP method discriminated between tumors positive or negative for EML4-ALK in a test set of specimens. Four tumors were also found to be positive for ALK in an archive of lung adenocarcinoma (n = 130) and another 4 among fresh cases analyzed in a diagnostic laboratory. These 8 tumors were found to include1with EML4-ALK variant1,1with variant 2, 3 with variant 3, and 2 with previously unidentified variants (designated variants 6 and 7). Inverse reverse transcription-PCR analysis revealed that the remaining tumor harbored a novel fusion in which intron 24 of KIF5B was ligated to intron 19 of ALK. Multiplex reverse transcription-PCR analysis of additional archival tumor specimens identified another case of lung adenocarcinoma positive for KIF5B-ALK. Conclusions: The iAEP method should prove suitable for immunohistochemical screening of tumors positive for ALK or ALK fusion proteins among pathologic archives. Coupling of PCR-based detection to the iAEP method should further facilitate the rapid identification of novel ALK fusion genes such as KIF5B-ALK.Gene fusion is a major mechanism of carcinogenesis in hematologic malignancies and sarcomas (1). Identification of the BCR-ABL fusion kinase, which is generated as a result of the balanced chromosome anomaly t(9;22)(q34;q11) in chronic myelogenous leukemia (2), has thus been followed by the discovery of many fusion-type oncogenes (3). In contrast, it has remained unclear whether such translocationdependent fusion-type oncogenes also play a major role in the pathogenesis of epithelial tumors. Recently, however, almost 50% of prostate cancer cases have been suggested to harbor gene fusions involving ETS transcription factor loci (4), and we have discovered a recurrent chromosome translocation, inv(2)(p21p23), in non -small cell lung cancer (NSCLC) that results in the production of an EML4-ALK fusion-type protein tyrosine kinase (PTK;.Forced expression of EML4-ALK in lung epithelial cells induced the rapid development of hundreds of lung cancer nodules in mice, and peroral administration of inhibitors of the PTK activity of EML4-ALK was shown to clear such tumors from the lungs, demonstrating the pivotal role of EML4-ALK in the pathogenesis of NSCLC positive for this fusion kinase (9). This latter observation also supports the clinical application of ALK

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

“…[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. Although 2p23 chromosomal abnormalities have not been previously emphasized as recurrent in renal cell carcinoma, a review of the literature and search of the Mitelman database (http://www.ncbi.nlm.nih.gov/cancerchromosomes) yielded four additional primary renal carcinomas and one renal carcinoma cell line exhibiting karyotypic aberrations at or near the ALK gene 5,[33][34][35][36] (Table 4).…”

“…[19][20][21][22] The predicted structure and function of VCL-ALK oncoprotein follows the archetypical pattern defined by the combined properties of the N-terminal partner (ubiquitous expression and homopolymerization) and the C-terminal kinase domain (constitutive activation). On the basis of the current data, we propose that ALK fusion genes represent an oncogenic driver in a subset of renal cell carcinomas, and that recognition of these cases may be useful in ascertaining a patient's eligibility for emerging tyrosine kinase-targeted therapies, the clinical efficacy of which has been recently reported in a trial of ALK-positive non-small cell lung carcinoma.…”

“…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.…”

Molecular pathology of lung cancer: key to personalized medicine

“…153 All seven variants are formed through the fusion of the intracellular tyrosine kinase domain of ALK with a variably truncated EML4 gene. 8,[154][155][156] Activated ALK is involved in the inhibition of apoptosis and the promotion of cellular proliferation through activation of downstream PIK3CA/AKT1-and MAPK1-signaling pathways. 157 Fusion of the EML4-ALK gene and its associated EML4-ALK product may further lead to constitutive activation of the RAS/RAF1/MAP2K1/MAPK1 pathway.…”

The Current Landscape of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer: Emerging Treatment Paradigms and Future Directions

“…Also Ou et al recently reported that the emergence of ALK -resistant mutations occurred more commonly in patients with variant 3 EML4-ALK rearrangement than in patients with variant 1 tumors [4]. Other rarer ALK fusions occur such as KIF5B-ALK [5], KLC1-ALK [6], and ALK-PTPN3 [7], but they collectively are less frequent than the ALK-EML4 rearrangement [8], and therefore little is known about their clinical significance with respects to different response to ALK TKIs. In general, patients with ALK -rearranged NSCLC tend to be younger, never smokers, and have lung adenocarcinoma, though rarely patients with other lung cancer histologies have also been found to harbor this mutation [9].…”