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

The Cancer Institute, Japanese Foundation for Cancer Research, Japan.
Clinical Cancer Research (Impact Factor: 8.19). 05/2009; 15(9):3143-9. DOI: 10.1158/1078-0432.CCR-08-3248
Source: PubMed

ABSTRACT 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.
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.
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 include 1 with EML4-ALK variant 1, 1 with 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.
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.

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    • ". The intercalated antibody-enhanced polymer method was used for the sensitive detection of ALK, as described previously (Takeuchi et al., 2009). "
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    ABSTRACT: Fetal lung interstitial tumor (FLIT) is a recently reported type of congenital lung lesion comprising solid and cystic components. The pathological features include unique interstitial mesenchyme-based cell proliferation, and differ from other neoplasms represented by pleuropulmonary blastoma or congenital peribronchial myofibroblastic tumor. FLIT is extremely rare and its gene expression profile has not yet been reported. We provide the first report of a novel chromosomal rearrangement resulting in α-2-macroglobulin (A2M) and anaplastic lymphoma kinase (ALK) gene fusion in a patient with FLIT. The tumor cells contained a t(2;12)(p23;p13) and were mesenchymal in origin (e.g., inflammatory myofibroblastic tumors), suggesting the involvement of ALK in this case of FLIT. Break apart fluorescence in situ hybridization demonstrated chromosomal rearrangement at ALK 2p23. Using 5'-rapid amplification of cDNA ends, we further identified a novel transcript fusing exon 22 of A2M to exon 19 of ALK, which was confirmed by reverse-transcription polymerase chain reaction. The corresponding chimeric gene was subsequently confirmed by sequencing, including the genomic break point between intron 22 and 18 of A2M and ALK, respectively. Discovery of A2M as a novel ALK fusion partner, together with the involvement of ALK, provides new insights into the pathogenesis of FLIT, and suggests the potential for new therapeutic strategies based on ALK inhibitors. © 2014 Wiley Periodicals, Inc.
    Genes Chromosomes and Cancer 10/2014; 53(10). DOI:10.1002/gcc.22199 · 3.84 Impact Factor
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    • "However, direct sequencing of the breakpoint on genomic DNA level requires either multiplex PCRs or enrichment-based library preparation using multiple oligomers covering the whole potential breakpoint region as baits. Both approaches are handicapped by the low amounts and short lengths of DNA fragments isolated from FFPE material compared to DNA from blood, for example, and by the broad variety of breakpoints among the 5`-fusion partner gene reported to date [2] [9] [10] [11] [12] [13] [14] [15] [16] [17]. To avoid these obstacles, we decided against direct confirmation of genomic breakpoints. "
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    ABSTRACT: Objectives Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in-situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. Materials and Methods We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (Multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5‘and 3‘ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analysed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. Results The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5‘and 3‘ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3‘ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. Conclusion Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.
    Lung cancer (Amsterdam, Netherlands) 06/2014; 84(3). DOI:10.1016/j.lungcan.2014.03.002 · 3.74 Impact Factor
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    • "The fusion is found about 2-7% in lung cancer [5] [6] [7] [8]. Other genes which can fuse with ALK had also been found, including KIF5B and TFG [7] [9] [10]. In NSCLC never/light smokers without EGFR mutation "
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    ABSTRACT: Whether Cell block (CB) samples are applicable to detect anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and ret proto-oncogene (RET) fusion genes in lung adenocarcinoma is still unknown. In this study, 108 cytological samples that contained lung adenocarcinoma cells were collected, and made into CB. The CB samples all contained at least 30% lung adenocarcinoma cells. In these patients, 48 harbored EGFR mutation. Among the 50 EGFR wild type patients who detected fusion genes, 14 carried EML4-ALK fusion (28%), 2 had TPM3-ROS1 fusion (4%), and 3 harbored KIF5B-RET fusion (6%). No double fusions were found in one sample. Patients with fusion genes were younger than those without fusion genes (p = 0.032), but no significant difference was found in sex and smoking status (p > 0.05). In the thirty-five patients who received first-line chemotherapy, patients with fusion gene positive had disease control rate (DCR) (72.7% VS 50%, p > 0.05) and objective response rate (ORR) (9.1% VS 4.2%, p > 0.05) compared with those having fusion gene negative. The median progression free survival (mPFS) were 4.0 and 2.7 months in patients harbored fusion mutations and wild type, respectively (p > 0.05). We conclude that CB samples could be used to detect ALK, ROS1 and RET fusions in NSCLC. The frequency distribution of three fusion genes is higher in lung adenocarcinoma with wild-type EGFR, compared with unselected NSCLC patient population. Patients with fusion genes positive are younger than those with fusion gene negative, but they had no significantly different PFS in first-line chemotherapy.
    Translational oncology 06/2014; 7(3):363–367. DOI:10.1016/j.tranon.2014.04.013 · 3.40 Impact Factor
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