Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Sanders, Heather; Li, Hairong; Bruey, Jean‐Marie; Scheerle, Jay; Meloni‐Ehrig, Aurelia; Kelly, John; Novick, Constance; Albitar, Mäher

doi:10.1016/j.cancergencyto.2010.08.024

Cited by 80 publications

(72 citation statements)

References 10 publications

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“…In addition to the original EML4-ALK fusion cDNA in which exon 13 of EML4 is fused to exon 20 of ALK in an inframe manner (designated the E13;A20 variant by analogy with karyotype nomenclature; see http://atlasgeneticsoncology.org/Tumors/inv2p21p23NSCCLungID5667.html), 14 different variants of EML4-ALK have been described (1,14,(21)(22)(23)(24)(25)(26)(27). Seven exons of EML4 are theoretically capable of in-frame fusion with exon 20 of ALK (Fig.…”

Section: Multiplex Rt-pcr Systemmentioning

confidence: 99%

A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer

Soda

Isobe

Inoue

et al. 2012

Clinical Cancer Research

113

100

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Purpose: EML4-ALK is a lung cancer oncogene, and ALK inhibitors show marked therapeutic efficacy for tumors harboring this fusion gene. It remains unsettled, however, how the fusion gene should be detected in specimens other than formalin-fixed, paraffin-embedded tissue. We here tested whether reverse transcription PCR (RT-PCR)-based detection of EML4-ALK is a sensitive and reliable approach.Experimental Design: We developed a multiplex RT-PCR system to capture ALK fusion transcripts and applied this technique to our prospective, nationwide cohort of non-small cell lung cancer (NSCLC) in Japan.Results: During February to December 2009, we collected 916 specimens from 853 patients, quality filtering of which yielded 808 specimens of primary NSCLC from 754 individuals. Screening for EML4-ALK and KIF5B-ALK with our RT-PCR system identified EML4-ALK transcripts in 36 samples (4.46%) from 32 individuals (4.24%). The RT-PCR products were detected in specimens including bronchial washing fluid (n ¼ 11), tumor biopsy (n ¼ 8), resected tumor (n ¼ 7), pleural effusion (n ¼ 5), sputum (n ¼ 4), and metastatic lymph node (n ¼ 1). The results of RT-PCR were concordant with those of sensitive immunohistochemistry with ALK antibodies.Conclusions: Multiplex RT-PCR was confirmed to be a reliable technique for detection of ALK fusion transcripts. We propose that diagnostic tools for EML4-ALK should be selected in a manner dependent on the available specimen types. FISH and sensitive immunohistochemistry should be applied to formalinfixed, paraffin-embedded tissue, but multiplex RT-PCR is appropriate for other specimen types.

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Section: Multiplex Rt-pcr Systemmentioning

confidence: 99%

A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer

Soda

Isobe

Inoue

et al. 2012

Clinical Cancer Research

113

100

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“…Large-scale screening of clinical specimens has revealed that the original EML4-ALK variant (referred to as E13;A20 or variant 1) together with a variant in which intron 6 of EML4 is fused to intron 19 of ALK (referred to as E6;A20 or variant 3; ref. 26) account for more than 90% of all EML4-ALKpositive cases of NSCLCs in Japan (M. Soda, personal communication), but many rare variants have also been identifi ed (17,26,27,30,31,34,37,38).…”

Section: Clinicopathologic Features and Diagnosis Of Eml4-alk-positivmentioning

confidence: 99%

ALKoma: A Cancer Subtype with a Shared Target

2012

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Anaplastic lymphoma kinase (ALK) is a receptor-type protein tyrosine kinase that is currently the focus of much attention in oncology. ALK is rendered oncogenic as a result of its fusion to NPM1 in anaplastic large cell lymphoma, to TPM3 or TPM4 in inflammatory myofibroblastic tumor, to EML4 in non–small cell lung carcinoma, and to VCL in renal medullary carcinoma. It is also activated as a result of missense mutations in neuroblastoma and anaplastic thyroid cancer. Whereas these various tumors arise in different organs, they share activated ALK, and a marked clinical efficacy with ALK inhibitors has already been shown for some of the tumors with ALK fusions. One of such compound, crizotinib, is now approved in the United States for the treatment of lung cancer positive for ALK rearrangement. I propose that tumors carrying abnormal ALK as an essential growth driver be collectively termed “ALKoma.” Significance: ALK acquires transforming ability through gene fusion or missense mutation in a wide range of human cancers. Some of these cancers, which I propose be collectively referred to as “ALKoma,” may all be effectively treated with small compounds or antibodies targeted to activated ALKs. Cancer Discov; 2(6); 495–502. © 2012 AACR.

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“…Multiple in-frame fusion variants have been reported to date, all of which contain the same breakpoint in ALK but different breakpoints in EML4. [2][3][4][5][6][7][8] The resulting EML4-ALK fusion proteins display constitutive kinase activity and are responsive to ALK tyrosine kinase inhibitors in vitro and in vivo. 2,3,9 Thus, ALK has emerged as a therapeutic target for patients with NSCLC who have EML4-ALK gene fusions.…”

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confidence: 99%

“…These tumors do not respond to EGFR antagonists but do respond to specific ALK kinase inhibitors. 6,10,8,19 Given the many EML4-ALK fusion variants identified to date, development of a sensitive and specific molecular diagnostic assay for formalin-fixed, paraffin-embedded tissue is not trivial. EML4-ALK fusion carcinomas can be identified by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or reverse transcriptionpolymerase chain reaction (RT-PCR).…”

mentioning

confidence: 99%

Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing

Wallander¹,

Geiersbach²,

Tripp³

et al. 2012

Archives of Pathology &Amp; Laboratory Medicine

105

104

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N Context.-Echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non-small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection.Objective.-To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions.Design.-Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b).Results.-EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation.Conclusions.-The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

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Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Cited by 80 publications

References 10 publications

A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer

A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer

ALKoma: A Cancer Subtype with a Shared Target

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