A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens

Lung, Jrhau; Lin, Yu Ching; Hung, Ming Szu; Jiang, Yuan; Lee, Kuan Der; Lin, Paul Y.; Tsai, Ying Huang

doi:10.1016/j.lungcan.2016.02.004

Cited by 5 publications

(4 citation statements)

References 35 publications

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“…It is important to note that IHC, like the RT-PCR assay used in this report, is not capable of differentiating ALK fusion-positive versus full-length ALK-positive NSCLC; thus, occasional cases of IHC-positive lung cancer could possibly express full-length ALK rather than the chimeric kinase although none of our 4 cases were IHC-positive. RT-PCR assays that assess the balance between expression of 5′ ALK (representing transcripts indicative of the full-length receptor tyrosine kinase) relative to 3′ ALK transcripts (that are common to both full-length and fusion forms of ALK ) have been designed for this discrimination [ 14 , 24 ]; however, the ALK RGQ RT-PCR Kit targets only the 3′ region of the ALK transcript which encodes the kinase domain. Amplification or increased copy number of full-length ALK has been reported in NSCLC tumors [ 25 ], but is not thought to be an oncogenic driver.…”

Section: Discussionmentioning

confidence: 99%

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Hout

Schweitzer

Lawrence

et al. 2017

Cancers

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Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

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Section: Discussionmentioning

confidence: 99%

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Hout

Schweitzer

Lawrence

et al. 2017

Cancers

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“…Nevertheless, various IHC platforms have differing sensitivity and specificity (Table 1) [29]. It has been proposed IHC and FISH complement one another and that combined testing may enhance the detection of ALK-positive cases [30, 31]. …”

Section: Methods Of Testingmentioning

confidence: 99%

ALK alterations and inhibition in lung cancer

Gerber

2017

Seminars in Cancer Biology

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The advent of precision medicine in non-small cell lung cancer has remarkably altered the direction of research and improved clinical outcomes. The identification of molecular subsets with differential response to targeted therapies began with the identification of epidermal growth factor receptor mutated tumors in subsets of non-small cell lung cancer (NSCLC). Emboldened by unprecedented response rates to kinase inhibitors seen in that subset, the oncologic community searched for other molecular subsets featuring oncogene addiction. An early result of this search was the discovery of NSCLC driven by activating rearrangements of the anaplastic lymphoma kinase (ALK) gene. In an astoundingly brief period following the recognition of ALK-positive NSCLC, details of the biology, clinicopathologic features, development of targeted inhibitors, mechanisms of therapeutic resistance, and new generations of treatment were elucidated. This review summarizes the current understanding of the pathologic features, diagnostic approach, treatment options, resistance mechanisms, and future research areas for ALK-positive NSCLC.

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“…After A-tailing, adaptor ligation, single primer extension, and streptavidin capture enrichment, 3 kb and 4.5 kb bands for LC-2/ad cells and H3122 cells were efficiently and successfully amplified in the first PCR reaction, respectively. The performance and reliability of the ligation-mediated PCR method for the identification of the chromosomal breakpoint of a fusion gene in clinical samples were also demonstrated using an ALK fusion gene-positive lung cancer sample identified in our earlier work [ 11 ] and a 5.1 kb amplicon was recovered ( Fig. 2 ).…”

Section: Resultsmentioning

confidence: 61%

An optimized ligation-mediated PCR method for chromosome walking and fusion gene chromosomal breakpoints identification

Lung,

Hung,

Chen

et al. 2024

Biology Methods and Protocols

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Molecular techniques that recover unknown sequences next to a known sequence region have been widely applied in various molecular studies, such as chromosome walking, identification of the insertion site of transposon mutagenesis, fusion gene partner, and chromosomal breakpoints, as well as targeted sequencing library preparation. Although various techniques have been introduced for efficiency enhancement, searching for relevant single molecular event present in a large-sized genome remains challenging. Here, the optimized ligation-mediated PCR method was developed and successfully identified chromosomal breakpoints far away from the exon of the new exon junction without the need for nested PCR. In addition to recovering unknown sequences next to a known sequence region, the high efficiency of the method could also improve the performance of targeted NGS sequencing.

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A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens

Cited by 5 publications

References 35 publications

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

ALK alterations and inhibition in lung cancer

An optimized ligation-mediated PCR method for chromosome walking and fusion gene chromosomal breakpoints identification

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