Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing

Moskalev, Evgeny A.; Frohnauer, Judith; Merkelbach‐Bruse, Sabine; Schildhaus, Hans–Ulrich; Dimmler, Arno; Schubert, Thomas; Boltze, Carsten; König, Helmut; Fuchs, Florian S.; Sı̂rbu, Horia; Rieker, Ralf J.; Agaimy, Abbas; Hartmann, Arndt; Haller, Florian

doi:10.1016/j.lungcan.2014.03.002

Cited by 26 publications

(18 citation statements)

References 39 publications

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“…These reasons have limited the use of the technique in clinical practice. Recently, a very sensitive RT-PCR-based method was devised to detect the overexpression of 3′ regions of fusion transcripts involving tumour genes constitutionally repressed or expressed at very low levels [53]; this approach has been successfully applied to ALK gene fusions in lung cancer [53, 54]. Unfortunately, this method cannot be easily applied to ROS1 , since the gene is also expressed in normal and hyperplastic lung tissue [15, 55].…”

Section: Detection Of Ros1 Gene Rearrangementsmentioning

confidence: 99%

Testing for ROS1 in non-small cell lung cancer: a review with recommendations

et al. 2016

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Rearrangements of the ROS1 gene occur in 1–2 % of non-small cell lung cancers (NSCLCs). Crizotinib, a highly effective inhibitor of ROS1 kinase activity, is now FDA-approved for the treatment of patients with advanced ROS1-positive NSCLC. Consequently, focus on ROS1 testing is growing. Most laboratories currently rely on fluorescence in situ hybridisation (FISH) assays using a dual-colour break-apart probe to detect ROS1 rearrangements. Given the rarity of these rearrangements in NSCLC, detection of elevated ROS1 protein levels by immunohistochemistry may provide cost-effective screening prior to confirmatory FISH testing. Non-in situ testing approaches also hold potential as stand-alone methods or complementary tests, including multiplex real-time PCR assays and next-generation sequencing (NGS) platforms which include commercial test kits covering a range of fusion genes. In order to ensure high-quality biomarker testing, appropriate tissue handling, adequate control materials and participation in external quality assessment programmes are essential, irrespective of the testing technique employed. ROS1 testing is often only considered after negative tests for EGFR mutation and ALK gene rearrangement, based on the assumption that these oncogenic driver events tend to be exclusive. However, as the use of ROS1 inhibitors becomes routine, accurate and timely detection of ROS1 gene rearrangements will be critical for the optimal treatment of patients with NSCLC. As NGS techniques are introduced into routine diagnostic practice, ROS1 fusion gene testing will be provided as part of the initial testing package.

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Section: Detection Of Ros1 Gene Rearrangementsmentioning

confidence: 99%

Testing for ROS1 in non-small cell lung cancer: a review with recommendations

et al. 2016

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“…Our test offers several advantages over published [39–43] assays assessing the unbalanced expression of the 3′ and 5′ portion of ALK transcript: i) it requires at least 5–10 times less input RNA; ii) it does not require expensive and proprietary technologies or specialist expertise (unlike NanoString and/or NGS platforms), and iii) it provides a quantitative assessment of 3′ and 5′ ALK mRNA positivity. Unlike traditional comparative (ΔΔCq) and/or 3′/5′ ratio methods derived thereof, our model provides confidence intervals for each point prediction.…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, more recent reverse transcription quantitative real-time PCR (RT-qPCR) assays based on the unbalanced expression of the 5′ and 3′ portions of the ALK transcript [39–41], which occurs when ALK is rearranged, require significant amounts of RNA (50–100 ng per PCR reaction) from FFPE tissues [40, 41]. Alternative technologies that could be applied to the detection of ALK rearrangements, i.e., NanoString (NanoString Technologies, Inc., Seattle, WA) and RNA massive parallel sequencing require, in addition to elevated amounts of total RNA, the availability of proprietary and cutting-edge platforms in pathology laboratories [42, 43]. To circumvent these problems, we describe herein, a simple quantitative PCR-based ALK predictive model fully optimized to work with low-quantity and low-quality RNA from FFPE samples.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer

et al. 2016

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Accurate detection of altered anaplastic lymphoma kinase (ALK) expression is critical for the selection of lung cancer patients eligible for ALK-targeted therapies. To overcome intrinsic limitations and discrepancies of currently available companion diagnostics for ALK, we developed a simple, affordable and objective PCR-based predictive model for the quantitative measurement of any ALK fusion as well as wild-type ALK upregulation. This method, optimized for low-quantity/−quality RNA from FFPE samples, combines cDNA pre-amplification with ad hoc generated calibration curves. All the models we derived yielded concordant predictions when applied to a cohort of 51 lung tumors, and correctly identified all 17 ALK FISH-positive and 33 of the 34 ALK FISH-negative samples. The one discrepant case was confirmed as positive by IHC, thus raising the accuracy of our test to 100%. Importantly, our method was accurate when using low amounts of input RNA (10 ng), also in FFPE samples with limited tumor cellularity (5–10%) and in FFPE cytology specimens. Thus, our test is an easily implementable diagnostic tool for the rapid, efficacious and cost-effective screening of ALK status in patients with lung cancer.

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“…Amplicon targeting requires primer design at the 5′ and 3′ ends of a transcript, and allows quantitative analysis of fusion events. This strategy was recently employed for the detection of ALK fusion events in lung cancer samples . Two similar amplicon‐based methods have been developed and commercialized by NuGene and ArcherDX, where expected fusion events were examined by two sequence‐specific primers together with a common primer that targets adapter sequence.…”

Section: Rna‐seq Methods For Specific Goalsmentioning

confidence: 99%

RNA‐Seq methods for transcriptome analysis

Hrdličková

Toloue

Tian³

2016

WIREs RNA

476

340

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Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions.

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Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing

Cited by 26 publications

References 39 publications

Testing for ROS1 in non-small cell lung cancer: a review with recommendations

Testing for ROS1 in non-small cell lung cancer: a review with recommendations

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer

RNA‐Seq methods for transcriptome analysis

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