Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing

Lam, Suk Wai; Cleton‐Jansen, Anne‐Marie; Cleven, Arjen H.G.; Ruano, Dina; Wezel, Tom van; Szuhai, Károly; Bovée, Judith V.M.G.

doi:10.1016/j.jmoldx.2018.05.007

Cited by 89 publications

(66 citation statements)

References 23 publications

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“…AMP-based NGS has been already used by other authors [16] to reveal the presence or absence of gene fusion in the background of unusual signals during FISH testing of ALK and ROS1. The method allows detection of ALK gene rearrangements even without prior knowledge of the partner gene [17,18]. As both new and known fusion variants can be detected with AMP, ambiguously positive FISH cases can be sorted out confidently in this way.…”

Section: Discussionmentioning

confidence: 99%

Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

et al. 2020

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Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy. Materials and Methods: Tissue and cytology samples from nearly one thousand patients with advanced stage nonsmall cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases. Results: Fifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3′) signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3′ signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively. Conclusion: Approximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3′ signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC.

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Section: Discussionmentioning

confidence: 99%

Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

et al. 2020

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“…Others have reported a slightly lower concordance using the Archer FusionPlex kit compared with pre‐existing techniques; however, this was a larger series that tested a greater diversity of fusions genes—and it is worth noting that most of their discrepancies were reconciled by independent testing that ultimately demonstrated the NGS technique was superior to conventional FISH and RT‐PCR . The authors of this study reported an overall failure rate of 14% with the Archer FusionPlex kit; this was attributed to both decalcification and heterogeneity in tissue and fixation conditions . In our hands, likewise using FusionPlex, we encountered a fairly similar percentage of test failures when evaluating this platform for possible implementation, especially with samples demonstrating poor quality RNA.…”

Section: Introductionmentioning

confidence: 54%

“…23 The authors of this study reported an overall failure rate of 14% with the Archer FusionPlex kit; this was attributed to both decalcification and heterogeneity in tissue and fixation conditions. 23 In our hands, likewise using FusionPlex, we encoun-…”

Section: 31 | Selection Of An Assaymentioning

confidence: 82%

“…The slide was soaked in xylene to remove the coverslip, then scraped into an Eppendorf tube, and the RNA extracted. F, Despite the poor quality of RNA, RNA-Seq nevertheless identified the presence of a diagnostic FN1-ALK fusion product slightly lower concordance using the Archer FusionPlex kit compared with pre-existing techniques 23 ; however, this was a larger series that tested a greater diversity of fusions genes-and it is worth noting that most of their discrepancies were reconciled by independent testing that ultimately demonstrated the NGS technique was superior to conventional FISH and RT-PCR. 23 The authors of this study reported an overall failure rate of 14% with the Archer FusionPlex kit; this was attributed to both decalcification and heterogeneity in tissue and fixation conditions.…”

Section: 31 | Selection Of An Assaymentioning

confidence: 96%

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Targeted RNA sequencing: A routine ancillary technique in the diagnosis of bone and soft tissue neoplasms

Dickson

Swanson

2018

Genes Chromosomes & Cancer

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The past decade has witnessed remarkable progress in delineating the molecular pathogenesis of many mesenchymal neoplasms. This, in large part, is attributable to the application of next‐generation sequencing. As these techniques decrease in cost, and increasingly support the use of routine clinical specimens—such as formalin‐fixed paraffin‐embedded tissue and cytology samples—they are beginning to be routinely implemented in diagnostic pathology laboratories. The breadth of testing possible by next‐generation sequencing makes this a useful adjunct for pathologists, particularly with the emergence of targeted therapies. The intent of this article is to share our experience, over 2 years, as an early adopter of targeted RNA sequencing as an ancillary diagnostic technique for fusion gene detection in bone and soft tissue neoplasms.

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“…This translated into more accurate classification and also prognostication of soft tissue tumors . At present, the two novel molecular multiplex methods commonly used in Dutch sarcoma centers are the anchored multiplex PCR (AMP)‐based NGS (Archer FusionPlex Sarcoma assay) and the NanoString nCounter platform . The Archer AMP PCR method targets exons of 26 genes commonly involved in fusion genes of soft tissue tumors, whereas the NanoString assay is a high‐throughput hybridization technique, which uses specific probes that target 174 unique gene fusion junctions in 22 soft tissue tumor types …”

Section: Introductionmentioning

confidence: 99%

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors

Song

Platteel

Suurmeijer

et al. 2020

Genes Chromosomes & Cancer

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Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor‐defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre‐defined gene fusion transcripts in formalin‐fixed and paraffin‐embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor‐defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost‐effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel.

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Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing

Cited by 89 publications

References 23 publications

Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

Targeted RNA sequencing: A routine ancillary technique in the diagnosis of bone and soft tissue neoplasms

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors

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