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TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC

B. Robesova, M. Bajerova, K. Liskova, J. Skrickova, M. Tomiskova, S. Pospisilova, J. Mayer, D. Dvorakova,

. 2014 ; 85 (1) : 25-30.

Jazyk angličtina Země Irsko

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc15014338

OBJECTIVES: Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). MATERIALS AND METHODS: This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). RESULTS: EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. CONCLUSION: Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.

Citace poskytuje Crossref.org

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$a OBJECTIVES: Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). MATERIALS AND METHODS: This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). RESULTS: EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. CONCLUSION: Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.
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$a Bajerova, Monika $u Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic.
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$a Tomiskova, Marcela $u Department of Respiratory Diseases and Tuberculosis, Masaryk University and University Hospital Brno, Brno, Czech Republic.
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$a Pospisilova, Sarka $u Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
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$a Mayer, Jiri $u Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
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$a Dvorakova, Dana $u Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
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