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RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance.
BMC Cancer 2018; 18(1):1158BC

Abstract

BACKGROUND

Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma.

METHODS

We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series.

RESULTS

Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript.

CONCLUSIONS

The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.

Authors+Show Affiliations

Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany. claudia.vollbrecht@charite.de. German Cancer Consortium (DKTK), partner site Berlin, Germany. claudia.vollbrecht@charite.de. Geman Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. claudia.vollbrecht@charite.de.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany. German Cancer Consortium (DKTK), partner site Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.Medical Department, Division of Infectiology and Pneumology, Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Augustenburger Platz 1, 13353, Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany. Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charité Comprehensive Cancer Center, Virchowweg 23, 10117, Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.Johannes Wesling Klinikum Minden, Institute for Pathology, Hans-Nolte-Straβe 1, 32429, Minden, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany. maximilian.von-laffert@charite.de. Berlin Institute of Health (BIH), Anna-Louisa-Karsch-Straβe 2, 10178, Berlin, Germany. maximilian.von-laffert@charite.de.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30466405

Citation

Vollbrecht, Claudia, et al. "RNA-based Analysis of ALK Fusions in Non-small Cell Lung Cancer Cases Showing IHC/FISH Discordance." BMC Cancer, vol. 18, no. 1, 2018, p. 1158.
Vollbrecht C, Lenze D, Hummel M, et al. RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance. BMC Cancer. 2018;18(1):1158.
Vollbrecht, C., Lenze, D., Hummel, M., Lehmann, A., Moebs, M., Frost, N., ... von Laffert, M. (2018). RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance. BMC Cancer, 18(1), p. 1158. doi:10.1186/s12885-018-5070-6.
Vollbrecht C, et al. RNA-based Analysis of ALK Fusions in Non-small Cell Lung Cancer Cases Showing IHC/FISH Discordance. BMC Cancer. 2018 Nov 22;18(1):1158. PubMed PMID: 30466405.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance. AU - Vollbrecht,Claudia, AU - Lenze,Dido, AU - Hummel,Michael, AU - Lehmann,Annika, AU - Moebs,Markus, AU - Frost,Nikolaj, AU - Jurmeister,Philipp, AU - Schweizer,Leonille, AU - Kellner,Udo, AU - Dietel,Manfred, AU - von Laffert,Maximilian, Y1 - 2018/11/22/ PY - 2018/06/14/received PY - 2018/11/08/accepted PY - 2018/11/24/entrez PY - 2018/11/24/pubmed PY - 2019/3/7/medline KW - Anaplastic lymphoma kinase (ALK) KW - Fluorescence in-situ hybridization (FISH) KW - Immunohistochemistry (IHC) KW - Massive parallel sequencing (MPS) KW - NanoString KW - Non-small cell lung cancer (NSCLC) SP - 1158 EP - 1158 JF - BMC cancer JO - BMC Cancer VL - 18 IS - 1 N2 - BACKGROUND: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. METHODS: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. RESULTS: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. CONCLUSIONS: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods. SN - 1471-2407 UR - https://www.unboundmedicine.com/medline/citation/30466405/RNA_based_analysis_of_ALK_fusions_in_non_small_cell_lung_cancer_cases_showing_IHC/FISH_discordance_ L2 - https://bmccancer.biomedcentral.com/articles/10.1186/s12885-018-5070-6 DB - PRIME DP - Unbound Medicine ER -