Amir Momeni-Boroujeni1, Elham Yousefi2, Sounak Gupta3, Ryma Benayed4, Michael F Berger1, Marc Ladanyi1, Robert Monroe5, Jeff Kim5, Achim Jungbluth1, Britta Weigelt1, Kay J Park6. 1. Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 2. Department of Pathology, Vancouver General Hospital, Vancouver, BC Canada. 3. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA. 4. AstraZeneca, Precision Medicine and Biosamples, New York, NY, USA. 5. Advanced Cell Diagnostics, San Jose, CA, USA. 6. Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address: parkk@mskcc.org.
Abstract
BACKGROUND: Telomerase reverse transcriptase (TERT) activation has been shown to be an important cancer hallmark; the activation and expression of TERT has been documented in >90% of tumors and TERT activation has been touted as a prognostic marker in many cancers. However, there is currently no simple testing modality to detect TERT mRNA expression in surgical pathology specimens. In this study we aim to evaluate and validate the utility and reliability of the TERT RNAscope® in-situ hybridization (ISH) assay for the detection of TERT mRNA expression in formalin-fixed, paraffin embedded tissue. METHODS AND MATERIALS: RNAscope® detection for TERT was performed on a Leica Biosystems BOND III research staining robot using the Hs-TERT-O1 (ACD, 481968) probe. Twenty three samples containing 48 tissue types were assessed. TERT genomic alterations were determined by targeted next generation sequencing (NGS), while TERT mRNA expression was determined by both targeted RNA-sequencing and TERT RNAscope® and the results compared. Manual vs automated TERT expression quantification methodologies were evaluated for the ISH assay. The expression levels in normal vs. neoplastic tissues were also compared. RESULTS: The RNAscope® assay showed high TERT expression in neoplastic tissues, while most normal tissues have no or very low expression levels (p-value= 0.0001, AUC: 0.99). In addition, there was good correlation of TERT expression between the RNAscope® assay and RNA-sequencing. For RNAscope® quantification, manual calculation of TERT signal/cell ratio based on a count of 100 cells was superior compared to automated signal detection. CONCLUSION: TERT RNAscope® assay is a simple and reliable tool for the evaluation of TERT mRNA expression. TERT signal/cell ratio based on a count of 100 cells is a reproducible and accurate interpretation approach for evaluation of TERT expression.
BACKGROUND: Telomerase reverse transcriptase (TERT) activation has been shown to be an important cancer hallmark; the activation and expression of TERT has been documented in >90% of tumors and TERT activation has been touted as a prognostic marker in many cancers. However, there is currently no simple testing modality to detect TERT mRNA expression in surgical pathology specimens. In this study we aim to evaluate and validate the utility and reliability of the TERT RNAscope® in-situ hybridization (ISH) assay for the detection of TERT mRNA expression in formalin-fixed, paraffin embedded tissue. METHODS AND MATERIALS: RNAscope® detection for TERT was performed on a Leica Biosystems BOND III research staining robot using the Hs-TERT-O1 (ACD, 481968) probe. Twenty three samples containing 48 tissue types were assessed. TERT genomic alterations were determined by targeted next generation sequencing (NGS), while TERT mRNA expression was determined by both targeted RNA-sequencing and TERT RNAscope® and the results compared. Manual vs automated TERT expression quantification methodologies were evaluated for the ISH assay. The expression levels in normal vs. neoplastic tissues were also compared. RESULTS: The RNAscope® assay showed high TERT expression in neoplastic tissues, while most normal tissues have no or very low expression levels (p-value= 0.0001, AUC: 0.99). In addition, there was good correlation of TERT expression between the RNAscope® assay and RNA-sequencing. For RNAscope® quantification, manual calculation of TERT signal/cell ratio based on a count of 100 cells was superior compared to automated signal detection. CONCLUSION: TERT RNAscope® assay is a simple and reliable tool for the evaluation of TERT mRNA expression. TERT signal/cell ratio based on a count of 100 cells is a reproducible and accurate interpretation approach for evaluation of TERT expression.
Authors: Klaus G Griewank; Rajmohan Murali; Joan Anton Puig-Butille; Bastian Schilling; Elisabeth Livingstone; Miriam Potrony; Cristina Carrera; Tobias Schimming; Inga Möller; Marion Schwamborn; Antje Sucker; Uwe Hillen; Celia Badenas; Josep Malvehy; Lisa Zimmer; André Scherag; Susana Puig; Dirk Schadendorf Journal: J Natl Cancer Inst Date: 2014-09-13 Impact factor: 13.506