A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer Patrizia Viola, MD, Manisha Maurya,

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A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer Patrizia Viola, MD, Manisha Maurya, PhD, James Croud, MSc, Jana Gazdova, BSc, Nadia Suleman, BSc, Eric Lim, FRCS(CTh), Tom Newsom-Davis, FRCP, Nick Plowman, MD, Alexandra Rice, FRCPath, M. Angeles Montero, MD, David Gonzalez de Castro, PhD, Sanjay Popat, FRCP, Andrew G. Nicholson, DM Journal of Thoracic Oncology Volume 11, Issue 7, Pages 1029-1039 (July 2016) DOI: 10.1016/j.jtho.2016.03.019 Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 (A) A cell block from a transbronchial needle aspirate of a lymph node shows a micropapillary architecture. (B) Immunohistochemical analysis for ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) protein shows variably granular cytoplasmic staining of mainly moderate intensity in 80% of cells within micropapillary clusters. (C) A biopsy specimen shows an adenocarcinoma showing focal signet ring cell morphologic features. (D) This case shows moderate staining in 90% of cells for ROS1 protein, with focal submembranous accentuation of staining. (E) A nodal biopsy specimen shows replacement by an adenocarcinoma with mixed micropapillary and cribriform patterns. (F) This case shows strong staining in 90% of cells for ROS1 protein. Journal of Thoracic Oncology 2016 11, 1029-1039DOI: (10.1016/j.jtho.2016.03.019) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 (A) Fluorescence in situ hybridization testing of case 5 shows a signal pattern characteristic of an unbalanced ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) translocation resulting in loss of the red probe. (B) Fluorescence in situ hybridization testing shows 3’ (red) and 5’ (green) regions of the ROS1 gene separated by rearrangement. Journal of Thoracic Oncology 2016 11, 1029-1039DOI: (10.1016/j.jtho.2016.03.019) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Mediastinal lymph nodes (A–C) and tumor in the left hilum (D–F) show persistent a response to crizotinib over 2.5 years. Journal of Thoracic Oncology 2016 11, 1029-1039DOI: (10.1016/j.jtho.2016.03.019) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Tumor cells are negative for ROS proto-oncogene 1, receptor tyrosine kinase protein, but there is positive staining of background pneumocytes. Journal of Thoracic Oncology 2016 11, 1029-1039DOI: (10.1016/j.jtho.2016.03.019) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions