Molecular Classification of Neuroendocrine Tumors of the Thymus Helen Dinter, Hanibal Bohnenberger, MD, Julia Beck, PhD, Kirsten Bornemann-Kolatzki, Ekkehard Schütz, MD, Stefan Küffer, Lukas Klein, Teri J. Franks, MD, Anja Roden, MD, Alexander Emmert, MD, Marc Hinterthaner, MD, Mirella Marino, MD, Luka Brcic, MD, Helmut Popper, MD, Cleo-Aron Weis, MD, Giuseppe Pelosi, MD, Alexander Marx, MD, Philipp Ströbel, MD  Journal of Thoracic Oncology  Volume 14, Issue 8, Pages 1472-1483 (August 2019) DOI: 10.1016/j.jtho.2019.04.015 Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 (A) Chromosomal gains (red) and losses (blue) in thymic as detected by shallow whole genome sequencing. Y axis shows percentage of cases with a given chromosomal alteration. (B) Unsupervised clustering of whole-genome sequencing data versus WHO histologic subtypes resulted in three major molecular clusters. Cluster 1 contained most of the typical (TCs) and atypical carcinoids (ACs), but also a few large cell neuroendocrine carcinomas (later reclassified as NET-G3). Cluster 2 contained mostly AC, but also an NET G3 and an LCNEC and one TC. Cluster 3 contained all small-cell carcinomas (SCC) and most of the LCNECs, but also three ACs. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Prognostic significance and identification of relevant subtypes by morphologic versus molecular TNET classifications. WHO, ENETS, and molecular classification using copy number instability (CNI) score identified three major subtypes (the difference between typical carcinoids, [TC] and atypical carcinoids [AC] in the WHO classification was not statistically significant, log rank test p = 0.39), whereas application of the ENETS system resulted only in two subgroups. The table within Figure 2 shows range of mitotic counts, Ki-67 indices, and histologic subtypes according to the three morphologic classifications. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 1 Morphological and molecular findings in two extreme outliers. a-d: Atypical carcinoid with classical “carcinoid” morphology (a+b) and low ki67 index (c), but highly aberrant chromosomal alterations with gains and losses of several chromosomes (d) and a CNI score of 97.7. e-h: Large cell neuroendocrine carcinoma/tumor with still recognizable carcinoid morphology with vascular invasion, high mitotic count, small foci of necrosis (a+b), and intermediate ki67 index (c), but only very few chromosomal alterations with a CNI score of 3.1. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 2 Molecular and morphological findings in four cases where more than one material was available In case #1, the primary tumor (PT) was a LCNEC (later re-classified as NET-G3), while a synchronous metastasis (MT) was classified as AC. In case #2, the PT was an AC, while a metastasis 3 years later was classified as LCNEC (later re-classified as NET G3). Case #3 had two samples taken from the biopsy (PT-1 and PT-2) and one MT after 5 years. PT1 was classified as TC, PT2 was classified as AC, and MT was classified as LCNEC (later re-classified as NET G3). Case #4 had two samples taken from the PT (PT-1 and PT-2), both were classified as LCNEC. a) genomic comparison of tumor manifestations within the same patient shows similar profiles in case #1 and case #4, and slightly different profiles in case #2 and #3. Morphological comparison of the same tumor manifestations in two exemplary cases. In case #2, the initial tumor was classified as AC (b), while the metastasis 3 years later was classified as LCNEC (and here re-classified as NET G3) (c). Direct comparison shows the classic “carcinoid” morphology in the PT, while the MT showed confluent sheets of large and markedly more pleomorphic tumor cells. The differentiation between NET G3 and LCNEC would not have possible in this case by morphology or mitotic count/ki67 index alone (c). In case #3, PT2 was classified as AC (d), while a MT 5 years later was classified as LCNEC (and here re-classified as NET G3) (e). Again, comparison of the two manifestations shows slight morphological progression with more confluent and slightly more pleomorphic tumor cells in MT. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 3 Summary of features that differentiated cases initially categorized as LCNEC according to WHO but falling into the CNIlow and CNIint (“NET G3”) from cases falling into the CNIhigh cluster (LCNEC). Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 4 Overexpression of EZH2 was a strong predictor of poor prognosis in thymic neuroendocrine tumors. Weak and strong staining were both counted as positive for statistical evaluation. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 5 Massive overlap of Ki-67 indices between thymic neuroendocrine tumors when classified according to WHO criteria. Journal of Thoracic Oncology 2019 14, 1472-1483DOI: (10.1016/j.jtho.2019.04.015) Copyright © 2019 International Association for the Study of Lung Cancer Terms and Conditions