Won-Chul Lee^1,2†, Alexandre Reuben^2*†, Xin Hu^1,2†, Nicholas McGranahan^3†, Runzhe Chen^1,2, Ali Jalali⁴, Marcelo V. Negrao², Shawna M. Hubert^1,2, Chad Tang⁵, Chia-Chin Wu¹, Anthony San Lucas⁶, Whijae Roh⁷, Kenichi Suda⁸, Jihye Kim⁹, Aik-Choon Tan¹⁰, David H. Peng¹¹, Wei Lu¹², Ximing Tang¹², Chi-Wan Chow¹², Junya Fujimoto¹², Carmen Behrens², Neda Kalhor¹³, Kazutaka Fukumura¹², Marcus Coyle¹, Rebecca Thornton¹, Curtis Gumbs¹, Jun Li¹, Chang-Jiun Wu¹, Latasha Little¹, Emily Roarty², Xingzhi Song¹, J. Jack Lee¹⁴, Erik P. Sulman¹⁵, Ganesh Rao¹⁶, Stephen Swisher¹⁷, Lixia Diao¹⁸, Jing Wang¹⁸, John V. Heymach², Jason T. Huse¹³, Paul Scheet⁶, Ignacio I. Wistuba^12†, Don L. Gibbons^2†, P. Andrew Futreal^1†, Jianhua Zhang^1†, Daniel Gomez^5,19*† and Jianjun Zhang^1,2*†

¹Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ²Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ³Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK. ⁴Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA. ⁵Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ⁶Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ⁷Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA. ⁸Department of Thoracic Surgery, Kindai University Faculty of Medicine, Osaka-Sayama, Japan. ⁹Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. ¹⁰Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, USA. ¹¹NYU Langone Health, New York, NY, USA. ¹²Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹³Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹⁴Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹⁵New York University Langone School of Medicine, New York, NY, USA. ¹⁶Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹⁷Department of Thoracic Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹⁸Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ¹⁹Current Address: Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

^†Won-Chul Lee, Alexandre Reuben, Xin Hu, and Nicholas McGranahan contributed equally to this work

^*Correspondence

Background
Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary.

Results
We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases.

Conclusions
Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.