Focal oncogene amplification and rearrangements drive tumor growth and evolution in multiple cancer types. We present AmpliconArchitect (AA), a tool to reconstruct the fine structure of focally amplified regions using whole genome sequencing (WGS) and validate it extensively on multiple simulated and real datasets, across a wide range of coverage and copy numbers. Analysis of AA-reconstructed amplicons in a pan-cancer dataset reveals many novel properties of copy number amplifications in cancer. These findings support a model in which focal amplifications arise due to the formation and replication of extrachromosomal DNA. Applying AA to 68 viral-mediated cancer samples, we identify a large fraction of amplicons with specific structural signatures suggestive of hybrid, human-viral extrachromosomal DNA. AA reconstruction, integrated with metaphase fluorescence in situ hybridization (FISH) and PacBio sequencing on the cell-line UPCI:SCC090 confirm the extrachromosomal origin and fine structure of a Forkhead box E1 (FOXE1)-containing hybrid amplicon.

The topographical organization of collagen within the tumor microenvironment has been implicated in modulating cancer cell migration and independently predicts progression to metastasis. Here, we show that collagen matrices with small pores and short fibers, but not Matrigel, trigger a conserved transcriptional response and subsequent motility switch in cancer cells resulting in the formation of multicellular network structures. The response is not mediated by hypoxia, matrix stiffness, or bulk matrix density, but rather by matrix architecture-induced β1-integrin upregulation. The transcriptional module associated with network formation is enriched for migration and vasculogenesis-associated genes that predict survival in patient data across nine distinct tumor types. Evidence of this gene module at the protein level is found in patient tumor slices displaying a vasculogenic mimicry (VM) phenotype. Our findings link a collagen-induced migration program to VM and suggest that this process may be broadly relevant to metastatic progression in solid human cancers.

Summary: The anti-cancer immune response against mutated peptides of potential immunological relevance (neoantigens) is primarily attributed to MHC-I-restricted cytotoxic CD8+ T-cell responses. MHC-II-restricted CD4+ T-cells also drive anti-tumor responses, but their relation to neoantigen selection and tumor evolution has not been systematically studied. Modeling the potential of an individual’s MHC-II genotype to present 1,018 driver mutations in 5,942 tumors, we demonstrate that the MHC-II genotype constrains the mutational landscape during tumorigenesis in a manner complementary to MHC-I. Mutations poorly bound to MHC-II are positively selected during tumorigenesis, even more than mutations poorly bound to MHC-I. This emphasizes the importance of CD4+ T-cells in anti-tumor immunity. In addition, we observed less inter-patient variation in mutation presentation for MHC-II than for MHC-I. These differences were reflected by age at diagnosis, which was correlated with presentation by MHC-I only. Collectively, our results emphasize the central role of MHC-II presentation in tumor evolution.

Individual MHC genotype constrains the mutational landscape during tumorigenesis. Immune checkpoint inhibition reactivates immunity against tumors that escaped immune surveillance in approximately 30% of cases. Recent studies demonstrated poorer response rates in female and younger patients. Although immune responses differ with sex and age, the role of MHCbased immune selection in this context is unknown. We find that tumors in younger and female individuals accumulate more poorly presented driver mutations than those in older and male patients, despite no differences in MHC genotype. Younger patients show the strongest effects of MHC-based driver mutation selection, with younger females showing compounded effects and nearly twice as much MHC-II based selection. This study presents evidence that strength of immune selection during tumor development varies with sex and age, and may influence the availability of mutant peptides capable of driving effective response to immune checkpoint inhibitor therapy.