Immune-checkpoint immunotherapy is revolutionizing cancer treatment with clinical activity observed in at least 20 malignancies. Anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) and anti-programmed death 1/ligand (PD1/L1) antibodies have shown robust response rates with some patients experiencing durable cancer control. Though an important step forward, many patients do not benefit and mechanisms to explain why some patients benefit are not well described. A model for understanding the spontaneous anti-tumor immune priming observed in some tumors is known as the T cell-inflamed tumor microenvironment. Our group has defined this phenotype using gene expression profiling whereby multiple markers of T cell-based inflammation can be identified. Importantly, patients experiencing clinical response to checkpoint immunotherapy predominately show the T cell-inflamed phenotype suggesting that gene expression profiling may be predictive biomarker for response to these treatments. Conversely the non-T cell-inflamed phenotype does not show these elements. An essential question to the field of cancer immunotherapy then surrounds elucidating the molecular mechanisms that drive the non-T cell-inflamed tumor phenotype.

To pursue this research we initially took a broad bioinformatic approach in which the RNAseq data for 30 cancers from The Cancer Genome Atlas (TCGA) were segregated into T cell-inflamed and non-T cell- inflamed cohorts using a 160 gene signature. Each tumor sample was scored as high, intermediate, or low for the T-cell signature, and the tumor types were ranked according to degree of inflammation. The range of T-cell-inflamed tumors within each cancer type varied significantly with the highest levels seen in clear cell kidney cancer while little T cell-inflammation was observed in paragangliomas and low grade gliomas. We then analyzed the associated exome sequencing data for these samples to assess the number of nonsynonymous mutations in each tumor type. While mutations ranged substantially between tumor types, there was no significant differences observed between T cell-inflamed and non-T cell-inflammed tumors (either between or within tumor types). These data suggest that mutational load in cancer is unlikely to explain the T-cell-inflamed tumor microenvironment. These data were published in the United States Proceedings of the National Academy of Sciences in November 2016.

After categorizing the presence or absence of a T cell-inflamed tumor microenvironment in different tumor types, we then went forward to assess whether particular molecular pathways may mediate immune exclusion leading to the non-T cell-inflamed tumor type. Given the prior work of our group surrounding WNT/β-catenin (Spranger et al. Nature 2015), we initially investigated this pathway across tumor types. To do this we again separated tumors within TCGA by T-cell inflamed and non-T cell-inflamed RNAseq data. We then interrogated the associated exomic sequencing data of the non-T cell-inflamed tumors for mutations in the β-catenin pathway. We also performed pathway analysis to identify gene expression changes predicted to activate the β-catenin signaling pathway. Immunohistochemistry was performed on human tumor specimens of multiple tumor histologies to correlate the presence of β-catenin with the lack of a T cell infiltrate. We observed activating mutations in CTNNB1 or inactivating mutations in Axin1, Axin2, APC1, APC2 in 13 tumor types with varying frequencies. Pathway prediction analysis identified a further 12 tumor types with increased expression of β-catenin pathway elements such as WNT ligands. Only 3 tumors (pancreatic, thyroid, paraganglioma) showed no activation correlating with absence of immune signatures. The immunohistochemical experiments demonstrated inverse correlations between β-catenin and CD8+T cells. These data suggest that the WNT/β-catenin pathway is associated with immune exclusion across many tumor types. These data were presented in a plenary session at both the American Society of Clinical Oncology and the European Society of Medical Oncology as well as several smaller conferences.

Having performed this work, we were intrigued when we became aware of a case report of a patient with nonseminomatous germ cell tumor who responded to anti-PD1 antibody treatment. In addition to describing this case report, we applied our bioinformatic approaches described above specifically to the testicular germ cell tumor cohort of TCGA. We reported that gene expression data revealed a T-cell- inflamed tumor microenvironment in 47% of testicular GCTs, including 83% of the seminoma subtype as compared with 17% of the nonseminoma subtypes. Further we observed that expression of alpha- fetoprotein RNA was inversely correlated with the T-cell signature as well as expression of several interferon-γ associated genes. These data were the first report of a response of a germ cell tumor to PD1 antibody and our novel gene expression profiling investigation has supported further development of immunotherapy for germ cell tumors. This research was published in Cancer Immunology Research in November of 2016.

Moving forward, we are in the process of investigating multiple molecular pathways beyond β-catenin across tumor types in extension of our prior TCGA analyses. In additionally we have launched a prospective observational biobanking protocol (University of Chicago IRB#15-0837) to collect human biospecimens in order to perform similar investigations connected to clinical outcomes.

Funding

These funds were used to support bioinformatic projects through the University of Chicago Comprehensive Cancer Center Bioinformatic Core, investigating The Cancer Genome Atlas of the National Cancer Institute, as well as tissue staining of human specimens within the Human Tissue Research Center. Funds were also used to support publication charges.

Expansion Plans

We have expanded this research into a prospective biobanking study (UC IRB#15-0837) which will facilitate our ability to expand the concepts and hypotheses contained within this work. Multiple career development and collaborative/program grants building off of this research area under review at governmental or private agencies at the time of this update. We are additionally expanding our biobanking efforts/analysis internationally to compare factors associated with the non-T cell-inflamed tumor phenotype across patients with cancer of various tumor types.

Summary of Project Expansion

Our research investigates molecular mechanisms mediating the non-T cell-inflamed tumor microenvironment. Our work to date is describing the presence of the T cell-inflammed and non-T cell- inflamed tumor microenvironment across cancers. We have additionally expanded our investigations into the role of the WNT/β-catenin pathway mediating immune exclusion beyond melanoma to all solid tumors. We have further applied these techniques to individual tumor types (i.e. – testicular germ cell tumors) suggesting further avenues of for immunotherapy development. Moving forward, we continue to investigate the role of pathways beyond B-catenin in mediating immune exclusion across tumor types. We additionally are pursuing prospective tissue based analysis based on research from this project and are also in the planning phase of human clinical trials based on our results.