Dynamic changes in the T cell receptor repertoire during treatment with radiotherapy combined with an immune checkpoint inhibitor.

Previous studies have indicated a synergistic effect between radiotherapy and immunotherapy. A better understanding of how this combination affects the immune system can help to clarify its role in the treatment of metastatic cancer. We performed T cell receptor (TCR) sequencing on 46 sequentially collected samples from 15 patients with stage IV non-small cell lung cancer, receiving stereotactic body radiotherapy combined with a programmed cell death ligand-1 (PD-L1) inhibitor. TCR repertoire diversity was assessed using Rényi diversity curves and the Shannon diversity index. TCR clones were tracked over time. We found decreasing or stable diversity in the best responders, and an increase in diversity at progression in patients with an initial response. Expansion of TCR clones was more often seen in responders. Several patients also developed new clones of high abundance. This seemed to be more related to radiotherapy than to immune checkpoint blockade. In summary, we observed similar dynamics in the TCR repertoire as have been described with immunotherapy alone. In addition, the occurrence of new unique clones of high abundance after radiotherapy may indicate that radiotherapy functions as a personalized cancer vaccine.

The blood transcriptome prior to ovarian cancer diagnosis: A case-control study in the NOWAC postgenome cohort.

Epithelial ovarian cancer (EOC) has a 5-year relative survival of 50%, partly because markers of early-stage disease are not available in current clinical diagnostics. The aim of the present study was to investigate whether EOC is associated with transcriptional profiles in blood collected up to 7 years before diagnosis. For this, we used RNA-stabilized whole blood, which contains circulating immune cells, from a sample of EOC cases from the population-based Norwegian Women and Cancer (NOWAC) postgenome cohort. We explored case-control differences in gene expression in all EOC (66 case-control pairs), as well as associations between gene expression and metastatic EOC (56 pairs), serous EOC (45 pairs, 44 of which were metastatic), and interval from blood sample collection to diagnosis (≤3 or >3 years; 34 and 31 pairs, respectively). Lastly, we assessed differential expression of genes associated with EOC in published functional genomics studies that used blood samples collected from newly diagnosed women. After adjustment for multiple testing, this nested case-control study revealed no significant case-control differences in gene expression in all EOC (false discovery rate q>0.96). With the exception of a few probes, the log2 fold change values obtained in gene-wise linear models were below ±0.2. P-values were lowest in analyses of metastatic EOC (80% of which were serous EOC). No common transcriptional profile was indicated by interval to diagnosis; when comparing the 100 genes with the lowest p-values in gene-wise tests in samples collected ≤3 and >3 years before EOC diagnosis, no overlap in these genes was observed. Among 86 genes linked to ovarian cancer in previous publications, our data contained expression values for 42, and of these, tests of LIME1, GPR162, STAB1, and SKAP1, resulted in unadjusted p<0.05. Although limited by sample size, our findings indicated less variation in blood gene expression between women with similar tumor characteristics.

Each pregnancy linearly changes immune gene expression in the blood of healthy women compared with breast cancer patients.

BACKGROUND: There is a large body of evidence demonstrating long-lasting protective effect of each full-term pregnancy (FTP) on the development of breast cancer (BC) later in life, a phenomenon that could be related to both hormonal and immunological changes during pregnancies. In this work, we studied the pregnancy-associated differences in peripheral blood gene expression profiles between healthy women and women diagnosed with BC in a prospective design. METHODS: Using an integrated system epidemiology approach, we modeled BC incidence as a function of parity in the Norwegian Women and Cancer (NOWAC) cohort (165,000 women) and then tested the resulting mathematical model using gene expression profiles in blood in a nested case-control study (460 invasive case-control pairs) of women from the NOWAC postgenome cohort. Lastly, we undertook a gene set enrichment analysis for immunological gene sets. RESULTS: A linear trend fitted the dataset precisely showing an 8% decrease in risk of BC for each FTP, independent of stratification on other risk factors and lasting for decades after a woman's last FTP. Women with six children demonstrated 48% reduction in the incidence of BC compared to nulliparous. When we looked at gene expression, we found that 756 genes showed linear trends in cancer-free controls (false discovery rate [FDR] 5%), but this was not the case for any of the genes in BC cases. Gene set enrichment analysis of immunologic gene sets (C7 collection in Molecular Signatures Database) revealed 215 significantly enriched human gene sets (FDR 5%). CONCLUSION: We found marked differences in gene expression and enrichment profiles of immunologic gene sets between BC cases and healthy controls, suggesting an important protective effect of the immune system on BC risk.

Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo.

Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer. In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1ß increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection. This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.

The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis.

BACKGROUND: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. METHODS: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. RESULTS: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. CONCLUSION: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma.

Agnoprotein of polyomavirus BK interacts with proliferating cell nuclear antigen and inhibits DNA replication.

BACKGROUND: The human polyomavirus BK expresses a 66 amino-acid peptide referred to as agnoprotein. Though mutants lacking agnoprotein are severely reduced in producing infectious virions, the exact function of this peptide remains incompletely understood. To elucidate the function of agnoprotein, we searched for novel cellular interaction partners. METHODS: Yeast-two hybrid assay was performed with agnoprotein as bait against human kidney and thymus libraries. The interaction between agnoprotein and putative partners was further examined by GST pull down, co-immunoprecipitation, and fluorescence resonance energy transfer studies. Biochemical and biological studies were performed to examine the functional implication of the interaction of agnoprotein with cellular target proteins. RESULTS: Proliferating cell nuclear antigen (PCNA), which acts as a processivity factor for DNA polymerase δ, was identified as an interaction partner. The interaction between agnoprotein and PCNA is direct and occurs also in human cells. Agnoprotein exerts an inhibitory effect on PCNA-dependent DNA synthesis in vitro and reduces cell proliferation when ectopically expressed. Overexpression of PCNA restores agnoprotein-mediated inhibition of cell proliferation. CONCLUSION: Our data suggest that PCNA is a genuine interaction partner of agnoprotein and the inhibitory effect on PCNA-dependent DNA synthesis by the agnoprotein may play a role in switching off (viral) DNA replication late in the viral replication cycle when assembly of replicated genomes and synthesized viral capsid proteins occurs.