ABSTRACT
Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (Nâ¯=â¯28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (pâ¯=â¯0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (pâ¯=â¯0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (pâ¯=â¯0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (pâ¯=â¯0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (pâ¯=â¯0.014) or lower levels of Th2 cells were associated with lower toxicity (pâ¯=â¯0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (pâ¯=â¯0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.
ABSTRACT
RATIONALE: The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. OBJECTIVES: To determine the role of Rora in smoke-induced emphysema. METHODS: Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. MEASUREMENTS AND MAIN RESULTS: Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. CONCLUSIONS: Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.
Subject(s)
DNA Damage/physiology , Lung/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 1/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Emphysema/metabolism , Animals , Biomarkers/metabolism , Cells, Cultured , DNA Repair , Disease Models, Animal , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Oligonucleotide Array Sequence Analysis , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Emphysema/etiology , Pulmonary Emphysema/genetics , Tobacco Smoke Pollution/adverse effectsABSTRACT
Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.
