Previously Unidentified Changes in Renal Cell Carcinoma Gene Expression Identified by Parametric Analysis of Microarray Data

BACKGROUND. Renal cell carcinoma is a common malignancy that often presents as a metastatic-disease for which there are no effective treatments. To gain insights into the mechanism of renal cell carcinogenesis, a number of genome-wide expression profiling studies have been performed. Surprisingly, there is very poor agreement among these studies as to which genes are differentially regulated. To better understand this lack of agreement we profiled renal cell tumor gene expression using genome-wide microarrays (45,000 probe sets) and compare our analysis to previous microarray studies. METHODS. We hybridized total RNA isolated from renal cell tumors and adjacent normal tissue to Affymetrix U133A and U133B arrays. We removed samples with technical defects and removed probesets that failed to exhibit sequence-specific hybridization in any of the samples. We detected differential gene expression in the resulting dataset with parametric methods and identified keywords that are overrepresented in the differentially expressed genes with the Fisher-exact test. RESULTS. We identify 1,234 genes that are more than three-fold changed in renal tumors by t-test, 800 of which have not been previously reported to be altered in renal cell tumors. Of the only 37 genes that have been identified as being differentially expressed in three or more of five previous microarray studies of renal tumor gene expression, our analysis finds 33 of these genes (89%). A key to the sensitivity and power of our analysis is filtering out defective samples and genes that are not reliably detected. CONCLUSIONS. The widespread use of sample-wise voting schemes for detecting differential expression that do not control for false positives likely account for the poor overlap among previous studies. Among the many genes we identified using parametric methods that were not previously reported as being differentially expressed in renal cell tumors are several oncogenes and tumor suppressor genes that likely play important roles in renal cell carcinogenesis. This highlights the need for rigorous statistical approaches in microarray studies.National Institutes of Healt

APOBEC-related mutagenesis and neo-peptide hydrophobicity: implications for response to immunotherapy.

Tumor-associated neo-antigens are mutated peptides that allow the immune system to recognize the affected cell as foreign. Cells carrying excessive mutation load often develop mechanisms of tolerance. PD-L1/PD-1 checkpoint immunotherapy is a highly promising approach to overcome these protective signals and induce tumor shrinkage. Yet, the nature of the neo-antigens driving those beneficial responses remains unclear. Here, we show that APOBEC-related mutagenesis - a mechanism at the crossroads between anti-viral immunity and endogenous nucleic acid editing - increases neo-peptide hydrophobicity (a feature of immunogenicity), as demonstrated by in silico computation and in the TCGA pan-cancer cohort, where APOBEC-related mutagenesis was also strongly associated with immune marker expression. Moreover, APOBEC-related mutagenesis correlated with immunotherapy response in a cohort of 99 patients with diverse cancers, and this correlation was independent of the tumor mutation burden (TMB). Combining APOBEC-related mutagenesis estimate and TMB resulted in greater predictive ability than either parameter alone. Based on these results, further investigation of APOBEC-related mutagenesis as a marker of response to anti-cancer checkpoint blockade is warranted

Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs

t(4;10)(q12;q23) PDGFRA/TNKS2

Comprehensive genomic profiling identifies a novel PDGFRA-TNKS2 gene fusion in a female case of myeloid neoplasm with eosinophilia. The patient was treated with imatinib, and showed a dramatic and ongoing response with no evidence of diseas

MHC-I genotype and tumor mutational burden predict response to immunotherapy.

BackgroundImmune checkpoint blockade (ICB) with antibodies inhibiting cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) (or its ligand (PD-L1)) can stimulate immune responses against cancer and have revolutionized the treatment of tumors. The influence of host germline genetics and its interaction with tumor neoantigens remains poorly defined. We sought to determine the interaction between tumor mutational burden (TMB) and the ability of a patient's major histocompatibility complex class I (MHC-I) to efficiently present mutated driver neoantigens in predicting response ICB.MethodsComprehensive genomic profiling was performed on 83 patients with diverse cancers treated with ICB to determine TMB and human leukocyte antigen-I (HLA-I) genotype. The ability of a patient's MHC-I to efficiently present mutated driver neoantigens (defined by the Patient Harmonic-mean Best Rank (PHBR) score (with lower PHBR indicating more efficient presentation)) was calculated for each patient.ResultsThe median progression-free survival (PFS) for PHBR score < 0.5 vs. ≥ 0.5 was 5.1 vs. 4.4 months (P = 0.04). Using a TMB cutoff of 10 mutations/mb, the stable disease > 6 months/partial response/complete response rate, median PFS, and median overall survival (OS) of TMB high/PHBR high vs. TMB high/PHBR low were 43% vs. 78% (P = 0.049), 5.8 vs. 26.8 months (P = 0.03), and 17.2 months vs. not reached (P = 0.23), respectively. These findings were confirmed in an independent validation cohort of 32 patients.ConclusionsPoor presentation of driver mutation neoantigens by MHC-I may explain why some tumors (even with a high TMB) do not respond to ICB

Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.Susan WhiteheadHillel and Liliana Bachrac

Hunting for dark halo substructure using submilliarcsecond-scale observations of macrolensed radio jets

Dark halo substructure may reveal itself through secondary, small-scale gravitational lensing effects on light sources that are macrolensed by a foreground galaxy. Here, we explore the prospects of using Very Long Baseline Interferometry (VLBI) observations of multiply-imaged quasar jets to search for submilliarcsecond-scale image distortions produced by various forms of dark substructures in the 1e3-1e8 Msolar mass range. We present lensing simulations relevant for the angular resolutions attainable with the existing European VLBI Network (EVN), the global VLBI array, and an upcoming observing mode in which the Atacama Large Millimeter Array (ALMA) is connected to the global VLBI array. While observations of this type would not be sensitive to standard cold dark matter subhalos, they can be used to detect more compact forms of halo substructure predicted in alternative structure formation scenarios. By mapping ~5 strongly lensed systems, it should be possible to detect or robustly rule out primordial black holes in the 1e3-1e6 Msolar mass range if they constitute >1% percent of the dark matter in these lenses. Ultracompact minihalos are harder to detect using this technique, but 1e6-1e8 Msolar ultracompact minihalos could in principle be detected if they constitute >10% of the dark matter.Comment: 13 pages, 8 figures; v.2 accepted for publication in MNRA