Discovering high-resolution patterns of differential DNA methylation that correlate with gene expression changes.

Methylation of the CpG-rich region (CpG island) overlapping a gene's promoter is a generally accepted mechanism for silencing expression. While recent technological advances have enabled measurement of DNA methylation and expression changes genome-wide, only modest correlations between differential methylation at gene promoters and expression have been found. We hypothesize that stronger associations are not observed because existing analysis methods oversimplify their representation of the data and do not capture the diversity of existing methylation patterns. Recently, other patterns such as CpG island shore methylation and long partially hypomethylated domains have also been linked with gene silencing. Here, we detail a new approach for discovering differential methylation patterns associated with expression change using genome-wide high-resolution methylation data: we represent differential methylation as an interpolated curve, or signature, and then identify groups of genes with similarly shaped signatures and corresponding expression changes. Our technique uncovers a diverse set of patterns that are conserved across embryonic stem cell and cancer data sets. Overall, we find strong associations between these methylation patterns and expression. We further show that an extension of our method also outperforms other approaches by generating a longer list of genes with higher quality associations between differential methylation and expression.

Prognostic Significance of Immune Cell Infiltration in Muscle-invasive Bladder Cancer Treated with Definitive Chemoradiation: A Secondary Analysis of RTOG 0524 and RTOG 0712.

Chemoradiation therapy (CRT) is a treatment for muscle-invasive bladder cancer (MIBC). Using a novel transcriptomic profiling panel, we validated prognostic immune biomarkers to CRT using 70 pretreatment tumor samples from prospective trials of MIBC (NRG/RTOG 0524 and 0712). Disease-free survival (DFS) and overall survival (OS) were estimated via the Kaplan-Meier method and stratified by genes correlated with immune cell activation. Cox proportional-hazards models were used to assess group differences. Clustering of gene expression profiles revealed that the cluster with high immune cell content was associated with longer DFS (hazard ratio [HR] 0.53, 95% confidence interval [CI] 0.26-1.10; p = 0.071) and OS (HR 0.48, 95% CI 0.24-0.97; p = 0.040) than the cluster with low immune cell content. Higher expression of T-cell infiltration genes (CD8A and ICOS) was associated with longer DFS (HR 0.40, 95% CI 0.21-0.75; p = 0.005) and OS (HR 0.49, 95% CI 0.25-0.94; p = 0.033). Higher IDO1 expression (IFNγ signature) was also associated with longer DFS (HR 0.44, 95% CI 0.24-0.88; p = 0.021) and OS (HR 0.49, 95% CI 0.24-0.99; p = 0.048). These findings should be validated in prospective CRT trials that include biomarkers, particularly for trials incorporating immunotherapy for MIBC. PATIENT SUMMARY: We analyzed patient samples from two clinical trials (NRG/RTOG 0524 and 0712) of chemoradiation for muscle-invasive bladder cancer using a novel method to assess immune cells in the tumor microenvironment. Higher expression of genes associated with immune activation and high overall immune-cell content were associated with better disease-free survival and overall survival for patients treated with chemoradiation.

Multidimensional biomarker predicts disease control in response to immunotherapy in recurrent or metastatic head and neck squamous-cell carcinoma.

PURPOSE: Anti-PD-1 therapy provides clinical benefit in 40-50% of patients with relapsed and/or metastatic head and neck squamous cell carcinoma (RM-HNSCC). Selection of anti- PD-1 therapy is typically based on patient PD-L1 immunohistochemistry (IHC) which has low specificity for predicting disease control. Therefore, there is a critical need for a clinical biomarker that will predict clinical benefit to anti-PD-1 treatment with high specificity. METHODS: Clinical treatment and outcomes data for 103 RM-HNSCC patients were paired with RNA-sequencing data from formalin-fixed patient samples. Using logistic regression methods, we developed a novel biomarker classifier based on expression patterns in the tumor immune microenvironment to predict disease control with monotherapy PD-1 inhibitors (pembrolizumab and nivolumab). The performance of the biomarker was internally validated using out-of-bag methods. RESULTS: The biomarker significantly predicted disease control (65% in predicted non-progressors vs. 17% in predicted progressors, p < 0.001) and was significantly correlated with overall survival (OS; p = 0.004). In addition, the biomarker outperformed PD-L1 IHC across numerous metrics including sensitivity (0.79 vs 0.64, respectively; p = 0.005) and specificity (0.70 vs 0.61, respectively; p = 0.009). CONCLUSION: This novel assay uses tumor immune microenvironment expression data to predict disease control and OS with high sensitivity and specificity in patients with RM-HNSCC treated with anti-PD-1 monotherapy.

T cell subtype profiling measures exhaustion and predicts anti-PD-1 response.

Anti-PD-1 therapy can provide long, durable benefit to a fraction of patients. The on-label PD-L1 test, however, does not accurately predict response. To build a better biomarker, we created a method called T Cell Subtype Profiling (TCSP) that characterizes the abundance of T cell subtypes (TCSs) in FFPE specimens using five RNA models. These TCS RNA models are created using functional methods, and robustly discriminate between naïve, activated, exhausted, effector memory, and central memory TCSs, without the reliance on non-specific, classical markers. TCSP is analytically valid and corroborates associations between TCSs and clinical outcomes. Multianalyte biomarkers based on TCS estimates predicted response to anti-PD-1 therapy in three different cancers and outperformed the indicated PD-L1 test, as well as Tumor Mutational Burden. Given the utility of TCSP, we investigated the abundance of TCSs in TCGA cancers and created a portal to enable researchers to discover other TCSP-based biomarkers.

Analytical Performance of an Immunoprofiling Assay Based on RNA Models.

As immuno-oncology drugs grow more popular in the treatment of cancer, better methods are needed to quantify the tumor immune cell component to determine which patients are most likely to benefit from treatment. Methods such as flow cytometry can accurately assess the composition of infiltrating immune cells; however, they show limited use in formalin-fixed, paraffin-embedded (FFPE) specimens. This article describes a novel hybrid-capture RNA sequencing assay, ImmunoPrism, that estimates the relative percentage abundance of eight immune cell types in FFPE solid tumors. Immune health expression models were generated using machine learning methods and used to uniquely identify each immune cell type using the most discriminatively expressed genes. The analytical performance of the assay was assessed using 101 libraries from 40 FFPE and 32 fresh-frozen samples. With defined samples, ImmunoPrism had a precision of ±2.72%, a total error of 2.75%, and a strong correlation (r2 = 0.81; P < 0.001) to flow cytometry. ImmunoPrism had similar performance in dissociated tumor cell samples (total error of 8.12%) and correlated strongly with immunohistochemistry (CD8: r2 = 0.83; P < 0.001) in FFPE samples. Other performance metrics were determined, including limit of detection, reportable range, and reproducibility. The approach used for analytical validation is shared here so that it may serve as a helpful framework for other laboratories when validating future complex RNA-based assays.

The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells.

Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP disclosed gap junction-mediated ICW which diffused more slowly and involved not more than five to six cells. After 2 months in culture, ICW appeared slower and wave propagation was much less inhibited by P2Y desensitization, suggesting an increase in gap junction-mediated ICW. After 4 months in culture cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells.

DNMT gene expression and methylome in Marek's disease resistant and susceptible chickens prior to and following infection by MDV.

Marek's disease (MD) is characterized as a T cell lymphoma induced by a cell-associated α-herpesvirus, Marek's disease virus type 1 (MDV1). As with many viral infectious diseases, DNA methylation variations were observed in the progression of MD; these variations are thought to play an important role in host-virus interactions. We observed that DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) were differentially expressed in chicken MD-resistant line 6 3 and MD-susceptible line 7 2 at 21 d after MDV infection. To better understand the role of methylation variation induced by MDV infection in both chicken lines, we mapped the genome-wide DNA methylation profiles in each line using Methyl-MAPS (methylation mapping analysis by paired-end sequencing). Collectively, the data sets collected in this study provide a more comprehensive picture of the chicken methylome. Overall, methylation levels were reduced in chickens from the resistant line 6 3 after MDV infection. We identified 11,512 infection-induced differential methylation regions (iDMRs). The number of iDMRs was larger in line 7 2 than in line 6 3, and most of iDMRs found in line 6 3 were overlapped with the iDMRs found in line 7 2. We further showed that in vitro methylation levels were associated with MDV replication, and found that MDV propagation in the infected cells was restricted by pharmacological inhibition of DNA methylation. Our results suggest that DNA methylation in the host may be associated with disease resistance or susceptibility. The methylation variations induced by viral infection may consequentially change the host transcriptome and result in diverse disease outcomes.

Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts.

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

The pathophysiology of disease in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by the expansion of a hematopoietic progenitor cell that has acquired a mutation in the X-linked PIGA gene. PNH occurs on the background of bone marrow failure. Bone marrow failure and the presence of the abnormal cells account for the clinical phenotype of patients with PNH including hemolysis, cytopenia, and thrombophilia. PIGA is essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor molecules. PNH blood cells are therefore deficient in all proteins that use such an anchor molecule for attachment to the cell membrane. Two of these proteins regulate complement activation on the cell surface. Their deficiency therefore explains the exquisite sensitivity of PNH red blood cells to complement-mediated lysis. Complement-mediated lysis of red blood cells is intravascular, and intravascular hemolysis contributes significantly to the morbidity and mortality in patients with this condition. PNH is an outstanding example of how an increased understanding of pathophysiology may directly improve the diagnosis, care, and treatment of disease.

P2 receptors in macrophage fusion and osteoclast formation.

Cells of the mononuclear phagocyte lineage fuse to form multinucleated giant cells and osteoclasts. Several lines of evidence suggest that P2 receptors, in particular P2X7, are involved in this process, although P2X7 is not absolutely required for fusion because P2X7-null mice form multinucleated osteoclasts. Extracellular ATP may be an important regulator of macrophage fusion.

ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells.

Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X(7) is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X(7), induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X(7) function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-kappaB ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X(7) protein, but little P2X(7) was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X(7) on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X(7) or another protein that is downregulated in concert with P2X(7) is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X(7) may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.