Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set

Charting a dynamic DNA methylation landscape of the human genome

DNA methylation is a defining feature of mammalian cellular identity and essential for normal development1,2. Most cell types, except germ cells and pre-implantation embryos3–5, display relatively stable DNA methylation patterns with 70–80% of all CpGs being methylated6. Despite recent advances we still have a too limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in depth analysis of 42 whole genome bisulfite sequencing (WGBS) data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, a majority of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription factor binding sites (TFBS), which allow identification of key lineage specific regulators. In addition, differentially methylated regions (DMRs) often harbor SNPs associated with cell type related diseases as determined by GWAS. The results also highlight the general inefficiency of WGBS as 70–80% of the sequencing reads across these data sets provided little or no relevant information regarding CpG methylation. To further demonstrate the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore our selected DMRs can serve as a starting point to help guide novel, more effective reduced representation approaches to capture the most informative fraction of CpGs as well as further pinpoint putative regulatory elements

Discontinuation of BRAF/MEK-Directed Targeted Therapy after Complete Remission of Metastatic Melanoma—A Retrospective Multicenter ADOReg Study

The advent of BRAF/MEK inhibitors (BRAFi/MEKi) has significantly improved progressionfree (PFS) and overall survival (OS) for patients with advanced BRAF-V600-mutant melanoma. Long-term survivors have been identified particularly among patients with a complete response (CR) to BRAF/MEK-directed targeted therapy (TT). However, it remains unclear which patients who achieved a CR maintain a durable response and whether treatment cessation might be a safe option in these patients. Therefore, this study investigated the impact of treatment cessation on the clinical course of patients with a CR upon BRAF/MEK-directed-TT. We retrospectively selected patients with BRAF-V600-mutant advanced non-resectable melanoma who had been treated with BRAFi ± MEKi therapy and achieved a CR upon treatment out of the multicentric skin cancer registry ADOReg. Data on baseline patient characteristics, duration of TT, treatment cessation, tumor progression (TP) and response to second-line treatments were collected and analyzed. Of 461 patients who received BRAF/MEK-directed TT 37 achieved a CR. TP after initial CR was observed in 22 patients (60%) mainly affecting patients who discontinued TT (n = 22/26), whereas all patients with ongoing TT (n = 11) maintained their CR. Accordingly, patients who discontinued TT had a higher risk of TP compared to patients with ongoing treatment (p < 0.001). However, our data also show that patients who received TT for more than 16 months and who discontinued TT for other reasons than TP or toxicity did not have a shorter PFS compared to patients with ongoing treatment. Response rates to second-line treatment being initiated in 21 patients, varied between 27% for immune-checkpoint inhibitors (ICI) and 60% for BRAFi/MEKi rechallenge. In summary, we identified a considerable number of patients who achieved a CR upon BRAF/MEK-directed TT in this contemporary realworld cohort of patients with BRAF-V600-mutant melanoma. Sustained PFS was not restricted to ongoing TT but was also found in patients who discontinued TT

Immune Checkpoint Blockade for Metastatic Uveal Melanoma: Re-Induction following Resistance or Toxicity

Re-induction with immune checkpoint blockade (ICB) needs to be considered in many patients with uveal melanoma (UM) due to limited systemic treatment options. Here, we provide hitherto the first analysis of ICB re-induction in UM. A total of 177 patients with metastatic UM treated with ICB were included from German skin cancer centers and the German national skin cancer registry (ADOReg). To investigate the impact of ICB re-induction, two cohorts were compared: patients who received at least one ICB re-induction (cohort A, n = 52) versus those who received only one treatment line of ICB (cohort B, n = 125). In cohort A, a transient benefit of overall survival (OS) was observed at 6 and 12 months after the treatment start of ICB. There was no significant difference in OS between both groups (p = 0.1) with a median OS of 16.2 months (cohort A, 95% CI: 11.1–23.8) versus 9.4 months (cohort B, 95% CI: 6.1–14.9). Patients receiving re-induction of ICB (cohort A) had similar response rates compared to those receiving ICB once. Re-induction of ICB may yield a clinical benefit for a small subgroup of patients even after resistance or development of toxicities

Brain metastasis and survival outcomes after first-line therapy in metastatic melanoma: a multicenter DeCOG study on 1704 patients from the prospective skin cancer registry ADOREG

Background Despite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis and survival outcomes dependent on the type of first-line therapy.Methods Patients with metastatic, non-resectable melanoma (AJCCv8 stage IIIC–V) without brain metastasis at start of first-line therapy (1L-therapy) were identified from the prospective multicenter real-world skin cancer registry ADOREG. Study endpoints were incidence of brain metastasis, brain metastasis-free survival (BMFS), progression-free survival (PFS), and overall survival (OS).Results Of 1704 patients, 916 were BRAF wild-type (BRAFwt) and 788 were BRAF V600 mutant (BRAFmut). Median follow-up time after start of 1L-therapy was 40.4 months. BRAFwt patients received 1L-therapy with immune checkpoint inhibitors (ICI) against CTLA-4+PD-1 (n=281) or PD-1 (n=544). In BRAFmut patients, 1L-therapy was ICI in 415 patients (CTLA-4+PD-1, n=108; PD-1, n=264), and BRAF+MEK targeted therapy (TT) in 373 patients. After 24 months, 1L-therapy with BRAF+MEK resulted in a higher incidence of brain metastasis compared with PD-1±CTLA-4 (BRAF+MEK, 30.3%; CTLA-4+PD-1, 22.2%; PD-1, 14.0%). In multivariate analysis, BRAFmut patients developed brain metastases earlier on 1L-therapy with BRAF+MEK than with PD-1±CTLA-4 (CTLA-4+PD-1: HR 0.560, 95% CI 0.332 to 0.945, p=0.030; PD-1: HR 0.575, 95% CI 0.372 to 0.888, p=0.013). Type of 1L-therapy, tumor stage, and age were independent prognostic factors for BMFS in BRAFmut patients. In BRAFwt patients, tumor stage was independently associated with longer BMFS; ECOG Performance status (ECOG-PS), lactate dehydrogenase (LDH), and tumor stage with OS. CTLA-4+PD-1 did not result in better BMFS, PFS, or OS than PD-1 in BRAFwt patients. For BRAFmut patients, multivariate Cox regression revealed ECOG-PS, type of 1L-therapy, tumor stage, and LDH as independent prognostic factors for PFS and OS. 1L-therapy with CTLA-4+PD-1 led to longer OS than PD-1 (HR 1.97, 95% CI 1.122 to 3.455, p=0.018) or BRAF+MEK (HR 2.41, 95% CI 1.432 to 4.054, p=0.001), without PD-1 being superior to BRAF+MEK.Conclusions In BRAFmut patients 1L-therapy with PD-1±CTLA-4 ICI resulted in a delayed and less frequent development of brain metastasis compared with BRAF+MEK TT. 1L-therapy with CTLA-4+PD-1 showed superior OS compared with PD-1 and BRAF+MEK. In BRAFwt patients, no differences in brain metastasis and survival outcomes were detected for CTLA-4+PD-1 compared with PD-1

The Crowdsourced Replication Initiative: Investigating Immigration and Social Policy Preferences. Executive Report.

In an era of mass migration, social scientists, populist parties and social movements raise concerns over the future of immigration-destination societies. What impacts does this have on policy and social solidarity? Comparative cross-national research, relying mostly on secondary data, has findings in different directions. There is a threat of selective model reporting and lack of replicability. The heterogeneity of countries obscures attempts to clearly define data-generating models. P-hacking and HARKing lurk among standard research practices in this area.This project employs crowdsourcing to address these issues. It draws on replication, deliberation, meta-analysis and harnessing the power of many minds at once. The Crowdsourced Replication Initiative carries two main goals, (a) to better investigate the linkage between immigration and social policy preferences across countries, and (b) to develop crowdsourcing as a social science method. The Executive Report provides short reviews of the area of social policy preferences and immigration, and the methods and impetus behind crowdsourcing plus a description of the entire project. Three main areas of findings will appear in three papers, that are registered as PAPs or in process

Influence of One Specific Carbon–Carbon Bond on the Quality, Stability, and Photovoltaic Performance of Hybrid Organic–Inorganic Bismuth Iodide Materials

Hybrid organic–inorganic halide perovskites are promising materials for thin-film solar cells. However, the toxicity and instability of best-in-class lead–halide perovskite materials make them nonideal. To combat these issues, we replaced lead with bismuth and explored the sensitivity of these new lead-free materials to the valency and bonding of their cationic organic groups. Specifically, we synthesized and characterized the materials properties and photophysical properties of hexane-1,6-diammonium bismuth pentaiodide ((HDA^{2+}Bil_{5}) and compared them to an analogue containing a more volatile organic group with half the number of carbon and nitrogen atoms in the form of n-propylammonium ((PA+)_{x}Bil_{3+x}, where 1<x<3). The full crystallographic structures of (HDA^{2+})Bil_{5} and (PA^{+})_{x}Bil_{3+x} were resolved by single-crystal X-ray diffraction. (HDA^{2+})Bil_{5} was shown to be pure-phase and have a one-dimensional structure, whereas (PA^{+})_{x}Bil_{3+x} was shown to be a mix of one-dimensional and zero-dimensional phases. Structures of the materials were confirmed by synchrotron X-ray diffraction of powders. Both (HDA^{2+})Bil_{5} and (PA^{+})_{x}Bil_{3+x} exhibit steady-state photoluminescence at room temperature. Density functional theory calculations of (HDA^{2+})Bil_{5} predict electronic absorption features and a ∼2 eV bandgap that are consistent with those observed experimentally. Structure–property relationships of the materials were examined, and moisture tolerance and film quality were found to be superior for dication-containing (HDA^{2+})Bil_{5} in relation to monocation-containing (PA^{+})_{x}Bil_{3+x}. We hypothesize that these trends are in part due to a molecular bridging effect enabled by the presence of the dicationic hexanediammonium groups in (HDA^{2+})Bil_{5}. Solar cells fabricated using (HDA^{2+})Bil_{5} as the photoactive layer exhibited photovoltaic action while those containing (PA^{+})_{x}Bil_{3+x} did not, suggesting that organic dicationic groups are beneficial to light-absorber morphology and ultimately solar-cell performance