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\"Set in a Madison, WI, vintage clothing shop, where every garment has a story, just like the women who meet there--a pregnant teenager, an Indian housewife, and a divorcee looking for love--who over the course of a few months come to transform the store and each other\"-- Provided by publisher.

Background Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. Results Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×10 6 -86×10 6 unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. Conclusions Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Background Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), are universally fatal pediatric brain tumors with no effective treatments. DIPG tumors actively utilize mitochondrial oxidative phosphorylation (OXPHOS). Inhibition of Complex I (a core OXPHOS component) by phenformin radiosensitizes DIPG in vitro and in vivo. However, phenformin’s clinical application is limited by its risk of lactic acidosis. We investigated whether co-administration of the pyruvate-dehydrogenase-kinase (PDK) inhibitor dichloroacetate (DCA) can mitigate phenformin-induced acidosis while enhancing its anti-tumor activity. Methods Patient-derived DIPG cells (SU-DIPG17, HSJD-DIPG007, SU-DIPG-VI) were treated with phenformin (0.625 mM), DCA (25 mM) or both. Mitochondrial and glycolytic flux (oxygen consumption rate (OCR) / extracellular acidification rate (ECAR)), ATP production, Reactive Oxygen Species (ROS), cell-cycle and apoptosis were quantified alongside RNA-seq and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS)/MS metabolomics. Hypoxia was measured in neurospheres using fluorescence. Radiosensitization was assessed by γ-H2AX foci and clonogenic survival. In vivo, HSJD-DIPG007 orthotopic xenografts received phenformin (125 mg/kg/day) and DCA (250 mg/kg/day) by oral gavage for 4 weeks, alone or with focal brain-stem irradiation (2 Gy × 10); tumor immunohistochemistry and survival were recorded. Results DCA alone shifted glucose metabolism from glycolysis to oxidative phosphorylation (OXPHOS), reducing ECAR and intracellular lactate. When combined with phenformin, DCA significantly suppressed phenformin-induced glycolysis and ECAR while further reducing ATP. In vitro, this combination induced synergistic cell-cycle arrest, apoptosis, and ROS-induced DNA damage. Multi-omics integration revealed coordinated repression of glycolytic/ hypoxia-inducible factor (HIF) programs and diversion of glucose into redox-supportive pentose-phosphate pathways. Hypoxic staining confirmed reduced hypoxia and HIF-1α in 3D neurospheroids. The combination produced additional in vitro radiosensitization, with the phenformin + DCA + radiation triple regimen achieving the greatest γ-H2AX persistence and clonogenic kill. In mice, the triple combination schedule incurred systemic toxicity, reflected from weight loss, and did not extend survival over 4 weeks treatment. Conclusions DCA effectively counteracts phenformin-induced lactic acidosis in vitro yet heightens metabolic stress and radiosensitization in DIPG cells, providing proof-of-concept that a carefully chosen metabolic partner can boost tumor control. However, weight loss in vivo limited the length of treatment schedule and optimizing dose and schedule, or selecting safer mitochondrial inhibitors with PDK blockade, will be essential next steps before determining in vivo efficacy of this metabolic strategy. Simple summary Diffuse midline gliomas, including diffuse intrinsic pontine glioma, are aggressive childhood brain tumors with no effective treatments. These tumors depend on energy produced by mitochondria, making mitochondrial metabolism a promising target for new therapies. However, drugs like phenformin, which block mitochondrial metabolism, can cause dangerous lactic acid buildup, limiting their clinical use. To address this, our study combined phenformin with dichloroacetate, a drug that targets glucose metabolism and can reduce lactic acidosis. This combination successfully lowered acid buildup, disrupted tumor cell metabolism, and enhanced cancer cell sensitivity to radiation in vitro, a key treatment for these tumors. However, a pilot in vivo study highlights the challenges that will need to be overcome in dosing and toxicity when combining multiple metabolic drugs with radiation.

Background The four-transmembrane MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but these had remained unconfirmed. MAL2 binds tumor protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of MAL2 and TPD52 overexpression was unknown. Methods Immunohistochemical analyses of MAL2 and TPD52 expression were performed using tissue microarray sections including benign, borderline and malignant epithelial ovarian tumours. Inmmunohistochemical staining intensity and distribution was assessed both visually and digitally. Results MAL2 and TPD52 were significantly overexpressed in high-grade serous carcinomas compared with serous borderline tumours. MAL2 expression was highest in serous carcinomas relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. MAL2 expression was not related to patient survival, however high-level TPD52 staining was significantly associated with improved overall survival in patients with stage III serous ovarian carcinoma (log-rank test, p < 0.001; n = 124) and was an independent predictor of survival in the overall carcinoma cohort (hazard ratio (HR), 0.498; 95% confidence interval (CI), 0.34-0.728; p < 0.001; n = 221), and in serous carcinomas (HR, 0.440; 95% CI, 0.294-0.658; p < 0.001; n = 182). Conclusions MAL2 is frequently overexpressed in ovarian carcinoma, and TPD52 overexpression is a favourable independent prognostic marker of potential value in the management of ovarian carcinoma patients.

The use of vaginal immobilization balloons placed into the vagina for immobilization of tandem and ovoid (T+O) applicator during high-dose-rate (HDR) brachytherapy delivery has been used at our institution, and seems to have improved our patient comfort, decreased procedure time, and minimized applicator misplacement. We aimed to show that these balloons, while originally marketed for single-day use, are safe and maintain applicator positioning/dosimetry when left overnight for treatment delivery on sequential days. Forty-two paired computed tomography (CT) scans from thirteen patients who underwent T+O HDR treatments on sequential days with vaginal immobilization balloons overnight were retrospectively compared to calculate mean change of balloon volumes and balloon/T+O distance to bony landmarks. Dosimetric planning was retroactively performed on day 2 using CT scan of each pair, and the change in estimated radiation delivery to the bladder and rectum was compared. No statistically significant overnight changes were found in balloon volumes or anterior balloon positioning. The posterior balloon shifted -0.29 ±0.46 cm ( = 0.03) to the anterior public symphysis and 0.32 ±0.50 cm ( = 0.01) to the right femoral head. The tandem shifted 0.37 ±0.39 cm ( = 0.002) to the pubic symphysis. There was no significant difference found in radiation delivered to the bladder or rectum between the paired scans. This study showed minimal change in balloon volumes, balloons/T+O positioning, or in radiation dose to bladder and rectum when the applicator remained overnight. These findings support that inflatable vaginal immobilization balloons remaining overnight for additional HDR T+O treatments on sequential days, is safe and provides stable dosimetry.

Background We previously identified that the CpG island-associated promoter of the novel lincRNA ZNF300P1 (also known as LOC134466 ) is frequently hypermethylated and silenced in ovarian cancer tissues. However, the function of ZNF300P1 was unknown. In this report we demonstrate that ZNF300P1 is involved in the regulation of key cell cycle and cell motility networks in human ovarian surface epithelial cells, and may play a role in promoting metastasis in ovarian cancer cells. Methods We applied methylated DNA immunoprecipitation on whole genome promoter tiling arrays and Sequenom assays to examine methylation status of ZNF300P1 in multiple ovarian cancer cell lines, as well as in normal ovarian and ovarian tumor tissues. Transcript profiling was used to investigate the effects of ZNF300P1 suppression in ovarian cancer cells. We utilized siRNA knockdown in normal ovarian surface epithelial cells and performed cellular proliferation, migration and adhesion assays to validate and explore the profiling results. Results We demonstrate that ZNF300P1 is methylated in multiple ovarian cancer cell lines. Loss of ZNF300P1 results in decreased cell proliferation and colony formation. In addition, knockdown of the ZNF300P1 transcript results in aberrant and less persistent migration in wound healing assays due to a loss of cellular polarity. Using an ex vivo peritoneal adhesion assay, we also reveal a role for ZNF300P1 in the attachment of ovarian cancer cells to peritoneal membranes, indicating a potential function of ZNF300P1 expression in metastasis of ovarian cancer cells to sites within the peritoneal cavity. Conclusion Our findings further support ZNF300P1 as frequently methylated in ovarian cancer and reveal a novel function for ZNF300P1 lincRNA expression in regulating cell polarity, motility, and adhesion and loss of expression may contribute to the metastatic potential of ovarian cancer cells.