Somatic intronic microsatellite loci differentiate glioblastoma from lower-grade gliomas

Genomic studies of glioma sub-types have amassed new disease specific mutations, yet these only partially explain how mutations are linked to predisposition or progression. We hypothesized that microsatellite variation could expand the understanding of glioma etiology. Furthermore, germline markers for gliomas are typically undetectable; therefore we also hypothesize that the predictability of cancer-associated microsatellite loci in germline DNA may support the current hypothesis of a glioma cell of origin. In this study, “normal” germline exome sequenced DNA from the 1000 Genomes Project (n=390) were compared with exome sequences from germlines of subjects with WHO grade II and III lower-grade glioma (LGG, n=136) and WHO grade IV glioblastoma (GBM, n=252) from The Cancer Genome Atlas to identify microsatellite loci non-randomly associated with glioma. From germline data, we identified 48 GBM-specific loci, 42 Lower-grade glioma specific loci and 29 loci that distinguish GBM from LGG (p≤ 0.01). We then attempted to distinguish WHO grade II glioma (n=67) from GBM resulting in 8 informative loci. Significantly, in all glioma grades, comparisons between tumor and matched germline sequences demonstrated no significant differences in these variants (p≥ 0.01). Therefore, these microsatellite loci are considered to be components of grade-specific signatures for glioma which distinguish germline sequences of individuals with cancer from those of individuals that are “normal”. In order to better understand the significance of these loci, we identified biological processes enriched in genes with these variants. Most strikingly, six helicase genes were enriched in the GBM cohort (p≤ 1.0 x10-3). The preservation of these glioma-specific loci could therefore serve as valuable diagnostic and therapeutic markers; especially since the heterogeneity of tumor cell populations can obscure the identification of mutations preceding a metastatic phenotype

Correction: A novel epigenetic modulating agent sensitizes pancreatic cells to a chemotherapy agent.

[This corrects the article DOI: 10.1371/journal.pone.0199130.]

A novel epigenetic modulating agent sensitizes pancreatic cells to a chemotherapy agent.

Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second leading cause of cancer mortality by 2030. PDAC remains resistant to the majority of systemic chemotherapies. In this paper, we explore if epigenetic sensitization can improve chemotherapy response in PDAC. Multiple PDAC cell lines were tested with serial concentrations of the epigenetic modulators 5-azacitidine (Aza) and guadecitabine (SGI-110). Guadecitabine was effective at inhibiting the expression of DNA Methyltransferase 1 (DNMT1) and in decreasing cell viability at nanomolar concentrations. We also report that guadecitabine has increased efficacy following a delay period or as we reference, a 'rest period'. Sensitization with guadecitabine improved response to the chemotherapeutic agent-Irinotecan- as measured by decreased cell viability and accompanied by an increase in caspase activity. Additional studies are needed to understand the mechanism of action

Variable response of pancreatic cell lines to epigenetic modulator <i>in vitro</i>.

<p>Cells were treated daily with an epigenetic modulator at different concentrations for up to 5 days (Fig 1A–1E). For guadecitabine, doses in nanomolar range are magnified to demonstrate the effectiveness of this modulator at low doses. At the end of the treatment, a standard MTT assay was used to determine cell viability. The percent viability was calculated by dividing each data point by its respective control and designated by percentages. Data shown represents mean ± SEM.</p

Guadecitabine sensitized PDAC cells to chemotherapy.

<p>Miapaca-2 (a) and Panc1 (b) were treated with 0.14x10^-3 μM for 3 days. Cells were treated with varying concentrations of Irinotecan either immediately or after 5 days of rest. Cells were incubated in Irinotecan for an additional 5 days. Viability was measured using a standard MTT assay. Each data point was compared to the control respectively, and represented as percent cell viability. Statistical analysis was performed using a non-parametric t test. Data shown represents mean ± SEM.</p

Pre-sensitized cells by guadecitabine upregulated apoptosis.

<p>Miapaca-2 (a) and Panc1 (b) cells were pretreated with 0.14x10^-3 μM of guadecitabine for 3 days. The cells were either treated immediately or given rest for 5 days. Afterwards, the cells were treated with different concentrations of Irinotecan for 5 days. Caspase activity was measured and statistical analyses was done as mentioned earlier. A non-parametric t test was used for statistical analysis. Data shown represents mean ± SEM.</p

A phase 2 trial of gemcitabine and docetaxel in patients with metastatic colorectal adenocarcinoma with methylated checkpoint with forkhead and ring finger domain promoter and/or microsatellite instability phenotype

Abstract We previously reported CHFR methylation in a subset of colorectal cancer (CRC; ∼30%) with high concordance with microsatellite instability (MSI). We also showed that CHFR methylation predicted for sensitivity to docetaxel, whereas the MSI‐high phenotypes were sensitive to gemcitabine. We hypothesized that this subset of patients with CRC would be selectively sensitive to gemcitabine and docetaxel. We enrolled a Phase 2 trial of gemcitabine and docetaxel in patients with MSI‐high and/or CHFR methylated CRC. The primary objective was Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 response rate. Enrolled patients were treated with gemcitabine 800 mg/m2 on days 1 and 8 and docetaxel 70 mg/m2 on day 8 of each 21‐day cycle. A total of 6 patients with CHFR‐methylated, MSI‐high CRC were enrolled from September 2012 to August 2016. The study was closed in September of 2017 due to poor accrual prior to reaching the first interim assessment of response rate, which would have occurred at 10 patients. No RECIST criteria tumor responses were observed, with 3 patients (50%) having stable disease as best response, 1 lasting more than 9 months. Median progression‐free survival (PFS) was 1.79 months (95% confidence interval [CI] = 1.28, not available [NA]) and median overall survival (OS) was 15.67 months (95% CI = 4.24, NA). Common grade 3 toxicities were lymphopenia (67%), leukopenia (33%), and anemia (33%). Although negative, this study establishes a proof‐of‐concept for the implementation of epigenetic biomarkers (CHFR methylation/MSI) as inclusion criteria in a prospective clinical trial to optimize combinatorial strategies in the era of personalized medicine. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? CHFR silencing via DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumors. The frequent association of CHFR methylation with microsatellite instability (MSI) suggested a possible combination therapy with gemcitabine, because the MSI phenotype may result in sensitivity to nucleoside analogues. WHAT QUESTION DID THIS STUDY ADDRESS? We hypothesized that metastatic colorectal cancer (mCRC), which have CHFR methylation and MSI phenotype were sensitive to gemcitabine and docetaxel, and have designed this Phase 2 trial in biomarker‐selected mCRC to test this prediction. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The study enrolled a molecularly defined subgroup of patients with colorectal cancer (CRC) and showed that the combination is safe in this population. Nevertheless, due to poor enrollment and early termination, no conclusions on the primary and secondary end points could be made. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? This study supports the feasibility of implementing DNA methylation markers in a prospective clinical trial and further efforts toward their application as predictive biomarkers for therapeutic agents in defined subsets of patients are warranted

Effect of guadecitabine on DNA Methyltransferase 1, and its delayed response.

<p>Miapaca-2 and Panc1 were treated with guadecitabine (0–8 μM) for three days. The two cell lines were fixed and further stained with Propidium Iodide and analyzed using flow cytometry methods to evaluate cell cycle arrest. The pink color represents G0G1 phase, dark blue -S phase, and light blue -G2_M phase, respectively. The data was analyzed using DIVA software (a). Pancreatic cells were treated with guadecitabine at 0.14x10^-3 μM (b-f) for 3 days. Levels of DNMT1 expression were measured via Western blotting following treatment (b). To quantify apoptotic activity, Caspase 3/7 levels were measured after 0, 5, or 10 days of rest. Each experimental value was compared with control and plotted in terms of percentages using Graph Pad Prism (c).The amount of necrosis was calculated using a LDH detection kit following treatment with no rest or rest (d). Images were taken for treated and untreated cells for Miapaca-2 and Panc1 at 10X magnification using EVOS cell imaging system (e-f).Statistical analysis was performed using a one-way ANOVA and significance measured with a Tukey’s multiple comparison test (c) or utilizing a non-parametric t test (d). Data represented shows mean ± SEM (2c-2d).</p