Epigenetic reprogramming of pancreatic cancer cells as a new therapeutic option

Large-scale gene expression analyses have demonstrated that pancreatic ductal adenocarcinoma can be classified into different molecular subtypes with clinical significance (Collisson et al., 2011, Moffitt et al., 2016, Bailey et al., 2016). So far, great effort has been put into unveiling the factors responsible for tumor heterogeneity in pancreatic cancer. Since epigenetic modifiers are, besides the four driver gene mutations KRAS, p16, p53, and SMAD4, among the most frequently mutated genes in PDAC, this study aimed at investigating the role of epigenetic changes in the two molecular PDAC cancer subtypes, represented by a classical and basal phenotype, as well as their therapeutic potential in pancreatic cancer cell lines (Bailey et al., 2016). The data showed that subtype-specific gene expression of cellular differentiation marker genes, such as EpCAM and GATA6, is epigenetically regulated. Chromatin-immunoprecipitation results demonstrated that the expression of these epithelial differentiation marker genes is activated through histone acetylation marks in the classical subtype. In contrast, their expression is repressed in the basal or quasimesenchymal subtype through increased levels of histone ubiquitination as well as a loss of histone acetylation marks. DNA methylation seemed to only play a minor part in regulating subtype-specific gene expression profiles of EpCAM and GATA6. Despite subtype-specific histone acetylation levels, single-drug treatment with chemical inhibitors targeting histone acetylation and deacetylation marks only showed limited effects in vitro. Classical and basal PDAC cell lines were almost completely resistant to HAT inhibitor treatment with A485. Only one of the basal cell lines, MIAPaca-2, reached a 50 % survival rate at the maximum dosage of 10 µM A485 (see Figure 12A, left panel). High doses of the HDAC inhibitor Vorinostat were able to inhibit cell survival to a greater extent, but the response was independent of the transcriptomic subtypes. It is possible that a compensatory upregulation of other epigenetic modifications limits the therapeutic effects. Hence, a multiplex CRISPR/Cas9 knockout plasmid targeting a combination of epigenetic modifiers (HDAC2, DNMT3A, RING1B) was constructed to induce simultaneous genetic knockout of all three target genes. However, transfection of a basal pancreatic cancer cell line with this plasmid did not yield a successful knockout cell line. Most likely, the combinatory knockout impaired critical cellular functions to such an extent that cell death occurred. To overcome the limitations of a multiplex CRISPR/Cas9 knockout strategy, a successive knockout of one target gene after the other might be a more successful strategy to analyze the effect of a combinatory loss of different epigenetic modifiers. Furthermore, a selection marker should be included in the plasmids to ensure successful transfection. The generated knockout cell line can then be used for transcriptome analysis by RNA sequencing as well as for basic cell assays and drug sensitivity tests. In order to translate preclinical data with epigenetic inhibitors into successful clinical trials, further studies are needed to determine subtype-specific changes after epigenetic targeting. For instance, unpublished data within the working group showed that HAT inhibitor treatment of cell lines with a classical PDAC subtype strongly downregulated the expression of GATA6 and decreased Gemcitabine drug sensitivity indicating a poorer outcome. These results emphasize the importance of establishing patient stratification systems in order to maximize the success of current treatment strategies. Overall, this thesis showed that the transcriptomic profiles defining molecular PDAC subtypes are in part regulated through epigenetic modifications. Although the targeting of single epigenetic modifiers showed some success in tumor cell reprogramming, the therapeutic targeting with epigenetic drugs remains limited. Thus, the precise effects of combination therapies with multiple epigenetic inhibitors need further investigation

Immune Cell and Stromal Signature Associated with Progression-free Survival of Patients with Resected Pancreatic Ductal Adenocarcinoma

Background & Aims: Changes to the microenvironment of pancreatic ductal adenocarcinomas (PDACs) have been associated with poor outcomes of patients. We studied the associations between composition of the pancreatic stroma (fibrogenic, inert, dormant, or fibrolytic stroma) and infiltration by inflammatory cells and times of progression-free survival (PFS) of patients with PDACs after resection. Methods: We obtained 1824 tissue microarray specimens from 385 patients included in the European Study Group for Pancreatic Cancer trial 1 and 3 and performed immunohistochemistry to detect alpha smooth muscle actin, type 1 collagen, CD3, CD4, CD8, CD68, CD206, and neutrophils. Tumors that expressed high and low levels of these markers were compared with patient outcomes using Kaplan-Meier curves and multivariable recursive partitioning for discrete-time survival tree analysis. Prognostic index was delineated by a multivariable Cox proportional hazards model of immune cell and stromal markers and PFS. Findings were validated using 279 tissue microarray specimens from 93 patients in a separate cohort. Results: Levels of CD3, CD4, CD8, CD68, and CD206 were independently associated with tumor recurrence. Recursive partitioning for discrete-time survival tree analysis identified a high level of CD3 as the strongest independent predictor for longer PFS. Tumors with levels of CD3 and high levels of CD206 associated with a median PFS time of 16.6 months and a median prognostic index of –0.32 (95% confidence interval [CI] –0.35 to –0.31), whereas tumors with low level of CD3 cell and low level of CD8 and high level of CD68 associated with a median PFS time of 7.9 months and a prognostic index of 0.32 (95% CI 0.050–0.32); we called these patterns histologic signatures. Stroma composition, when unassociated with inflammatory cell markers, did not associate significantly with PFS. In the validation cohort, the histologic signature resulted in an error matrix accuracy of predicted response of 0.75 (95% CI 0.64–0.83; accuracy P < .001). Conclusions: In an analysis of PDAC tissue microarray specimens, we identified and validated a histologic signature, based on leukocyte and stromal factors, that associates with PFS times of patients with resected PDACs. Immune cells might affect the composition of the pancreatic stroma to affect progression of PDAC. These findings provide new insights into the immune response to PDAC

Influence of spectral shaping and tube voltage modulation in ultralow-dose computed tomography of the abdomen

Abstract Purpose Unenhanced abdominal CT constitutes the diagnostic standard of care in suspected urolithiasis. Aiming to identify potential for radiation dose reduction in this frequent imaging task, this experimental study compares the effect of spectral shaping and tube voltage modulation on image quality. Methods Using a third-generation dual-source CT, eight cadaveric specimens were scanned with varying tube voltage settings with and without tin filter application (Sn 150, Sn 100, 120, 100, and 80 kVp) at three dose levels (3 mGy: standard; 1 mGy: low; 0.5 mGy: ultralow). Image quality was assessed quantitatively by calculation of signal-to-noise ratios (SNR) for various tissues (spleen, kidney, trabecular bone, fat) and subjectively by three independent radiologists based on a seven-point rating scale (7 = excellent; 1 = very poor). Results Irrespective of dose level, Sn 100 kVp resulted in the highest SNR of all tube voltage settings. In direct comparison to Sn 150 kVp, superior SNR was ascertained for spleen (p ≤ 0.004) and kidney tissue (p ≤ 0.009). In ultralow-dose scans, subjective image quality of Sn 100 kVp (median score 3; interquartile range 3–3) was higher compared with conventional imaging at 120 kVp (2; 2–2), 100 kVp (1; 1–2), and 80 kVp (1; 1–1) (all p < 0.001). Indicated by an intraclass correlation coefficient of 0.945 (95% confidence interval: 0.927–0.960), interrater reliability was excellent. Conclusions In abdominal CT with maximised dose reduction, tin prefiltration at 100 kVp allows for superior image quality over Sn 150 kVp and conventional imaging without spectral shaping