Plasma ctDNA enables early detection of temozolomide resistance mutations in glioma.

Background: Liquid biopsy based on circulating tumor DNA (ctDNA) is a novel tool in clinical oncology, however, its use has been limited in glioma to date, due to low levels of ctDNA. In this study, we aimed to demonstrate that sequencing techniques optimized for liquid biopsy in glioma patients can detect ctDNA in plasma with high sensitivity and with potential clinical utility. Methods: We investigated 10 glioma patients with tumor tissue available from at least 2 surgical operations, who had 49 longitudinally collected plasma samples available for analysis. Plasma samples were sequenced with CAPP-seq (AVENIO) and tissue samples with TSO500. Results: Glioma-derived ctDNA mutations were detected in 93.8% of plasma samples. 25% of all mutations detected were observed in plasma only. Mutations of the mismatch repair (MMR) genes MSH2 and MSH6 were the most frequent circulating gene alterations seen after temozolomide treatment and were frequently observed to appear in plasma prior to their appearance in tumor tissue at the time of surgery for recurrence. Conclusions: This pilot study suggests that plasma ctDNA in glioma is feasible and may provide sensitive and complementary information to tissue biopsy. Furthermore, plasma ctDNA detection of new MMR gene mutations not present in the initial tissue biopsy may provide an early indication of the development of chemotherapy resistance. Additional clinical validation in larger cohorts is needed.

Plasma ctDNA liquid biopsy of IDH1, TERTp, and EGFRvIII mutations in glioma.

Background: Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene mutations IDH1, TERTp, and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy. Methods: We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient). DNA was isolated from plasma and analyzed for IDH1, TERTp, and EGFRvIII mutations using ddPCR. Results: Total cfDNA was significantly associated with tumor grade, tumor volume, and both overall and progression-free survival for all gliomas as well as the grade 4 glioblastoma subgroup, but was not reliably associated with changes in tumor volume/progression during the patients' postoperative time course. IDH1 mutation was detected with 84% overall sensitivity across all plasma samples and 77% in the preoperative samples alone; however, IDH1 mutation plasma levels were not associated with tumor progression or survival. IDH1m plasma levels were not associated with pre- or postsurgery progression or survival. The TERTp C228T mutation was detected in the plasma ctDNA in 88% but the C250T variant in only 49% of samples. The EGFRvIII mutation was detected in plasma in 5 out of 7 patients (71%) with tissue EGFRvIII mutations in tumor tissue. Conclusions: Plasma ctDNA mutations detected with ddPCR provide excellent diagnostic sensitivity for IDH1, TERTp-C228T, and EGFRvIII mutations in glioma patients. Total cfDNA may also assist with prognostic information. Further studies are needed to validate these findings and the clinical role of ctDNA in glioma.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

Fast Quantitation of TGF-ß Signaling Using Adenoviral Reporter.

The transforming growth factor-ß (TGF-ß) is a multifunctional cytokine critical for embryogenesis and tissue homeostasis. Alterations in TGF-ß signaling pathway are observed in several types of malignant tumors and often related with cancer progression and metastasis. TGF-ß signaling is transduced across the plasma membrane after ligand-receptor binding and consequent phosphorylation of the intracellular effectors SMAD2/3 by TGF-ß receptors. Phosphorylated SMAD2/3 accumulates in the nucleus after complex formation with SMAD4 to act as transcription factors and regulate the expression of genes critically associated with cell proliferation and differentiation. Traditional methodologies used to assess TGF-ß signaling pathway lack accuracy and/or show poor scalability, limiting in vitro experiments and almost excluding their use in vivo. Here, we describe a fast method to quantitate TGF-ß signaling pathway activity in vitro and in vivo by using adenoviral reporters. Its implementation in vitro allows quantitating cell response to TGF-ß at concentrations as low as pictograms/mL. Additionally, the use of an in vivo imaging system (IVIS) enables quantitating and monitoring TGF-ß signaling pathway activity over time during cancer progression, eliminating the requirement of animal euthanasia at multiple time points for this purpose. Importantly, this protocol has been consistently used in different models and effectively led to the visualization and measurement of TGF-ß activity levels. Improving the sensitivity, specificity, and scalability of methods focused on characterizing this and other molecular pathways will result in a better understanding of their biology in physiological and pathological processes.