Abstract
With the approval of new therapies targeting the PI3K pathway, the detection of PIK3CA mutations has become a key factor in treatment management for HR+/HER2− metastatic breast cancer (MBC). ...We developed multiplex digital PCR (dPCR) assays to detect and quantify PIK3CA mutations. A first screening assay allows the detection of 21 mutations, with a drop-off system targeting the 542–546 hotspot mutations combined with the simultaneous detection of N345K, C420R, H1047L and H1047R mutations. In the case of a positive result, a sequential strategy based on other assays that we have developped allows for precise mutation identification. Clinical validity was determined by analyzing plasma circulating free DNA (cfDNA) from 213 HR+/HER2− MBC samples, as well as DNA extracted from 97 available matched tumors from 89 patients. Our assays have shown reliable specificity, accuracy and reproducibility, with limits of blank of three and four droplets for the screening assay. Sixty-eight patients (32%) had at least one PIK3CA mutation detectable in their plasma, and we obtained 83.1% agreement between the cfDNA analysis and the corresponding tumors. The high sensitivity and robustness of these new dPCR assays make them well-suited for rapid and cost-effective detection of PIK3CA mutations in the plasma of MBC patients.
Cellular stress induced by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) is a central feature of secretory cells and is observed in many tissues in various diseases, ...including cancer, diabetes, obesity, and neurodegenerative disorders. Cellular adaptation to ER stress is achieved by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that transmits information about the protein folding status at the ER to the cytosol and nucleus to restore proteostasis. In the past decade, ER stress has emerged as a major pathway in remodeling gene expression programs that either prevent transformation or provide selective advantage in cancer cells. Controlled by the formation of a dynamic scaffold onto which many regulatory components assemble, UPR signaling is a highly regulated process that leads to an integrated reprogramming of the cell. In this Review, we provide an overview of the regulatory mechanisms underlying UPR signaling and how this pathway modulates cancer progression, particularly the aggressiveness and chemotherapeutic resistance exhibited by glioblastoma, a form of brain cancer. We also discuss the emerging cross-talk between the UPR and related metabolic processes to ensure maintenance of proteostasis, and we highlight possible therapeutic opportunities for targeting the pathway with small molecules.
Changes in promoter DNA methylation pattern of genes involved in key biological pathways have been reported in glioblastoma. Genome-wide assessments of DNA methylation levels are now required to ...decipher the epigenetic events involved in the aggressive phenotype of glioblastoma, and to guide new treatment strategies.
We performed a whole-genome integrative analysis of methylation and gene expression profiles in 40 newly diagnosed glioblastoma patients. We also screened for associations between the level of methylation of CpG sites and overall survival in a cohort of 50 patients uniformly treated by surgery, radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter of which was differentially expressed in a concordant way. Thirteen of the genes with concordant CpG sites displayed an inverse correlation between promoter methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival analysis identified six CpG sites associated with overall survival. SOX10 promoter methylation status (two CpG sites) stratified patients similarly to MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p-value < 5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters identified patients with MGMT-methylated tumors that did not respond to STUPP treatment (p-value < 1e-04).
This study provides the first genome-wide integrative analysis of DNA methylation and gene expression profiles obtained from the same GBM cohort. We also present a methylome-based survival analysis for one of the largest uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We have identified genes whose expression may be tightly regulated by epigenetic mechanisms and markers that may guide treatment decisions.
Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality ...control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1‐dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1‐dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.
Synopsis
The IRE1 arm of the Unfolded Protein Response (UPR) plays a major role in cancer development. Dissecting IRE1 signals in human glioblastoma tumors, primary and established cell lines reveals the dual role of XBP1 mRNA splicing and RIDD in tumor aggressiveness.
GBM tumors cluster into two groups exhibiting low or high IRE1 activity.
XBP1s elicits pro‐tumorigenic signals and promotes angiogenesis and macrophage recruitment to the tumor.
RIDD dampens angiogenesis and tumor cell migration.
Patients bearing tumors with high XBP1s low RIDD features show lower survival than those with low XBP1s high RIDD, thereby providing potential therapeutic avenues.
The IRE1 arm of the Unfolded Protein Response (UPR) plays a major role in cancer development. Dissecting IRE1 signals in human glioblastoma tumors, primary and established cell lines reveals the dual role of XBP1 mRNA splicing and RIDD in tumor aggressiveness.
A very small proportion of patients diagnosed with glioblastoma (GBM) survive more than 3 years. Isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations define a small subgroup of GBM patients with ...favourable prognosis. However, it remains controversial whether long-term survivors (LTS) are found among those IDH1/2 mutated patients.
We retrospectively analyzed 207 GBM patients followed at Lariboisière Hospital (Paris) between 2005 and 2010. Clinical parameters were obtained from medical records. Mutations of IDH1/2 were analyzed in these patients, by immunohistochemistry for the R132H mutation of IDH1 and by high-resolution melting-curve analysis, followed by Sanger sequencing for IDH1 and IDH2 exon 4 mutations. Mutation rates in LTS and non-LTS groups were compared by Chi square Pearson test.
Seventeen patients with survival >3 years were identified (8.2% of the total series). The median overall survival in long-term survivors was 4.6 years. Subgroup analysis found that the median age at diagnosis was significantly higher for non long-term survivors (non-LTS) compared to LTS (60 versus 51 years, p <0.03). The difference in the rate of IDH mutation between non-LTS and LTS was statistically not significant (1.16% versus 5.9%, p = 0.144). Among LTS, 10 out of 16 tumors presented a methylation of MGMT promoter.
This study confirms that long-term survival in GBM patients is if at all only weakly correlated to IDH-mutation.
Bevacizumab (Bv) remains frequently prescribed in glioblastoma (GBM) patients, especially at recurrence. We conducted a prospective clinical trial with 29 recurrent GBM patients treated with Bv alone ...with a longitudinal follow-up of different circulating immune cells complete blood count, myeloid-derived suppressor cells (MDSCs), classical, intermediate, non-classical and Tie2 monocytes, VEGFR1+ and regulatory T cells (Treg). We observed a significant increase for leucocytes, neutrophils, eosinophils and classical monocytes and a decrease for the fraction of Treg during the treatment. The best prognostic values for survival under Bv were obtained for basal neutrophils and Treg. Counts below 3.9 G/L for neutrophils and above 0.011 G/L for Treg were associated with an overall survival of 17.5 and 19.9 months, respectively, as compared with 5.4 and 5.6 months, respectively, for counts above and below these cutoffs (
p
= 0.004 and
p
< 0.001). No prognostic impact was observed for neutrophils in a retrospective cohort of 26 patients treated with nitrosoureas alone. In another retrospective validation cohort of 61 GBM patients treated at recurrence with a Bv-containing regimen, an interaction was observed between neutrophils and corticosteroid intake. The predictive value of neutrophils on survival under Bv was lost in patients treated with corticosteroids, when steroid-free patients with a low neutrophil count had a particularly long median survival of 3.4 years. These two simply accessible criteria (basal neutrophils and steroid intake) could be used to reserve this relatively costly treatment for patients likely to be the most responsive to Bv and prevent unnecessary side effects in others.
Glioblastoma (GB) is a highly invasive primary brain tumor that nearly always systematically recurs at the site of resection despite aggressive radio-chemotherapy. Previously, we reported a gene ...expression signature related to tumor infiltration. Within this signature, the EMX2 gene encodes a homeodomain transcription factor that we found was down regulated in glioblastoma. As EMX2 is reported to play a role in carcinogenesis, we investigated the impact of EMX2 overexpression in glioma-related cell lines.
For that purpose, we constructed tetracycline-inducible EMX2 expression lines. Transfected cell phenotypes (proliferation, cell death and cell cycle) were assessed in time-course experiments.
Restoration of EMX2 expression in U87 glioblastoma cells significantly inhibited cell proliferation. This inhibition was reversible after EMX2 removal from cells. EMX2-induced proliferative inhibition was very likely due to cell cycle arrest in G1/S transition and was not accompanied by signs of cell death.
Our results suggest that EMX2 may constitute a putative therapeutic target for GB treatment. Further studies are required to decipher the gene networks and transduction signals involved in EMX2's effect on cell proliferation.
Lack of appropriate diagnostic/prognostic tools for glioblastoma (GB) is considered one of the major setbacks in the early diagnosis and treatment of this deadly brain tumor. The current gold ...standard for its diagnosis and staging still relies on invasive biopsy followed by histological examination as well as molecular profiling. Nevertheless, noninvasive approaches are being explored and one example is through the investigation of extracellular vesicles (EVs) in the biofluids of GB patients. EVs are known to carry molecular cargoes such as DNA, mRNA, miRNA, proteins and lipids in almost every type of body fluids. Thus, molecular signature of GB may be present in the EVs derived from these patients. This review focuses on the diagnostic/prognostic potential of EVs in GB, through presenting recent studies on (i) molecular components of EVs, (ii) links between EVs and GB tumor microenvironment, and (iii) clinical potential of EV biomarkers, together with the technical shortcomings researchers need to consider for future studies.
Recent studies have highlighted the heterogeneity of gliomas and demonstrated that molecular and genetic analysis could help in their classification and in the design of treatment protocols. In a ...previous study we have identified a 4-gene signature highly correlated with survival of glioma patients. The aim of this study is to confirm and extend these findings by investigating the expression of these genes at the protein level and their association with outcome of patients with high grade gliomas.
Immunohistochemical staining for EDN/RB, HJURP, p60/CAF-1 and PDLI4 was studied on archive materials from 96 patients (64 glioblastomas and 32 grade III gliomas). The levels of all four proteins differed significantly between grade III and grade IV tumours. The levels of the EDN/RB, HJURP and p60/CAF-1 proteins were strongly associated with overall survival (p<0.001, p<0.001 and p=0.002, respectively), whereas the one of PDLI4 was not (P=0.11). A risk criterion defined as high levels of at least two of the EDN/RB, HJURP and p60/CAF-1 proteins accurately predicted the prognosis of patients. Multivariate analysis confirmed that this criterion was an independent negative prognostic marker (hazard ratio = 2.225; 95% CI, 1.248 to 3.966, p=0.007).
The expression of the EDN/RB, HJURP, p60/CAF-1 and PDLI4 proteins is disrupted in high grade gliomas and increases in the levels of these proteins are closely linked to tumour aggressiveness and poor outcome.
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