Foci of Tumor Heterogeneity in Diffuse Low-Grade Gliomas
Condition: Glioma
MeSH terms: Glioma
Study type: Observational

Summary: 
      Background:

      Diffuse low-grade gliomas (DLGG) are slow-growing primary-cancer of the brain and spinal
      cord. They represent up to 15% of the developing tumors in those organs with fatal outcome
      for the patients because of their evolution. The reasons for this transformation towards more
      malignant tumors still remain ill defined. Previously, the research team in neuro oncology at
      Montpellier University Hospital found foci of tumor heterogeneity within 20 to 30 % of the
      patients developing a DLGG and published their results. The investigators assumed that those
      foci represent the early beginning of the transformation from a diffuse low-grade glioma to a
      glioblastoma, tumor with highly malignant cells and a life expectancy of two years in average
      for the patient.

      Methods:

      The investigators selected adult patients with no prior surgery nor neuro oncology treatment
      when enrolled. They presented a specific mutation for an enzyme of the metabolism named IDH1,
      standing for Isocitrate Dehydrogenase 1, found in 70% of DLGG. Patients were also selected
      because they presented foci of tumor heterogeneity. After obtaining their consent, the
      investigators studied by immunohistochemistry the pathways deregulated between the DLGG and
      the foci. The investigators also extracted RNAs, molecules expressing the life and metabolism
      of tumor cells, and compared them to know what genes were differentially expressed between
      the DLGG and the foci. All RNAs were tested for quality control prior to be processed
      further. The investigators then studied 8 patients with compliance with ethics,
      authorizations and institutional guidelines. Genes of interest were studied in vitro to
      assess their functions. The investigators found a barely described enzyme of the catabolism
      of the phosphoethanolamines and discovered a new anti-proliferative tumor-role for it.

      •Discussion: The investigators first showed that foci have a higher percentage of p-STAT3+
      cells which indicates STAT3 pathway activation in these cells. Phosphorylated STAT3
      translocates to the cell nucleus to regulate many genes involved in proliferation, apoptosis
      and angiogenesis. As such, phosphorylation of STAT proteins, notably STAT3, is involved in
      the pathogenesis of many cancers, including GBM, by promoting cell cycle progression,
      stimulating angiogenesis, and impairing tumor immune surveillance.

      The investigators found that ETNPPL RNA and protein are reduced in foci cells and absent in
      glioblastomas. This is consistent with glioma database analyses showing that ETNPLL
      expression is inversely correlated to STAT3 and MKI67 whose expression are higher in foci and
      glioblastomas. In addition, Kaplan-Meier analysis shows that patients with low expression of
      ETNPPL have lower overall survival These observations suggested that this enzyme may oppose
      glioma cells proliferation. The investigators demonstrated this hypothesis by overexpressing
      ETNPPL in 3 glioblastoma cell cultures. Two were sensitive to ETNPPL overexpression which
      reduced their growth while no effect was detected in Gli4 cells. These glioblastoma-derived
      cultures have different types of mutations.
    

Detailed description: 
      IDH1-mutated gliomas are slow-growing brain tumors which progress into high-grade gliomas.
      The early molecular events causing this progression are ill-defined. Previous studies
      revealed that 20% of these tumors already have transformation foci. These foci offer
      opportunities to better understand malignant progression. The investigators used
      immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have
      higher p-STAT3 staining revealing activation of JAK/STAT signaling. They downregulate genes
      involved in Hippo/Yap pathway (AMOT, CCDC80, LIX1), Wnt signaling (CPE, DAAM2, GPR37, SFRP2),
      EGFR signaling (EPS15, MLC1), cytoskeleton and cell-cell communication (EZR, GJA1) while
      increasing SKA3, a kinetochore-associated protein. In addition, foci cells show reduced
      levels of the lipid metabolic ethanolamine-phosphate phospho-lyase (ETNPPL/AGXT2L1). This
      enzyme is involved in the catabolism of phosphoethanolamine involved in membrane synthesis.
      The investigators detected ETNPPL protein in glioma cells as well as in astrocytes in the
      human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL
      expression is inversely correlated to glioma grade and the investigators found no ETNPPL
      protein in glioblastomas.

      Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme
      opposes gliomagenesis. Collectively, these results suggest that a combined alteration in
      membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant
      progression.

      Tumors with foci of at least four millimeters in diameter, assessed by hematoxylin & eosin
      stainings, were selected. Four drills (two in foci and two in the other part of the tumor )
      were performed in the FFPE tumor blocks using a two millimetres punch from a Tissue Micro
      Array apparatus in RNAse-free conditions. After the punches, the adequate selection of tumor
      areas was checked by hematoxylin & eosin stainings of sections. Total RNA was extracted using
      the Qiagen RNeasy FFPE kit, quantified with Nanodrop 1000 (Thermo Fisher) and the RNA
      integrity number (RIN) was determined using a Bioanalyzer 2100. The RIN was on average 2.5
      but were still suitable for labelling and hybridization on DNA chips according to the
      Affymetrix technical department. After amplification and labelling with an Affymetrix WT Pico
      Kit, cDNA were hybridized on Human Gene 2.1 ST chips. Data were normalized with the
      Affymetrix Expression Console software (GC-RMA algorithm) and the RNA profiles were generated
      using the Affymetrix Transcriptome Analysis Console (3.1.0.5) software. Differentially
      expressed genes were selected on the basis of a linear fold change ≥ 1.1 and p-value ≤ 0.05
      (n=8 tumors). The data that support the findings of our study are openly available at the
      functional genomics data Gene Expression Omnibus.
    

Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        An individual must fulfill all of the following criteria in order to be eligible for study
        enrollment Aged between 18 and 70 years, Suffering from IDH1-mutated diffuse low-grade
        glioma, No pre operative nor oncology treatment prior to join the study.