<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT04423094</url>
  </required_header>
  <id_info>
    <org_study_id>RECHMPL19_0469</org_study_id>
    <nct_id>NCT04423094</nct_id>
  </id_info>
  <brief_title>Foci of Tumor Heterogeneity in Diffuse Low-Grade Gliomas</brief_title>
  <acronym>FDLGG</acronym>
  <official_title>Foci of Tumor Heterogeneity in IDH1-mutated Diffuse Low-Grade Gliomas Reveal STAT3 Activation and Downregulation of the Phosphoethanolamine Enzyme ETNPPL Acting as a Negative Regulator of Glioma Growth</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>University Hospital, Montpellier</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>University Hospital, Montpellier</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Background:&#xD;
&#xD;
      Diffuse low-grade gliomas (DLGG) are slow-growing primary-cancer of the brain and spinal&#xD;
      cord. They represent up to 15% of the developing tumors in those organs with fatal outcome&#xD;
      for the patients because of their evolution. The reasons for this transformation towards more&#xD;
      malignant tumors still remain ill defined. Previously, the research team in neuro oncology at&#xD;
      Montpellier University Hospital found foci of tumor heterogeneity within 20 to 30 % of the&#xD;
      patients developing a DLGG and published their results. The investigators assumed that those&#xD;
      foci represent the early beginning of the transformation from a diffuse low-grade glioma to a&#xD;
      glioblastoma, tumor with highly malignant cells and a life expectancy of two years in average&#xD;
      for the patient.&#xD;
&#xD;
      Methods:&#xD;
&#xD;
      The investigators selected adult patients with no prior surgery nor neuro oncology treatment&#xD;
      when enrolled. They presented a specific mutation for an enzyme of the metabolism named IDH1,&#xD;
      standing for Isocitrate Dehydrogenase 1, found in 70% of DLGG. Patients were also selected&#xD;
      because they presented foci of tumor heterogeneity. After obtaining their consent, the&#xD;
      investigators studied by immunohistochemistry the pathways deregulated between the DLGG and&#xD;
      the foci. The investigators also extracted RNAs, molecules expressing the life and metabolism&#xD;
      of tumor cells, and compared them to know what genes were differentially expressed between&#xD;
      the DLGG and the foci. All RNAs were tested for quality control prior to be processed&#xD;
      further. The investigators then studied 8 patients with compliance with ethics,&#xD;
      authorizations and institutional guidelines. Genes of interest were studied in vitro to&#xD;
      assess their functions. The investigators found a barely described enzyme of the catabolism&#xD;
      of the phosphoethanolamines and discovered a new anti-proliferative tumor-role for it.&#xD;
&#xD;
      •Discussion: The investigators first showed that foci have a higher percentage of p-STAT3+&#xD;
      cells which indicates STAT3 pathway activation in these cells. Phosphorylated STAT3&#xD;
      translocates to the cell nucleus to regulate many genes involved in proliferation, apoptosis&#xD;
      and angiogenesis. As such, phosphorylation of STAT proteins, notably STAT3, is involved in&#xD;
      the pathogenesis of many cancers, including GBM, by promoting cell cycle progression,&#xD;
      stimulating angiogenesis, and impairing tumor immune surveillance.&#xD;
&#xD;
      The investigators found that ETNPPL RNA and protein are reduced in foci cells and absent in&#xD;
      glioblastomas. This is consistent with glioma database analyses showing that ETNPLL&#xD;
      expression is inversely correlated to STAT3 and MKI67 whose expression are higher in foci and&#xD;
      glioblastomas. In addition, Kaplan-Meier analysis shows that patients with low expression of&#xD;
      ETNPPL have lower overall survival These observations suggested that this enzyme may oppose&#xD;
      glioma cells proliferation. The investigators demonstrated this hypothesis by overexpressing&#xD;
      ETNPPL in 3 glioblastoma cell cultures. Two were sensitive to ETNPPL overexpression which&#xD;
      reduced their growth while no effect was detected in Gli4 cells. These glioblastoma-derived&#xD;
      cultures have different types of mutations.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      IDH1-mutated gliomas are slow-growing brain tumors which progress into high-grade gliomas.&#xD;
      The early molecular events causing this progression are ill-defined. Previous studies&#xD;
      revealed that 20% of these tumors already have transformation foci. These foci offer&#xD;
      opportunities to better understand malignant progression. The investigators used&#xD;
      immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have&#xD;
      higher p-STAT3 staining revealing activation of JAK/STAT signaling. They downregulate genes&#xD;
      involved in Hippo/Yap pathway (AMOT, CCDC80, LIX1), Wnt signaling (CPE, DAAM2, GPR37, SFRP2),&#xD;
      EGFR signaling (EPS15, MLC1), cytoskeleton and cell-cell communication (EZR, GJA1) while&#xD;
      increasing SKA3, a kinetochore-associated protein. In addition, foci cells show reduced&#xD;
      levels of the lipid metabolic ethanolamine-phosphate phospho-lyase (ETNPPL/AGXT2L1). This&#xD;
      enzyme is involved in the catabolism of phosphoethanolamine involved in membrane synthesis.&#xD;
      The investigators detected ETNPPL protein in glioma cells as well as in astrocytes in the&#xD;
      human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL&#xD;
      expression is inversely correlated to glioma grade and the investigators found no ETNPPL&#xD;
      protein in glioblastomas.&#xD;
&#xD;
      Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme&#xD;
      opposes gliomagenesis. Collectively, these results suggest that a combined alteration in&#xD;
      membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant&#xD;
      progression.&#xD;
&#xD;
      Tumors with foci of at least four millimeters in diameter, assessed by hematoxylin &amp; eosin&#xD;
      stainings, were selected. Four drills (two in foci and two in the other part of the tumor )&#xD;
      were performed in the FFPE tumor blocks using a two millimetres punch from a Tissue Micro&#xD;
      Array apparatus in RNAse-free conditions. After the punches, the adequate selection of tumor&#xD;
      areas was checked by hematoxylin &amp; eosin stainings of sections. Total RNA was extracted using&#xD;
      the Qiagen RNeasy FFPE kit, quantified with Nanodrop 1000 (Thermo Fisher) and the RNA&#xD;
      integrity number (RIN) was determined using a Bioanalyzer 2100. The RIN was on average 2.5&#xD;
      but were still suitable for labelling and hybridization on DNA chips according to the&#xD;
      Affymetrix technical department. After amplification and labelling with an Affymetrix WT Pico&#xD;
      Kit, cDNA were hybridized on Human Gene 2.1 ST chips. Data were normalized with the&#xD;
      Affymetrix Expression Console software (GC-RMA algorithm) and the RNA profiles were generated&#xD;
      using the Affymetrix Transcriptome Analysis Console (3.1.0.5) software. Differentially&#xD;
      expressed genes were selected on the basis of a linear fold change ≥ 1.1 and p-value ≤ 0.05&#xD;
      (n=8 tumors). The data that support the findings of our study are openly available at the&#xD;
      functional genomics data Gene Expression Omnibus.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">November 1, 2016</start_date>
  <completion_date type="Actual">March 30, 2019</completion_date>
  <primary_completion_date type="Actual">March 1, 2019</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Retrospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>statistically significant increase in the number of tumor cells</measure>
    <time_frame>1 day</time_frame>
    <description>statistically significant increase in the number of tumor cells</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>determine predictive markers of this tumor development</measure>
    <time_frame>1 day</time_frame>
    <description>determine predictive markers of this tumor development</description>
  </secondary_outcome>
  <enrollment type="Actual">8</enrollment>
  <condition>Glioma</condition>
  <eligibility>
    <study_pop>
      <textblock>
        An individual must fulfill all of the following criteria in order to be eligible for study&#xD;
        enrollment Aged between 18 and 70 years, Suffering from IDH1-mutated diffuse low-grade&#xD;
        glioma, No pre operative nor oncology treatment prior to join the study.&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion criteria:&#xD;
&#xD;
        An individual must fulfill all of the following criteria in order to be eligible for study&#xD;
        enrollment:&#xD;
&#xD;
          -  Aged between 18 and 70 years.&#xD;
&#xD;
          -  Suffering from IDH1-mutated diffuse low-grade glioma.&#xD;
&#xD;
          -  No pre operative nor oncology treatment prior to join the study.&#xD;
&#xD;
          -  Signed informed consent form.&#xD;
&#xD;
        Exclusion criteria:&#xD;
&#xD;
          -  Subject unable to read or/and write&#xD;
&#xD;
          -  Grade 3 or 4 gliomas&#xD;
&#xD;
          -  Tumor with IDH1-WT status&#xD;
      </textblock>
    </criteria>
    <gender>Female</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>70 Years</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>VALERIE RIGAUX, MD, PhD</last_name>
    <role>Study Director</role>
    <affiliation>University Hospital, Montpellier</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Uh Montpellier</name>
      <address>
        <city>Montpellier</city>
        <zip>34295</zip>
        <country>France</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>France</country>
  </location_countries>
  <verification_date>November 2019</verification_date>
  <study_first_submitted>November 25, 2019</study_first_submitted>
  <study_first_submitted_qc>June 4, 2020</study_first_submitted_qc>
  <study_first_posted type="Actual">June 9, 2020</study_first_posted>
  <last_update_submitted>June 4, 2020</last_update_submitted>
  <last_update_submitted_qc>June 4, 2020</last_update_submitted_qc>
  <last_update_posted type="Actual">June 9, 2020</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Glioma</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>Undecided</sharing_ipd>
    <ipd_description>NC</ipd_description>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT04423094
org study id: RECHMPL19_0469
nct id: NCT04423094
lead sponsor: 
has dmc: No
is fda regulated drug: No
is fda regulated device: No
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.
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
measure: statistically significant increase in the number of tumor cells
time frame: 1 day
description: statistically significant increase in the number of tumor cells
measure: determine predictive markers of this tumor development
time frame: 1 day
description: determine predictive markers of this tumor development
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: Female
minimum age: 18 Years
maximum age: 70 Years
healthy volunteers: No
last name: VALERIE RIGAUX, MD, PhD
role: Study Director
affiliation: University Hospital, Montpellier
facility: 
country: France
responsible party type: Sponsor
mesh term: Glioma
sharing ipd: Undecided
ipd description: NC