<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/NCT03779399</url>
  </required_header>
  <id_info>
    <org_study_id>032018</org_study_id>
    <nct_id>NCT03779399</nct_id>
  </id_info>
  <brief_title>Treg, Th17 Cells, NKT in Epithelial Ovarian Tumor</brief_title>
  <official_title>Lymphocyte T Regulatory , Th17 and NKT in Epithelial Ovarian Tumor- Prognostic Assessment and Relationship With Clinical Marker</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Medical University of Lublin</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Medical University of Lublin</source>
  <oversight_info>
    <has_dmc>Yes</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>
      The aim of the study was to estimate the percentage and of Treg, Th17 and NKT in peripheral&#xD;
      blood and the tissue of the epithelial ovarian tumor and relationship with blood serum level&#xD;
      of HE4, CA125, as well as algorithm ROMA.&#xD;
&#xD;
      Material and methods Mononuclear cells (PBMCs) were isolated by density gradient&#xD;
      centrifugation obtained from peripheral blood and ovarian tissue of patient suffering ovarian&#xD;
      pathology. Patient from control group underwent surgery for unexplanied infertility. The&#xD;
      percentage of Treg and Th17 , NKT in peripheral blood and the tissue was assessed using the&#xD;
      flow cytometry method according to the manufacturer's instructions. The ROMA index was&#xD;
      calculated according to the levels of HE4 and CA-125 in serum.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Study groups. The study group consisted of 60 women. The patients were divided into 3&#xD;
      subgroups: a group of 24 women with malignant epithelial ovarian tumors&#xD;
      (cystadenocarcinomas), 25 women with benign ovarian tumors (cystadenomas) and 11 women with&#xD;
      borderline ovarian tumors (serous borderline tumors). The control group consisted of 20 women&#xD;
      without ovarian pathology who underwent surgery for unexplanied infertility. Patients were&#xD;
      admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland, between&#xD;
      2011 and 2014. All women with OCs were staged III or IV according to revised 2013 FIGO&#xD;
      classification (International Journal of Gynecology and Obstetrics, January, 2014) Isolation&#xD;
      of mononuclear cells from peripheral blood (PBMCs). Immediately after being taken from the&#xD;
      antecubital vein, mononuclear cells (PBMCs) were isolated by density gradient centrifugation&#xD;
      applying Gradisol L formulation of specific gravity 1.077g/ml (Aqua Medica, Łódź, Poland) for&#xD;
      20 min. at 700 x g. The pellet containing PBMC was washed twice in PBS and evaluated for the&#xD;
      number (using Neubauer chamber) and viability (trypan blue staining - 0.4% trypan Blue&#xD;
      Solution, Sigma-Aldrich, Munich, Germany). Viability of less than 95% was a disqualifying&#xD;
      criterion.&#xD;
&#xD;
      Isolation of mononuclear cells infiltrating the tumor and healthy tissues.&#xD;
&#xD;
      During the surgery, ovarian tissue (size of approximately 1cm3) not containing any visible&#xD;
      necrotic areas was collected. It was minced with a scalpel, suspended in 30 ml of RPMI 1640&#xD;
      medium (Biochrome, Holliston MA, USA) and subjected to cleavage in a mixture containing: 1&#xD;
      mg/ml collagenase type IA (Sigma-Aldrich, Munich, Germany), 1 mg/ml DNase type I&#xD;
      (Sigma-Aldrich, Munich, Germany), 0.1 mg/ml hyaluronidase (Sigma-Aldrich, Munich, Germany) at&#xD;
      37°C for 60 min., constantly vortexed. After cleavage, the suspension was filtered through a&#xD;
      strainer (70μm, BD Biosciences, San Jose CA, USA) and centrifuged for 5 min. at 700 x g. The&#xD;
      suspension cells were washed twice in RPMI 1640.&#xD;
&#xD;
      Evaluation of the percentage of Th17 cells.&#xD;
&#xD;
      The evaluation of the percentage of Th17 cells (secreting IL-17A) of peripheral blood&#xD;
      mononuclear cells, healthy tissue and tumor tissue was performed by flow cytometry using a&#xD;
      Th17 Cytokine Staining panel according to the manufacturer's recommendation (eBiosciences,&#xD;
      San Diego, USA) using a FACSCanto (BD Biosciences, San Jose CA,USA).&#xD;
&#xD;
      Establishment of PBMC culture and ovarian tissue (tumor or control) and stimulation with&#xD;
      ionomycin.&#xD;
&#xD;
      After 24-hour culture of the PBMCs and ovarian tissue was set up. The medium was prepared&#xD;
      consisting of 97% RPMI 1640 (Biochrome, Holliston MA,USA) supplemented with 2mM L-glutamine,&#xD;
      2% human albumin (ZLB Bioplasma, Bern, Switzerland) and antibiotics in an amount of 100 U/ml&#xD;
      penicillin (Sigma-Aldrich, Munich, Germany) and 100 mg/ml streptomycin (Sigma-Aldrich,&#xD;
      Munich, Germany). Cultivation was carried out in 6-well plates in a 5 ml culture medium. For&#xD;
      each patient, two cultures were established, one of the PBMC and the other from tumor cells&#xD;
      or normal ovarian tissue. The culture was conducted in an incubator under standard conditions&#xD;
      (5% CO2, 95% humidity, 37°C) for 4 hours. To individual wells were added: ionomycin&#xD;
      (Sigma-Aldrich, Munich, Germany) at a concentration of 1 ug/ml and PMA at a concentration of&#xD;
      25 ng/ml to stimulate cells for the production of cytokines and brefeldin at a concentration&#xD;
      10 μg/ml (Sigma-Aldrich, Munich, Germany) in order to inhibit the activity of the endoplasmic&#xD;
      reticulum, leading to retain the cytokine within the cell.&#xD;
&#xD;
      The determination of intracellular cytokines.&#xD;
&#xD;
      24-hour cultures after moving from the culture plate to two properly signed tubes were washed&#xD;
      twice in 2 ml of Flow Cytometry Staining Buffer (eBioscience, San Diego, USA) after&#xD;
      vortexing. Constant parameters used during each rinsing in this procedure are: run time 5&#xD;
      min., 700 x g. After removing the supernatant, 5μl of anti-CD4 (eFluor 450, eBioscience, San&#xD;
      Diego, USA) was added to each tube. The mixture was incubated for 20 min. in darkness. After&#xD;
      washing away, the excess of antibody in 2 ml of Flow Cytometry Staining Buffer (eBioscience,&#xD;
      San Diego,USA), 100μl IC Fixation Buffer (eBioscience, San Diego, USA) was added to each tube&#xD;
      in order to consolidate. Mixing/Vortexing this mixture was also incubated for 20 minutes in&#xD;
      darkness. Then it was washed twice with 2 ml of the permeabilization buffer (Permeabilization&#xD;
      Buffer, eBioscience, San Diego, USA). Preparing the buffer involved its 10-fold dilution with&#xD;
      PBS. After removing the supernatant, the cells were resuspended in 100μl of permeabilization&#xD;
      buffer and separated to previously prepared cytometry tubes into control and test samples.&#xD;
      Thereafter, 5μl antibodies were added to the test samples: anti-IL-17A (FITC). After&#xD;
      vortexing, all samples were incubated for 20 min. in darkness. They were washed twice and&#xD;
      after suspending the cells in the Flow Cytometry Staining Buffer, cytometric analysis was&#xD;
      carried out. Analysis was performed using flow cytometric BD FACSCanto II (BD Biosciences,&#xD;
      San Diego, USA). Measurements were performed using the software BDFACS Diva.&#xD;
&#xD;
      Estimation of the percentage of regulatory T lymphocytes.&#xD;
&#xD;
      Estimation of the percentage of regulatory T lymphocytes among peripheral blood mononuclear&#xD;
      cells and in healthy and neoplastic tissue was made with the flow cytometry method using the&#xD;
      Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488 / CD4 PE-Cy5/CD25 PE, BioLegend®, USA) with the&#xD;
      use of FACSCanto apparatus (BD Biosciences, San Diego,USA).&#xD;
&#xD;
      Determination of iNKT cell subpopulation by flow cytometry&#xD;
&#xD;
      Mononuclear cells isolated from peripheral blood, healthy tissue and epithelial ovarian&#xD;
      tumors were incubated with the appropriate volume (according to the procedures recommended by&#xD;
      the manufacturers) of the appropriate monoclonal antibodies: the anti-iNKT FITC (anti-Vα24)&#xD;
      (cat#558371, clone 6B11, BD Pharmingen, San Jose, CA, USA), anti-CD3 PE (Cat #554829, clone&#xD;
      G4. 18, BD Pharmingen, San Jose, CA, USA) and anti CD161-PE f(clone Dx12, Cat#550968, BD&#xD;
      Pharmingen, San Jose, CA, USA). Prepared samples were incubated for 20 min. at room&#xD;
      temperature in the dark. After twice washing in buffered physiological saline (PBS), the&#xD;
      cells were immediately subjected to cytometric analysis. Data acquisition and analysis were&#xD;
      performed using a FACS Calibur flow cytometer (Becton Dickinson, New Jersey, USA). Expressing&#xD;
      cells iNKT+/CD3+/CD161+ were rated among CD3+&#xD;
&#xD;
      Assessment of concentration of protein HE4, CA125 and algorithm ROMA.&#xD;
&#xD;
      Samples were collected from all the patients prior to surgery and 3 ml blood was collected.&#xD;
      Serum was centrifuged at 2000 × g and stored at -20 and -80°C until use. Serum level of&#xD;
      CA-125 and HE4 were detected using the full automatic chemiluminescence analyzer Cobs601 and&#xD;
      the test procedure was performed according to the manufacturer's instructions (Roche&#xD;
      Diagnostics, Indianapolis, IN, USA). Next, serum HE4 and CA-125 levels were calculated for&#xD;
      ROMA index value using the Roche ROMA index of ovarian cancer risk assessment software. Serum&#xD;
      HE4 and CA-125 reference range was &lt;140 pmol/l and &lt;32 U/ml, respectively.&#xD;
&#xD;
      Assessment of risk of epithelial ovarian carcinoma (ROMA) The ROMA index was calculated&#xD;
      according to the levels of HE4 and CA-125. HE4 and CA-125 amount were input to the ovarian&#xD;
      cancer risk assessment software, followed by automatic calculation of the corresponding ROMA&#xD;
      index. The premenopausal calculation formula of the ROMA index was: 12+2.38 × LN(HE4)+0.062 6&#xD;
      × LN(CA-125). The postmenopausal calculation formula of the ROMA index was: 8.09+1.04 ×&#xD;
      LN(HE4)+0.732 × LN(CA-125). Premenopausal women with a ROMA value ≥11.4, had a higher risk of&#xD;
      ovarian cancer. Postmenopausal women with ROMA value ≥29.9 had a higher risk of ovarian&#xD;
      cancer.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">December 1, 2011</start_date>
  <completion_date type="Actual">December 31, 2016</completion_date>
  <primary_completion_date type="Actual">May 31, 2014</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Case-Control</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>Percentage of Treg, Th17, NKT in peripheral blood and tissue</measure>
    <time_frame>3 days</time_frame>
    <description>Percentage of regulatory TREG, Th17, NKT among peripheral blood mononuclear cells and in healthy and neoplastic tissue was made with the flow cytometry</description>
  </primary_outcome>
  <primary_outcome>
    <measure>Value of ROMA in serum</measure>
    <time_frame>3 days</time_frame>
    <description>Assessment level of CA125 and HE4 in serum</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Prognostic assessment of T reg, Th17, NKT lymphocytes in the tissue and peripheral blood of patients with ovarian cancer</measure>
    <time_frame>3 years</time_frame>
    <description>Kaplan-Meier survival analysis was performed in the group of patients with ovarian cancer.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Association between Treg, Th17,NKTand clinical marker</measure>
    <time_frame>1 year</time_frame>
    <description>Spearman's rank correlation coefficient and its significance test was applied to assess relationships between two parameters.</description>
  </secondary_outcome>
  <number_of_groups>4</number_of_groups>
  <enrollment type="Actual">60</enrollment>
  <condition>Ovarian Cancer</condition>
  <condition>Borderline Ovarian Tumors</condition>
  <condition>Benign Ovarian Tumor</condition>
  <condition>Unexplained Infertility</condition>
  <arm_group>
    <arm_group_label>Patient with benign ovarian tumor</arm_group_label>
    <description>25 patients with benign ovarian tumor (cystadenoma) were admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland.</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Patient with borderline tumor</arm_group_label>
    <description>11 women with borderline ovarian tumor were admitted to IInd Department of Gynecology</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Patient with ovarian cancer</arm_group_label>
    <description>24 women with ovarian cancer were admitted to IInd Department of Gynecology</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Patient without ovarian pathology</arm_group_label>
    <description>20 patient with unexpleined infertility were admitted to IInd Department of Gynecology</description>
  </arm_group>
  <intervention>
    <intervention_type>Other</intervention_type>
    <intervention_name>Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.</intervention_name>
    <description>Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.</description>
    <arm_group_label>Patient with benign ovarian tumor</arm_group_label>
    <arm_group_label>Patient with borderline tumor</arm_group_label>
    <arm_group_label>Patient with ovarian cancer</arm_group_label>
    <arm_group_label>Patient without ovarian pathology</arm_group_label>
  </intervention>
  <biospec_retention>Samples With DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      peripheral blood, ovarian tissue, serum&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        The study group consisted of 60 women. The patients were divided into 3 subgroups: a group&#xD;
        of 24 women with malignant epithelial ovarian tumors (cystadenocarcinomas), 25 women with&#xD;
        benign ovarian tumors (cystadenomas) and 11 women with borderline ovarian tumors (serous&#xD;
        borderline tumors). The control group consisted of 20 women without ovarian pathology who&#xD;
        underwent surgery for unexplanied infertility. Patients were admitted to IInd Department of&#xD;
        Gynecology, Lublin Medical University, Lublin, Poland, between 2011 and 2014. All women&#xD;
        with OCs were staged III or IV according to revised 2013 FIGO classification (International&#xD;
        Journal of Gynecology and Obstetrics, January, 2014)&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  written informed consent&#xD;
&#xD;
          -  age 18-75&#xD;
&#xD;
          -  ovarian tumor&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  below 18 years old&#xD;
&#xD;
          -  necrosis in tumor&#xD;
      </textblock>
    </criteria>
    <gender>Female</gender>
    <gender_based>Yes</gender_based>
    <gender_description>18-75</gender_description>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>75 Years</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Izabela Winkler</last_name>
    <role>Principal Investigator</role>
    <affiliation>Medical University of Lublin</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>IInd Department of Gynecology</name>
      <address>
        <city>Lublin</city>
        <zip>20-954</zip>
        <country>Poland</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Poland</country>
  </location_countries>
  <verification_date>December 2018</verification_date>
  <study_first_submitted>December 14, 2018</study_first_submitted>
  <study_first_submitted_qc>December 17, 2018</study_first_submitted_qc>
  <study_first_posted type="Actual">December 19, 2018</study_first_posted>
  <last_update_submitted>December 17, 2018</last_update_submitted>
  <last_update_submitted_qc>December 17, 2018</last_update_submitted_qc>
  <last_update_posted type="Actual">December 19, 2018</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>Medical University of Lublin</investigator_affiliation>
    <investigator_full_name>Paweł Miotła</investigator_full_name>
    <investigator_title>Clinical Proffesor</investigator_title>
  </responsible_party>
  <keyword>Ovarian Cancer</keyword>
  <keyword>Borderline Ovarian Tumor</keyword>
  <keyword>Benign Ovarian Tumor</keyword>
  <keyword>lymphocyte T regulatory</keyword>
  <keyword>Natural Killer T cell</keyword>
  <keyword>ROMA</keyword>
  <keyword>HE4</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Neoplasms</mesh_term>
    <mesh_term>Ovarian Neoplasms</mesh_term>
    <mesh_term>Infertility</mesh_term>
  </condition_browse>
  <!-- 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/NCT03779399
org study id: 032018
nct id: NCT03779399
lead sponsor: 
has dmc: Yes
is fda regulated drug: No
is fda regulated device: No
The aim of the study was to estimate the percentage and of Treg, Th17 and NKT in peripheral
      blood and the tissue of the epithelial ovarian tumor and relationship with blood serum level
      of HE4, CA125, as well as algorithm ROMA.

      Material and methods Mononuclear cells (PBMCs) were isolated by density gradient
      centrifugation obtained from peripheral blood and ovarian tissue of patient suffering ovarian
      pathology. Patient from control group underwent surgery for unexplanied infertility. The
      percentage of Treg and Th17 , NKT in peripheral blood and the tissue was assessed using the
      flow cytometry method according to the manufacturer's instructions. The ROMA index was
      calculated according to the levels of HE4 and CA-125 in serum.
Study groups. The study group consisted of 60 women. The patients were divided into 3
      subgroups: a group of 24 women with malignant epithelial ovarian tumors
      (cystadenocarcinomas), 25 women with benign ovarian tumors (cystadenomas) and 11 women with
      borderline ovarian tumors (serous borderline tumors). The control group consisted of 20 women
      without ovarian pathology who underwent surgery for unexplanied infertility. Patients were
      admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland, between
      2011 and 2014. All women with OCs were staged III or IV according to revised 2013 FIGO
      classification (International Journal of Gynecology and Obstetrics, January, 2014) Isolation
      of mononuclear cells from peripheral blood (PBMCs). Immediately after being taken from the
      antecubital vein, mononuclear cells (PBMCs) were isolated by density gradient centrifugation
      applying Gradisol L formulation of specific gravity 1.077g/ml (Aqua Medica, Łódź, Poland) for
      20 min. at 700 x g. The pellet containing PBMC was washed twice in PBS and evaluated for the
      number (using Neubauer chamber) and viability (trypan blue staining - 0.4% trypan Blue
      Solution, Sigma-Aldrich, Munich, Germany). Viability of less than 95% was a disqualifying
      criterion.

      Isolation of mononuclear cells infiltrating the tumor and healthy tissues.

      During the surgery, ovarian tissue (size of approximately 1cm3) not containing any visible
      necrotic areas was collected. It was minced with a scalpel, suspended in 30 ml of RPMI 1640
      medium (Biochrome, Holliston MA, USA) and subjected to cleavage in a mixture containing: 1
      mg/ml collagenase type IA (Sigma-Aldrich, Munich, Germany), 1 mg/ml DNase type I
      (Sigma-Aldrich, Munich, Germany), 0.1 mg/ml hyaluronidase (Sigma-Aldrich, Munich, Germany) at
      37°C for 60 min., constantly vortexed. After cleavage, the suspension was filtered through a
      strainer (70μm, BD Biosciences, San Jose CA, USA) and centrifuged for 5 min. at 700 x g. The
      suspension cells were washed twice in RPMI 1640.

      Evaluation of the percentage of Th17 cells.

      The evaluation of the percentage of Th17 cells (secreting IL-17A) of peripheral blood
      mononuclear cells, healthy tissue and tumor tissue was performed by flow cytometry using a
      Th17 Cytokine Staining panel according to the manufacturer's recommendation (eBiosciences,
      San Diego, USA) using a FACSCanto (BD Biosciences, San Jose CA,USA).

      Establishment of PBMC culture and ovarian tissue (tumor or control) and stimulation with
      ionomycin.

      After 24-hour culture of the PBMCs and ovarian tissue was set up. The medium was prepared
      consisting of 97% RPMI 1640 (Biochrome, Holliston MA,USA) supplemented with 2mM L-glutamine,
      2% human albumin (ZLB Bioplasma, Bern, Switzerland) and antibiotics in an amount of 100 U/ml
      penicillin (Sigma-Aldrich, Munich, Germany) and 100 mg/ml streptomycin (Sigma-Aldrich,
      Munich, Germany). Cultivation was carried out in 6-well plates in a 5 ml culture medium. For
      each patient, two cultures were established, one of the PBMC and the other from tumor cells
      or normal ovarian tissue. The culture was conducted in an incubator under standard conditions
      (5% CO2, 95% humidity, 37°C) for 4 hours. To individual wells were added: ionomycin
      (Sigma-Aldrich, Munich, Germany) at a concentration of 1 ug/ml and PMA at a concentration of
      25 ng/ml to stimulate cells for the production of cytokines and brefeldin at a concentration
      10 μg/ml (Sigma-Aldrich, Munich, Germany) in order to inhibit the activity of the endoplasmic
      reticulum, leading to retain the cytokine within the cell.

      The determination of intracellular cytokines.

      24-hour cultures after moving from the culture plate to two properly signed tubes were washed
      twice in 2 ml of Flow Cytometry Staining Buffer (eBioscience, San Diego, USA) after
      vortexing. Constant parameters used during each rinsing in this procedure are: run time 5
      min., 700 x g. After removing the supernatant, 5μl of anti-CD4 (eFluor 450, eBioscience, San
      Diego, USA) was added to each tube. The mixture was incubated for 20 min. in darkness. After
      washing away, the excess of antibody in 2 ml of Flow Cytometry Staining Buffer (eBioscience,
      San Diego,USA), 100μl IC Fixation Buffer (eBioscience, San Diego, USA) was added to each tube
      in order to consolidate. Mixing/Vortexing this mixture was also incubated for 20 minutes in
      darkness. Then it was washed twice with 2 ml of the permeabilization buffer (Permeabilization
      Buffer, eBioscience, San Diego, USA). Preparing the buffer involved its 10-fold dilution with
      PBS. After removing the supernatant, the cells were resuspended in 100μl of permeabilization
      buffer and separated to previously prepared cytometry tubes into control and test samples.
      Thereafter, 5μl antibodies were added to the test samples: anti-IL-17A (FITC). After
      vortexing, all samples were incubated for 20 min. in darkness. They were washed twice and
      after suspending the cells in the Flow Cytometry Staining Buffer, cytometric analysis was
      carried out. Analysis was performed using flow cytometric BD FACSCanto II (BD Biosciences,
      San Diego, USA). Measurements were performed using the software BDFACS Diva.

      Estimation of the percentage of regulatory T lymphocytes.

      Estimation of the percentage of regulatory T lymphocytes among peripheral blood mononuclear
      cells and in healthy and neoplastic tissue was made with the flow cytometry method using the
      Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488 / CD4 PE-Cy5/CD25 PE, BioLegend®, USA) with the
      use of FACSCanto apparatus (BD Biosciences, San Diego,USA).

      Determination of iNKT cell subpopulation by flow cytometry

      Mononuclear cells isolated from peripheral blood, healthy tissue and epithelial ovarian
      tumors were incubated with the appropriate volume (according to the procedures recommended by
      the manufacturers) of the appropriate monoclonal antibodies: the anti-iNKT FITC (anti-Vα24)
      (cat#558371, clone 6B11, BD Pharmingen, San Jose, CA, USA), anti-CD3 PE (Cat #554829, clone
      G4. 18, BD Pharmingen, San Jose, CA, USA) and anti CD161-PE f(clone Dx12, Cat#550968, BD
      Pharmingen, San Jose, CA, USA). Prepared samples were incubated for 20 min. at room
      temperature in the dark. After twice washing in buffered physiological saline (PBS), the
      cells were immediately subjected to cytometric analysis. Data acquisition and analysis were
      performed using a FACS Calibur flow cytometer (Becton Dickinson, New Jersey, USA). Expressing
      cells iNKT+/CD3+/CD161+ were rated among CD3+

      Assessment of concentration of protein HE4, CA125 and algorithm ROMA.

      Samples were collected from all the patients prior to surgery and 3 ml blood was collected.
      Serum was centrifuged at 2000 × g and stored at -20 and -80°C until use. Serum level of
      CA-125 and HE4 were detected using the full automatic chemiluminescence analyzer Cobs601 and
      the test procedure was performed according to the manufacturer's instructions (Roche
      Diagnostics, Indianapolis, IN, USA). Next, serum HE4 and CA-125 levels were calculated for
      ROMA index value using the Roche ROMA index of ovarian cancer risk assessment software. Serum
      HE4 and CA-125 reference range was <140 pmol/l and <32 U/ml, respectively.

      Assessment of risk of epithelial ovarian carcinoma (ROMA) The ROMA index was calculated
      according to the levels of HE4 and CA-125. HE4 and CA-125 amount were input to the ovarian
      cancer risk assessment software, followed by automatic calculation of the corresponding ROMA
      index. The premenopausal calculation formula of the ROMA index was: 12+2.38 × LN(HE4)+0.062 6
      × LN(CA-125). The postmenopausal calculation formula of the ROMA index was: 8.09+1.04 ×
      LN(HE4)+0.732 × LN(CA-125). Premenopausal women with a ROMA value ≥11.4, had a higher risk of
      ovarian cancer. Postmenopausal women with ROMA value ≥29.9 had a higher risk of ovarian
      cancer.
observational model: Case-Control
time perspective: Prospective
measure: Percentage of Treg, Th17, NKT in peripheral blood and tissue
time frame: 3 days
description: Percentage of regulatory TREG, Th17, NKT among peripheral blood mononuclear cells and in healthy and neoplastic tissue was made with the flow cytometry
measure: Value of ROMA in serum
time frame: 3 days
description: Assessment level of CA125 and HE4 in serum
measure: Prognostic assessment of T reg, Th17, NKT lymphocytes in the tissue and peripheral blood of patients with ovarian cancer
time frame: 3 years
description: Kaplan-Meier survival analysis was performed in the group of patients with ovarian cancer.
measure: Association between Treg, Th17,NKTand clinical marker
time frame: 1 year
description: Spearman's rank correlation coefficient and its significance test was applied to assess relationships between two parameters.
arm group label: Patient with benign ovarian tumor
description: 25 patients with benign ovarian tumor (cystadenoma) were admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland.
arm group label: Patient with borderline tumor
description: 11 women with borderline ovarian tumor were admitted to IInd Department of Gynecology
arm group label: Patient with ovarian cancer
description: 24 women with ovarian cancer were admitted to IInd Department of Gynecology
arm group label: Patient without ovarian pathology
description: 20 patient with unexpleined infertility were admitted to IInd Department of Gynecology
intervention type: Other
intervention name: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.
description: Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.
arm group label: Patient with benign ovarian tumor
arm group label: Patient with borderline tumor
arm group label: Patient with ovarian cancer
arm group label: Patient without ovarian pathology
peripheral blood, ovarian tissue, serum
study pop: 
sampling method: Probability Sample
criteria: 
gender: Female
gender based: Yes
gender description: 18-75
minimum age: 18 Years
maximum age: 75 Years
healthy volunteers: Accepts Healthy Volunteers
last name: Izabela Winkler
role: Principal Investigator
affiliation: Medical University of Lublin
facility: 
country: Poland
responsible party type: Principal Investigator
investigator affiliation: Medical University of Lublin
investigator full name: Paweł Miotła
investigator title: Clinical Proffesor
mesh term: Neoplasms
mesh term: Ovarian Neoplasms
mesh term: Infertility