Treg, Th17 Cells, NKT in Epithelial Ovarian Tumor
Condition: Ovarian Cancer; Borderline Ovarian Tumors; Benign Ovarian Tumor; Unexplained Infertility
MeSH terms: Neoplasms; Ovarian Neoplasms; Infertility
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.
Intervention type(s): Other
Intervention description(s): Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        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)
      
Biospecimen description: 
      peripheral blood, ovarian tissue, serum
    
Keywords: Ovarian Cancer; Borderline Ovarian Tumor; Benign Ovarian Tumor; lymphocyte T regulatory; Natural Killer T cell; ROMA; HE4