Treg, Th17 Cells, NKT in Epithelial Ovarian Tumor

Lymphocyte T Regulatory , Th17 and NKT in Epithelial Ovarian Tumor- Prognostic Assessment and Relationship With Clinical Marker

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 Overview

• Status: Completed
• Conditions: Ovarian Cancer; Unexplained Infertility; Borderline Ovarian Tumors; Benign Ovarian Tumor
• Intervention / Treatment: Other: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.

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.

• Study Type: Observational
• Enrollment (Actual): 60

Contacts and Locations

Study Locations

• Poland
  • Lublin, Poland, 20-954
    • IInd Department of Gynecology

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 75 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: Female

• 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)

Description

Inclusion Criteria:

• written informed consent
• age 18-75
• ovarian tumor

Exclusion Criteria:

• below 18 years old
• necrosis in tumor

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Prospective

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Patient with benign ovarian tumor; 25 patients with benign ovarian tumor (cystadenoma) were admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland.	Other: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.; Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.
Patient with borderline tumor; 11 women with borderline ovarian tumor were admitted to IInd Department of Gynecology	Other: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.; Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.
Patient with ovarian cancer; 24 women with ovarian cancer were admitted to IInd Department of Gynecology	Other: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.; Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.
Patient without ovarian pathology; 20 patient with unexpleined infertility were admitted to IInd Department of Gynecology	Other: Assessment of percentage of Treg, Th17, NKT and serum level of HE4, CA125 and ROMA.; Treg, Th17, NKT cells in peripheral blood and ovarian tissue and serum level of HE4, CA125 and ROMA.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Percentage of Treg, Th17, NKT in peripheral blood and tissue	Percentage of regulatory TREG, Th17, NKT among peripheral blood mononuclear cells and in healthy and neoplastic tissue was made with the flow cytometry	3 days
Value of ROMA in serum	Assessment level of CA125 and HE4 in serum	3 days

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Prognostic assessment of T reg, Th17, NKT lymphocytes in the tissue and peripheral blood of patients with ovarian cancer	Kaplan-Meier survival analysis was performed in the group of patients with ovarian cancer.	3 years
Association between Treg, Th17,NKTand clinical marker	Spearman's rank correlation coefficient and its significance test was applied to assess relationships between two parameters.	1 year

Collaborators and Investigators

• Sponsor: Medical University of Lublin
• Investigators: Principal Investigator: Izabela Winkler, Medical University of Lublin

Publications and helpful links

General Publications

• Winkler I, Wos J, Bojarska-Junak A, Semczuk A, Rechberger T, Baranowski W, Markut-Miotla E, Tabarkiewicz J, Wolinska E, Skrzypczak M. An association of iNKT+/CD3+/CD161+ lymphocytes in ovarian cancer tissue with CA125 serum concentration. Immunobiology. 2020 Nov;225(6):152010. doi: 10.1016/j.imbio.2020.152010. Epub 2020 Aug 28.

Study record dates

Study Major Dates

• Study Start (Actual): December 1, 2011
• Primary Completion (Actual): May 31, 2014
• Study Completion (Actual): December 31, 2016

Study Registration Dates

• First Submitted: December 14, 2018
• First Submitted That Met QC Criteria: December 17, 2018
• First Posted (Actual): December 19, 2018

Study Record Updates

• Last Update Posted (Actual): December 19, 2018
• Last Update Submitted That Met QC Criteria: December 17, 2018
• Last Verified: December 1, 2018

More Information

Terms related to this study

• Keywords: Ovarian Cancer; HE4; ROMA; Borderline Ovarian Tumor; Benign Ovarian Tumor; lymphocyte T regulatory; Natural Killer T cell
• Additional Relevant MeSH Terms: Urogenital Neoplasms; Neoplasms by Site; Genital Neoplasms, Female; Endocrine System Diseases; Ovarian Diseases; Adnexal Diseases; Gonadal Disorders; Endocrine Gland Neoplasms; Neoplasms; Infertility; Ovarian Neoplasms
• Other Study ID Numbers: 032018

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No