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Progestin treatment decreases CD133 + cancer stem cell populations in endometrial cancer
Progestin Treatment Decreases CD133 + Cancer Stem Cell Populations in Endometrial Cancer Michael S. Guy, Lubna Qamar, Kian Behbakht, Miriam D. Post, Jeanelle Sheeder, Carol A. Sartorius, Monique A. Spillman PII: DOI: Reference:
S0090-8258(15)30226-2 doi: 10.1016/j.ygyno.2015.12.022 YGYNO 976155
To appear in:
Gynecologic Oncology
Received date: Revised date: Accepted date:
17 September 2015 22 December 2015 23 December 2015
Please cite this article as: Michael S. Guy, Lubna Qamar, Kian Behbakht, Miriam D. Post, Jeanelle Sheeder, Carol A. Sartorius, Monique A. Spillman, Progestin Treatment Decreases CD133 + Cancer Stem Cell Populations in Endometrial Cancer, Gynecologic Oncology (2015), doi: 10.1016/j.ygyno.2015.12.022
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ACCEPTED MANUSCRIPT Progestin Treatment Decreases CD133+ Cancer Stem Cell Populations in Endometrial Cancer
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Michael S. Guy, MDa, Lubna Qamar PhDb, Kian Behbakht, MDb, Miriam D. Post, MDc, Jeanelle Sheeder, MSPH, PhDb, Carol A. Sartorius, PhDc, Monique A. Spillman, MD, PhDd a
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Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Cincinnati School of Medicine Cincinnati OH 45219, USA b Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Colorado School of Medicine Aurora CO 80045, USA c Department of Pathology, University of Colorado Denver School of Medicine, Aurora CO 80045, USA d Texas Oncology, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas TX 75246, USA
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Disclosure Statement: All authors declare that there are no financial disclosures, nor conflicts of interest.
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Corresponding Author: Michael Guy, MD University of Cincinnati Department of OB/GYN, Division of Gynecologic Oncology 222 Piedmont Ave. Medical Arts Building, Suite 4100 Cincinnati, OH 45219 Phone: 513-475-8162 Fax: 513-475-8174 E-mail: [email protected] Suggested Reviewers 1. Michael Goodheart, MD 2. David Mutch, MD Permanent Address 337 Oak Forest Dr. Dayton, OH 45419
ACCEPTED MANUSCRIPT ABSTRACT
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Objectives: Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells.
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Methods: The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4uM medroxyprogesterone 17-acetate (MPA) for 6 days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression were scored and evaluated for change over time on serial biopsies.
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Results: CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment.
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Conclusion: CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells. Keywords: endometrial cancer, cancer stem cells, hormonal treatment, progestin, progesterone WORD COUNT: 235
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ACCEPTED MANUSCRIPT Highlights: 1. CD133+ cells are identified in endometrial cancer cell lines and patient samples.
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2. Hormonal treatment with progestins decreases CD133+ cells in vitro and in patient samples from a clinical project.
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ACCEPTED MANUSCRIPT Introduction Endometrial cancer is the most common gynecologic malignancy in the United States
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with an expected 54,870 cases in 2015[1]. The majority of cases are diagnosed after menopause, but up to 14% of cases are diagnosed before menopause including 4% diagnosed before the age
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of 40 [2-5]. A majority of cases in younger women are lower grade and earlier stage [2].
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Seventy to ninety percent of type 1 endometrial cancer patients have significant medical comorbidities that may complicate surgery [6]. Surgical staging including hysterectomy,
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bilateral salpingo-oophorectomy and sampling of lymph nodes is the standard treatment.
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However, conservative management utilizing hormonal therapy may be considered in those desiring fertility sparing treatment or those who have multiple medical comorbidities making
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them poor surgical candidates.
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Hormonal treatment, especially with progestins, has been examined in the context of
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advanced stage or recurrent endometrial cancer. The Gynecologic Oncology Group (GOG) protocol 81 demonstrated that medroxprogesterone acetate (MPA) is active against endometrial carcinoma with an overall response rate of 25%[7]. In a systematic review, 15-30% of women responded to progestin therapy [8], the highest response rates were noted in low grade, estrogen receptor (ER) positive tumors [9]. In a prospective study using MPA in women with early stage, well differentiated, endometrial cancer a 55% response rate to treatment was seen[10]. The factors that play a role in response are incompletely understood. GOG protocol 119 demonstrated that response to progestin treatment was seen even when tumors did not express ER suggesting that there are alternative pathways being activated by progestin therapy[9]. One of the proposed components of cancer progression and recurrence are cancer stem cells. Cancer stem cells (CSC) are defined as a small subpopulation of cells within a tumor that contribute to tumor maintenance and progression and are resistant to traditional
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ACCEPTED MANUSCRIPT chemotherapy[11-14]. These tumorigenic cells are defined by their ability to self renew and generate mature cells of a particular tissue through differentiation [13]. CSC have been
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described from several human solid cancers including breast [15], brain [16, 17], colon [18, 19]
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head and neck [20] and pancreatic cancer [21]. Cancer stem cells have also been identified in gynecologic malignancies using extracellular markers including clusters of differentiation (CD)
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133 [22-26], CD44 [27] and CD117 [28] in ovarian and endometrial cancer.
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Nakamura et al. have shown that overexpression of CD133 in endometrial cancer patients is an independent predictor of decreased overall survival[24]. Their group also demonstrated
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that when patient derived tumor cells were isolated and treated with cisplatin or paclitaxel they were resistant to chemotherapy[24]. CD133+ cells also have differential response to estrogen
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treatment. Rutella et al. demonstrated that CD133+ endometrial cancer cells respond with increased growth after treatment with estradiol indicating that this is also a hormonally
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responsive sub-population [23].
The response of CD133 cells to hormonal treatment with progestins has not been explored. We explored the hypothesis that CD133+ endometrial cancer cells express ER and PR and that progestin therapy differentially regulates those cells. Specifically, by treating ER+/PR+ and ER-/PR- cell lines with progestins we anticipated that we would see decreases in cell number, increases in apoptosis and decreases in this unique CD133+ cell population. Secondly, in a clinical project, we hypothesized a similar decrease in CD133+ cells would occur in patients who responded to treatment with progestin therapy.
Materials and Methods Cell Lines and cell culture
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ACCEPTED MANUSCRIPT The Ishikawa (endometrial cancer), KLE (endometrial cancer), MCF7 (breast cancer), LNCaP (prostate cancer), and 2008 (ovarian cancer) cell lines were cultured at 37°C and 5%
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CO2 in RPMI 1640 (Invitrogen, Grand Island, NY 11875-093) with 10% fetal bovine serum
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(Phoenix Research Products, Asheville, NC A704410-7007) and 5% Pen Strep (Invitrogen, Grand Island, NY 15140-22). Cell lines were authenticated by polymorphic short tandem repeat
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analysis at the University of Colorado DNA Sequencing and Analysis Core as previously
Cambridge, MA) were used in western blots.
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Drugs and Drug Treatment
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described[29]. Purchased lysates from the PC3 prostate cancer cell line (ab3954, Abcam,
In experiments involving flow cytometry and apoptosis assays, cells were cultured in
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phenol red free RPMI 1640 (Invitrogen, Grand Island, NY 11835-055) with charcoal stripped fetal bovine serum (DCC) (Phoenix Research Products Asheville, NC, FBS-500DC) and 5% Pen
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Strep. Cells were plated, and then treated after 24 hours. Treatments included a control vehicle of ethanol (Sigma-Aldrich, St. Louis, MO, E7023) or 30.4 uM medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich, St. Louis, MO, M1629) dissolved in ethanol. Final concentrations of ethanol in media were 0.3%. MPA dosing was derived from previously published IC50 for the Ishikawa cell line [30]. Media was changed every 48 hours, and treatment concluded 7 days after plating. Flow Cytometry Analysis 0.5x106 cells were suspended in a 1:20 solution of MACS BSA Stock Solution and autoMACS ™ Rinsing Solution (Mitenyi Biotec, Auburn, CA, 130-091-376). 20L of fragment crystallizable (Fc) receptor blocking antibody (Mitenyi Biotec, Auburn, CA, 130-059-901) was added followed by either 10uL of CD133/2 (293C3)-APC antibodies (Mitenyi Biotec, Auburn,
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ACCEPTED MANUSCRIPT CA, 130-090-854) or Mouse IgG1 isotype control antibodies (Mitenyi Biotec, Auburn, CA, 130092-214) and incubated for 10 minutes at 4°C. Cells were then washed, and resuspended. Flow
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cytometry analysis was completed with CyAn ADP 9 color Analyzer (Beckman Coulter, Inc.,
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Brea, CA). Cloning Efficiency Assay
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4x107 cells were labeled with CD133/2 (293C3)-APC antibodies as detailed above.
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Sorting into CD133+ and CD133- populations was completed on a Moflo XDP 100 (Beckman Coulter, Inc., Brea, CA). 10,000 KLE, or 2,500 Ishikawa cells were plated in 6 well plates.
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Media was changed every 72 hours. At days 3, 6 and 9 cell counts and images were collected. Cell counts were completed with a ViCELL® Cell Counter (Beckman Coulter, Brea, CA) using
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trypan blue exclusion. Images were captured with an Olympus CKX41 microscope fitted with an Olympus DD73 camera using cellSens digital imaging software (Olympus, Center Valley,
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PA). Immunocytochemistry (ICC)
3x105 cells were plated in single chamber slides. After 48 hours, cells were fixed with 70% acetone and 30% cold methanol for 5 minutes at room temperature. Immunocytochemistry was conducted according to standard protocols. Antibodies used were as follows: ER (1:200, SP1 clone RM9101; LabVision/NeoMarkers), PR (1:500, PgR1294 clone M3568; Dako) and AR (1:600, rabbit monoclonal D6F11, Cell Signaling). Secondary antibody was Alexa Flour 555 (1:200, A21429, Life Technologies). Vector Shield (Vector Laboratories) was utilized for DAPI counter stain and mounting media. Immunofluorescence images were captured at room temperature on a Olympus CKX41 fluorescence microscope equipped with an Olympus DD73 camera (Olympus, Center Valley, PA) using cellSens digital imaging software (Olympus, Center
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ACCEPTED MANUSCRIPT Valley, PA) with a 40X objective. Androgen receptor localization studies utilized the above protocol, but prior to fixation, 10nM testosterone was added to media and cells were stimulated
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for 2 hours as previously described by Li et al [31].
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Preparation of Whole Cell Lysates, Nuclear Extracts and Western Blotting
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Protein was extracted, and concentration was quantitated by Bradford Assay. 100 µg of nuclear extract and 50g of whole cell lysate per lane were resolved on 7.5% SDS-PAGE gels
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(Gels: 456-1023EDU, Bio-Rad, Hercules, CA; Blotter: 170-4070 Bio-Rad, Hercules, CA,). Proteins were transferred to nitrocellulose (88013 Thermo Scientific, Rockford, IL), blocked
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with 5% milk in 1x TBST (T9039, Sigma-Aldrich, St. Louis, MO), then probed with the respective primary antibodies, washed then labeled with secondary antibodies (Anti-mouse:
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7076S, Anti-rabbit: 7074S, Cell Signaling Technology, Danvers, MA) and visualized using enhanced chemiluminescence (Supersignal West Pico 34080 and SuperSignal West Femto
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34095, Pierce, Rockford, IL). Film (34091, Pierce Rockford, IL) was exposed and developed. PC3, a prostate derived cell line, whole cell lysates were purchased (ab3954, Abcam, Cambridge, MA) and used as a negative control for the androgen receptor. Apoptosis Assay
2.5x103 cells were plated in 8 chamber slides (C7182-1CS, Sigma-Aldrich, St. Louis, MO). Cells were treated with progestins as described above for 7 days. Apotag® In situ Apoptosis Detection Kit (S7100, Millipore, Billerica, MA) was utilized for detection of terminal deoxynucleotidyl transferase found in apoptotic cells after treatment with progestins. Images were captured utilizing equipment described in ICC methods. Total number of cells and apoptotic cells were counted in 3 separate 20X fields then averaged in triplicate experiments. Patient Samples and Immunohistochemistry
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ACCEPTED MANUSCRIPT Institutional review board approval was obtained through the Colorado Multiple Institutional Review Board (COMIRB Protocol 13-1563). Women ≥18 years of age with a
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diagnosis of endometrial adenocarcinoma that had been treated with progestins were identified
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through a review of the University of Colorado Tumor Registry, Gynecologic Oncology Tumor Board and relevant ICD-9 codes. Clinical variables abstracted from the medical record included
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age, race, body mass index (BMI), medical comorbidities, FIGO tumor grade and stage,
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histologic cell type, indication for treatment, menopausal status, type of hormonal treatment, duration of hormonal treatment and length of surveillance in patients undergoing hysterectomy.
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Tissue samples were retrieved from the pathology department. Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained
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for Androgen Receptor (Cell Signaling, Danvers, MA; rabbit monoclonal D6F11; cat#: 5153; Lot: #2; 1:500), Estrogen Receptor (Thermo Scientific, Pittsburgh, PA; rabbit monoclonal SP1:
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cat#: MA139540; 1:40), Progesterone Receptor (DAKO, Carpinteria, CA; mouse monoclonal 1294; cat#M3568; 1:100) and CD133 (Miltenyi Biotec, Auburn, CA; mouse monoclonal cat#: 130-090-422; 1:15). All antibodies were diluted in TBST + 1% BSA w/v + 0.05% ProClin 950. Antigens to AR, ER and PR were revealed in pH 9.5 BORG solution (Biocare Medical, Concord, CA) for 5 minutes at 125°C (22 psi; Decloaking chamber, Biocare) with a 10 minute ambient cool down. Immunodetection was performed on the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ) at an operating temperature of 37°C. Primary antibodies were incubated for 32 minutes and detected with the UltraView universal polymer kit (Ventana). Antigens to CD133 required modest retrieval for 15 minutes at 110°C in Target Retrieval Solution (DAKO, diluted 1:50). All staining steps were performed at ambient temperature. Endogenous peroxidase and non-specific staining were blocked with 3% hydrogen peroxide (aq.
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ACCEPTED MANUSCRIPT v/v; 10 minutes) and 2.5% normal goat serum (from Mouse ImmPress kit; Vector; 20 minutes), respectively. Diluted primary antibody was applied and incubated for 60 minutes in a
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humidified chamber. Then the antibody was detected with a Mouse ImmPress polymer kit
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(Vector, cat#: MP-7402; 30 minutes). The complexes were visualized with ImmPACT DAB (Vector: cat#: SK-4105; 5 minutes). All sections were counterstained in Harris hematoxylin for
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xylene and coverglass mounted using synthetic resin.
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2 minutes, blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in
Hemotoxylin and eosin (H+E) stained slides were reviewed to identify tumor. Then the
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IHC slides were assessed at 40X by three independent reviewers (MG, KB, MP) utilizing two variables (intensity and percent positive) that were multiplied to create a histoscore (H-score).
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Intensity was graded on a 0-3 scale based on positive and negative controls from non-sample tissue (0=no stain, 1=weak staining, 2=moderate staining, 3=strong staining). Percent positive
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was scored from 0 to 100 percent. H-scores were averaged for each sample to create the composite H-score for ER, PR and AR. Interclass correlation, or overall agreement between the three observers was calculated and reported as Cronbach’s alpha. Interclass correlations were poor if values less than 0.40, fair for values between 0.40 and 0.59, good for values between 0.60 and 0.74, and excellent for values between 0.75 and 1.0. A gynecologic pathologist (MP) reviewed the H+E slides at the time of scoring to assess for progestin effect. Patients who had a clinical response with regression of FIGO grade, complete resolution of cancer, or noted treatment effect were considered as “responders”. Patients who had no noted treatment effect, persistent tumor or worsening tumor grade were categorized as “non-responders”. Statistical Analysis
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ACCEPTED MANUSCRIPT Statistical analysis of continuous variables with student’s t-test was performed using Graphpad Prism 6 for Mac OS X. Interclass correlations were conducted with IBM SPSS
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Results Steroid receptor profiles differ in Ishikawa and KLE cell lines
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statistics version 21. Statistical significance was determined by P values less than 0.05.
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To set up a model for ER+ and ER- endometrial cancer, hormone receptor profiles were
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analyzed for the Ishikawa and KLE cell lines using both Western Blot and ICC. In Western blot the MCF7 breast cancer cell line served as ER and PR positive controls. The 2008 ovarian
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cancer cell line was a negative control for ER and PR. PC3 (ab3954, Abcam, Cambridge, MA) and LNCaP prostate cancer cell lines served as the AR positive and negative controls
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respectively. In ICC, MCF7 served as positive controls for ER, PR and AR. Ishikawa whole
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cell lysates and ICC expressed ER, PR and AR. KLE cell lysates and ICC were ER and PR
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negative and expressed AR (Figure 1). ICC negative IgG controls provided for all images in supplemental Figure 1.
CD133+ cells are present in endometrial cancer cell lines and demonstrate differential growth The ER+/PR+ Ishikawa and the ER-/PR- KLE endometrial cancer cell lines were assayed for the presence of CD133 populations by flow cytometry. CD133+ cells were detected in both cell lines, but with a higher proportion in the ER+/PR+ Ishikawa cell line. Repeated experiments demonstrated 6.5% of ER-/PR- KLE and 80.7% of ER+/PR+ Ishikawa cells were positive for CD133 (Figure 2, panel A). A clonogenic assay was used to demonstrate the differential proliferative potential between CD133- and CD133+ populations. Day 1 cell counts were the same between groups. By day 9, significant differential growth of the CD133+ cells was confirmed in the KLE (7.7 x
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ACCEPTED MANUSCRIPT 104 cells vs. 1.55 x 105 cells, P=0.001) and Ishikawa (3.38 x 105 cells vs. 5.74 x 105 cells, P<0.0001) cell lines (Figure 2, panel B).
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Progestin treatment differentially increases apoptosis in CD133+ cells Cell lines were treated with MPA for 6 days. Compared to ethanol treated controls, there
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was a significant reduction in viable cells in the MPA treated Ishikawa (100% vs. 86.4%,
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P=0.036) and KLE (100% vs. 45.6%, P=0.0002) cell lines (Figure 3A). Significant reductions in
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CD133+ populations were noted after treatment when compared to controls in Ishikawa (62.2% vs. 81.1%, P<0.0001; normalized to controls 79.4% vs. 100%) and KLE (3.0% vs. 6.4%
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P=0.0001; normalized to controls 46.8% vs. 100%) cell lines (Figure 3B). MPA treatment significantly increased the number of apoptotic cells in both cell lines compared to ethanol
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controls (Ishikawa 32.1% vs. 0.4%, P=0.01; KLE 28.4% vs. 0.7%, P=0.0006) (Figure 3C). Androgen Receptor present in CD133+ endometrial cancer cells
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Progestins such as MPA also activate androgen receptor (AR) [32] and AR has been identified previously in the Ishikawa cell line[33]. Because the MPA driven apoptosis of CD133+ cells was also detected in the ER-/PR- KLE cells, we postulated that these cells would contain the AR. After KLE and Ishikawa cells were sorted into CD133+ and CD133populations with flow cytometry, cells were stimulated with testosterone for two hours and fixed. ICC was then completed demonstrating that AR was present in both CD133+ and CD133populations of the Ishikawa and KLE cell lines (Figure 4). Progestin mediated decrease in CD133+ cells confirmed in clinical tissue samples Twelve patients were identified who were treated with progestins, and a total of 28 individual endometrial cancer samples were reviewed. Five patients only had a single specimen available for analysis and could not be compared over time. These patients began hormonal
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ACCEPTED MANUSCRIPT treatment at outside facilities and presented to our institution for hysterectomy. Their prior biopsies were not available for comparison.
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Demographics and clinicopathologic characteristics are detailed in table 1 of the
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remaining 7 patients. The majority of the population analyzed was post-menopausal, white, extremely obese (class III, BMI > 40), with multiple major medical co-morbidities and had well-
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differentiated endometrioid carcinomas. Documented indications for progestin treatment were
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related to increased surgical risk and concern for significant perioperative complications. All patients were treated with megestrol acetate (Megace), and 3 patients also had levonorgestrel
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IUDs placed as an additional therapeutic intervention.
The median length of hormonal treatment in the cohort was 21 months. Two patients
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(28.6%) had complete regression of cancer. One patient underwent radiation with curative intent. Four patients (57.1%) underwent definitive treatment with hysterectomy due to non-response to
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treatment or improvement in co-morbid conditions. In patients who underwent hysterectomy, median length of treatment with progestins prior to surgery was 8.5 months (range 1-21 months). The patients with hysterectomy have been followed for a median of 18.5 months after surgery. One post-hystectomy patient whose final pathology revealed a poorly differentiated mixed endometrial cancer that declined adjuvant chemotherapy and radiation had a pelvic recurrence 12 months after surgery. ER was expressed in 96.4% (n=27/28) of individual endometrial cancer specimens with a median H-score of 263 (95% CI 223.3-280.0). PR was expressed in 96.4% (n=27/28) samples, with a median H-score of 175.0 (95% CI 120.0-250.0). AR was expressed in 89.3% (n=25/28) samples, with a median H-score of 95.0 (95% CI 53.3-200.0). CD133 was expressed in 100% of samples (n=28), with a median H-score of 66.0 (95% CI 50.0-90.0) (Figure 5A). Interclass
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ACCEPTED MANUSCRIPT correlations average measures were calculated for ER (Cronbach’s alpha=0.961; 95% CI 0.930.98), PR (Cronbach’s alpha=0.965; 95% CI 0.94-0.98), AR (Cronbach’s alpha=0.853; 95% CI
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0.75-0.92) and CD133 (Cronbach’s alpha=0.846; 95% CI 0.72-0.92). Inter-rate reliability was
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excellent between 3 independent reviewers. For each patient, we assessed for changes in ER, PR and AR H-scores over time and no significant trends were identified.
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We further assessed the endometrial samples for changes in the proportion of CD133
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cells in response to progestins over time. Of the 7 patients with multiple samples available for comparison, there were 4 responders and 3 non-responders to progestins. In the responders,
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100% (n=4) showed a decrease in CD133 H-score between their first and last endometrial cancer samples (Figure 5B). In the three non-responders, changes in CD133 levels were mixed with no
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clear trends noted (Supplemental Figure 2). In responders and non-responders, samples were evaluated for loss or gain of ER/PR and AR expression and no significant change in H-scores
Discussion
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occurred during treatment.
Endometrial cancer is a hormonally responsive malignancy. The factors that play a role in response and progression are incompletely understood. CD133 is a cancer stem cell or tumorinitiating cell marker that has been defined in many cancers including gynecologic malignancies. We hypothesized that the CD133+ cell population in endometrial cancer was hormonally responsive and would differentially regulated by progestins. Our study demonstrates that CD133 cells are present in the ER+/PR+ Ishikawa and ER/PR- KLE endometrial cancer cell lines and patient endometrial cancer samples. When treated with progestins, CD133+ cells decreased in endometrial cancer cell lines as well as patient samples for patients who responded to progestin treatment. In the in vitro model, MPA
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ACCEPTED MANUSCRIPT treatment resulted in both decreased cell number and increased apoptosis in both ER+/PR+ and ER-/PR- cells.
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Our hypothesis and findings are supported by previous research that has demonstrated
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that CD133+ endometrial cancer cells are responsive to hormonal treatment with another steroid hormone, estrogen. After sorting patient derived endometrial cancer cells into CD133 positive
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and negative groups, Rutella et al. treated with estradiol for 72 hours and showed increased
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growth in only the CD133+ population [23]. Combining our results with these prior findings indicate that CD133+ endometrial cancer cells can be both positively and negatively growth
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regulated by hormonal treatment with estrogens and progestins respectively. Because progestin treatment effect was noted in both ER+/PR+ (Ishikawa) and ER-/PR-
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(KLE) cell lines it is likely that the decrease in proliferation, increase in apoptosis and decrease in CD133+ cells related to progestin treatment is multifactorial. In addition to direct interaction
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with the progesterone receptor, other mechanisms may involve non-classical signaling pathways utilizing the androgen receptor. In breast cancer, another hormonally responsive malignancy, Hackenburg et al. demonstrated in ER-/PR-/AR+ breast cancer and endometrial cancer cell lines that there is growth inhibition via AR with MPA[32]. To further support this possible interaction we demonstrated AR was not only present in the whole cell lysates of endometrial cancer cells, but was also present in sorted CD133+ sub-population. Furthermore, GOG 119, a phase II clinical trial that evaluated response to tamoxifen and intermittent MPA in patients with recurrent or advanced stage endometrial cancer, demonstrated highest response rates in patients with ER+ tumors, but response rates of 26% were also noted in patients with receptor negative tumors[34]. While AR was not assayed in this study, one may postulate that AR expression might account for this response. These data from other disease sites and the clinical trial
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ACCEPTED MANUSCRIPT information support the concept of complex hormonal interplay between progestin and multiple targets, both direct and indirect.
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Strengths of this study include the addition of a clinical component evaluating tumor
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specimens of patients treated with progestins at our institution. Their history, co-morbid conditions, treatment course and physician clinical decision-making was well documented.
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Patient follow-up since complete response or definitive treatment with surgery reaches a median
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of almost 2 years. Weaknesses of this study include the small sample size that limits statistical analysis and more definitive conclusions regarding the clinical utility of CD133 H-scores and its
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role in initial patient selection for progestin therapy, as well as its use for monitoring treatment. All patient samples in our study were ER+ so we were not able to parallel the in vitro ER+/PR+
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versus ER-/PR- model in the patient specimens. In future research, use of a larger endometrial cancer sequential biopsy set through a GOG ancillary study or a multi-institutional effort would
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be beneficial.
In conclusion, this work provides insight into the differential impact of progestin treatment on the hormonally responsive CD133+ endometrial cancer cells. We have shown that MPA treatment results in change in cell number through apoptosis and decrease in CD133+ cell number. This effect was noted in ER+/PR+ and ER-/PR- endometrial cancer cell lines and may be mediated by the androgen receptor. This exciting finding indicates that progestin therapy should be considered for treatment in both ER+ and ER- tumors, in patients who are not candidates for definitive surgery or who desire future fertility.
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Acknowledgements This study was supported by the Academic Enrichment Fund administered by the Department of Obstetrics and Gynecology at the University of Colorado School of Medicine. We thank Dr. Twila Jackson, PhD for generously providing the Ishikawa and KLE cell lines and Dr. Britta Jacobsen, PhD (Univ of Colorado School of Medicine and Anschutz Medical Campus) for the LNCaP cell line.
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Disclosure Statement: All authors declare that there are no financial disclosures, nor conflicts of interest.
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[1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5-29. [2] Duska LR, Garrett A, Rueda BR, Haas J, Chang Y, Fuller AF. Endometrial cancer in women 40 years old or younger. Gynecol Oncol. 2001;83:388-93. [3] Gitsch G, Hanzal E, Jensen D, Hacker NF. Endometrial cancer in premenopausal women 45 years and younger. Obstet Gynecol. 1995;85:504-8. [4] Lee NK, Cheung MK, Shin JY, Husain A, Teng NN, Berek JS, et al. Prognostic factors for uterine cancer in reproductive-aged women. Obstet Gynecol. 2007;109:655-62. [5] Tran BN, Connell PP, Waggoner S, Rotmensch J, Mundt AJ. Characteristics and outcome of endometrial carcinoma patients age 45 years and younger. Am J Clin Oncol. 2000;23:476-80. [6] von Gruenigen VE, Gil KM, Frasure HE, Jenison EL, Hopkins MP. The impact of obesity and age on quality of life in gynecologic surgery. Am J Obstet Gynecol. 2005;193:1369-75. [7] Thigpen JT, Brady MF, Alvarez RD, Adelson MD, Homesley HD, Manetta A, et al. Oral medroxyprogesterone acetate in the treatment of advanced or recurrent endometrial carcinoma: a dose-response study by the Gynecologic Oncology Group. J Clin Oncol. 1999;17:1736-44. [8] Decruze SB, Green JA. Hormone therapy in advanced and recurrent endometrial cancer: a systematic review. Int J Gynecol Cancer. 2007;17:964-78. [9] Singh M, Zaino RJ, Filiaci VJ, Leslie KK. Relationship of estrogen and progesterone receptors to clinical outcome in metastatic endometrial carcinoma: a Gynecologic Oncology Group Study. Gynecol Oncol. 2007;106:325-33. [10] Ushijima K, Yahata H, Yoshikawa H, Konishi I, Yasugi T, Saito T, et al. Multicenter phase II study of fertility-sparing treatment with medroxyprogesterone acetate for endometrial carcinoma and atypical hyperplasia in young women. J Clin Oncol. 2007;25:2798-803. [11] Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267-84. [12] Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med. 2006;355:1253-61. [13] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105-11. [14] Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell biology to cancer. Nat Rev Cancer. 2003;3:895-902. [15] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100:3983-8. [16] Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature. 2004;432:396-401. [17] Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004;64:7011-21. [18] O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445:106-10. [19] Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007;445:111-5. [20] Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A. 2007;104:973-8.
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[21] Li C, Lee CJ, Simeone DM. Identification of human pancreatic cancer stem cells. Methods Mol Biol. 2009;568:161-73. [22] Friel AM, Zhang L, Curley MD, Therrien VA, Sergent PA, Belden SE, et al. Epigenetic regulation of CD133 and tumorigenicity of CD133 positive and negative endometrial cancer cells. Reprod Biol Endocrinol. 2010;8:147. [23] Rutella S, Bonanno G, Procoli A, Mariotti A, Corallo M, Prisco MG, et al. Cells with characteristics of cancer stem/progenitor cells express the CD133 antigen in human endometrial tumors. Clin Cancer Res. 2009;15:4299-311. [24] Nakamura M, Kyo S, Zhang B, Zhang X, Mizumoto Y, Takakura M, et al. Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer. Hum Pathol. 2010;41:1516-29. [25] Kryczek I, Liu S, Roh M, Vatan L, Szeliga W, Wei S, et al. Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. Int J Cancer. 2012;130:29-39. [26] Baba T, Convery PA, Matsumura N, Whitaker RS, Kondoh E, Perry T, et al. Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells. Oncogene. 2009;28:209-18. [27] Steffensen KD, Alvero AB, Yang Y, Waldstrom M, Hui P, Holmberg JC, et al. Prevalence of epithelial ovarian cancer stem cells correlates with recurrence in early-stage ovarian cancer. J Oncol. 2011;2011:620523. [28] Luo L, Zeng J, Liang B, Zhao Z, Sun L, Cao D, et al. Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome. Exp Mol Pathol. 2011;91:596-602. [29] Korch C, Spillman MA, Jackson TA, Jacobsen BM, Murphy SK, Lessey BA, et al. DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecologic Oncology. 2012;127:241-8. [30] Moe BT, Vereide AB, Orbo A, Jaeger R, Sager G. Levonorgestrel, medroxyprogesterone and progesterone cause a concentration-dependent reduction in endometrial cancer (Ishikawa) cell density, and high concentrations of progesterone and mifepristone act in synergy. Anticancer Res. 2009;29:1047-52. [31] Li L, Yang G, Ebara S, Satoh T, Nasu Y, Timme TL, et al. Caveolin-1 mediates testosterone-stimulated survival/clonal growth and promotes metastatic activities in prostate cancer cells. Cancer Res. 2001;61:4386-92. [32] Hackenberg R, Schulz KD. Androgen receptor mediated growth control of breast cancer and endometrial cancer modulated by antiandrogen- and androgen-like steroids. J Steroid Biochem Mol Biol. 1996;56:113-7. [33] Apparao KB, Lovely LP, Gui Y, Lininger RA, Lessey BA. Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome. Biol Reprod. 2002;66:297-304. [34] Singh M, Zaino RJ, Filiaci VJ, Leslie KK. Relationship of estrogen and progesterone receptors to clinical outcome in metastatic endometrial carcinoma: a Gynecologic Oncology Group Study. Gynecologic Oncology. 2007;106:325-33.
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ACCEPTED MANUSCRIPT Tables and Figures
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Figure 1. Steroid receptor profiles in Ishikawa and KLE cell lines. (A) Immunocytochemistry demonstrating estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). MCF7 cells serve as positive controls for ER, PR and AR. DAPI (blue) highlights nuclei. Nuclear hormone receptor detected by red stain. Size bar is 20 μM. (B) Western blots demonstrating ER, PR and AR in nuclear extracts. Molecular weights are displayed on the lateral portions of images. MCF7 cells serve as positive controls for ER and PR. PC3 cells serve as positive control for AR. 2008 cells are negative control for ER and PR. LNCAP cells are the negative control for AR.
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Figure 2. CD133 expression and clonogenic assay in endometrial cancer cell lines. (A) Fluorescence-activated cell sorting analysis of the frequency of CD133+ cells in Ishikawa and KLE cell lines. Results representative of repeated experiments of FACS for CD133/2 (293C3). (B) Clonogenic assay demonstrates the proliferative differences between CD133+ and CD133populations in Ishikawa (P<0.001) and KLE (P =0.001) cell lines. After sorting, 10,000 KLE, or 2,500 Ishikawa cells were plated in 6 well plates. Viable cells were counted after exclusion of trypan blue-positive dead cells. Cell counts and photos were completed on days 3, 6 and 9. Photos are representative of triplicate experiments. Size bar is 100 μM. Graph represents mean cell counts ± SEM.
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Figure 3. MPA decreases total cell number, percent of CD133+ cells, and increases apoptosis. Ishikawa and KLE cell lines treated with MPA or ethanol control for a total of 6 days. (A) Cell counts, completed with typan blue exclusion. Results displayed as percent of control with SEM, *P < 0.05. (B) Cells were analyzed for CD133/2-APC using flow cytometry. MPA significantly decreases CD133+ fraction of cells. Results displayed as percent of control ± SEM, *P < .05. (C) MPA treatment increases apoptosis and decreases total cell number. Total number of cells and apoptotic cells were counted in triplicate. Photos are of MPA treated cells and ethanol controls, DAPI (blue) highlights nuclei, red is a secondary antibody marking apoptotic cells. Size bar is 100 μM. Figure 4. Androgen receptor is present in CD133+ and CD133- cells. After being sorted into CD133+ and CD133- fractions with flow cytometry, cells were plated, stimulated with testosterone and fixed, then cells were examined for AR using ICC. Nuclear stain is marked with DAPI (blue) and nuclear hormone receptor is red. Size bar is 20 μM. Figure 5. Progestin treatment decreases CD133 expression in patient samples over time. (A) Sample CD133 IHC demonstrating +3 intensity and 85% positive staining at 200X magnification. (B) Graph shows the responders change in H-score (histoscore) during hormonal treatment. Y-axis is the H-score. X-axis is time, far right is most recent biopsy or hysterectomy, graph moves backwards in time to first biopsy. Each line represents a unique patient (P4, P8, P10, P12) in the study.
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ACCEPTED MANUSCRIPT Table 1 Demographic and Clinicopathologic Characteristics Median (range) or % (n)
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n=7 Demographic
58 (44-65)
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Age (years) Caucasian
85.7 (6)
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African America Clinicopathologic BMI (kg/m2)
50.3 (22.4-65.4)
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Co-morbidities Diabetes, type 2 Cardiac
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Pulmonary Orthopedic Psychiatric
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FIGO Grade 1 3
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14.2 (1)
Endometrioid cell type
57.1 (4) 85.7 (6) 57.1 (4) 57.1 (4) 71.4 (4) 14.2 (1) 85.7 (6) 14.2 (1) 0 (0) 100 (7)
Reason for medical treatment MMP
85.7 (6)
Patient request
14.2 (1)
Post-menopausal
85.7 (6)
Hormonal Therapy*
Megestrol acetate
100 (7)
Levonorgestrel IUD
42.9 (3)
Response Complete regression
28.6 (2)
Definitive hysterectomy
57.1 (4)
Follow up Duration of hormonal treatment (months) Length of surveillance surgical patients (months)
21 (1-59) 18.5 (1-26)
BMI, body mass index; VTE, venous thromboembolus; FIGO, International Federation of Gynecology and Obstetrics; MMP, multiple medical problems; IUD, intrauterine device, *some patients had more than one treatment
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S0090-8258(15)30226-2 doi: 10.1016/j.ygyno.2015.12.022 YGYNO 976155
To appear in:
Gynecologic Oncology
Received date: Revised date: Accepted date:
17 September 2015 22 December 2015 23 December 2015
Please cite this article as: Michael S. Guy, Lubna Qamar, Kian Behbakht, Miriam D. Post, Jeanelle Sheeder, Carol A. Sartorius, Monique A. Spillman, Progestin Treatment Decreases CD133 + Cancer Stem Cell Populations in Endometrial Cancer, Gynecologic Oncology (2015), doi: 10.1016/j.ygyno.2015.12.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Progestin Treatment Decreases CD133+ Cancer Stem Cell Populations in Endometrial Cancer
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Michael S. Guy, MDa, Lubna Qamar PhDb, Kian Behbakht, MDb, Miriam D. Post, MDc, Jeanelle Sheeder, MSPH, PhDb, Carol A. Sartorius, PhDc, Monique A. Spillman, MD, PhDd a
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Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Cincinnati School of Medicine Cincinnati OH 45219, USA b Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Colorado School of Medicine Aurora CO 80045, USA c Department of Pathology, University of Colorado Denver School of Medicine, Aurora CO 80045, USA d Texas Oncology, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas TX 75246, USA
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Disclosure Statement: All authors declare that there are no financial disclosures, nor conflicts of interest.
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Corresponding Author: Michael Guy, MD University of Cincinnati Department of OB/GYN, Division of Gynecologic Oncology 222 Piedmont Ave. Medical Arts Building, Suite 4100 Cincinnati, OH 45219 Phone: 513-475-8162 Fax: 513-475-8174 E-mail: [email protected] Suggested Reviewers 1. Michael Goodheart, MD 2. David Mutch, MD Permanent Address 337 Oak Forest Dr. Dayton, OH 45419
ACCEPTED MANUSCRIPT ABSTRACT
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Objectives: Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells.
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Methods: The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4uM medroxyprogesterone 17-acetate (MPA) for 6 days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression were scored and evaluated for change over time on serial biopsies.
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Results: CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment.
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Conclusion: CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells. Keywords: endometrial cancer, cancer stem cells, hormonal treatment, progestin, progesterone WORD COUNT: 235
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ACCEPTED MANUSCRIPT Highlights: 1. CD133+ cells are identified in endometrial cancer cell lines and patient samples.
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2. Hormonal treatment with progestins decreases CD133+ cells in vitro and in patient samples from a clinical project.
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ACCEPTED MANUSCRIPT Introduction Endometrial cancer is the most common gynecologic malignancy in the United States
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with an expected 54,870 cases in 2015[1]. The majority of cases are diagnosed after menopause, but up to 14% of cases are diagnosed before menopause including 4% diagnosed before the age
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of 40 [2-5]. A majority of cases in younger women are lower grade and earlier stage [2].
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Seventy to ninety percent of type 1 endometrial cancer patients have significant medical comorbidities that may complicate surgery [6]. Surgical staging including hysterectomy,
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bilateral salpingo-oophorectomy and sampling of lymph nodes is the standard treatment.
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However, conservative management utilizing hormonal therapy may be considered in those desiring fertility sparing treatment or those who have multiple medical comorbidities making
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them poor surgical candidates.
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Hormonal treatment, especially with progestins, has been examined in the context of
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advanced stage or recurrent endometrial cancer. The Gynecologic Oncology Group (GOG) protocol 81 demonstrated that medroxprogesterone acetate (MPA) is active against endometrial carcinoma with an overall response rate of 25%[7]. In a systematic review, 15-30% of women responded to progestin therapy [8], the highest response rates were noted in low grade, estrogen receptor (ER) positive tumors [9]. In a prospective study using MPA in women with early stage, well differentiated, endometrial cancer a 55% response rate to treatment was seen[10]. The factors that play a role in response are incompletely understood. GOG protocol 119 demonstrated that response to progestin treatment was seen even when tumors did not express ER suggesting that there are alternative pathways being activated by progestin therapy[9]. One of the proposed components of cancer progression and recurrence are cancer stem cells. Cancer stem cells (CSC) are defined as a small subpopulation of cells within a tumor that contribute to tumor maintenance and progression and are resistant to traditional
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ACCEPTED MANUSCRIPT chemotherapy[11-14]. These tumorigenic cells are defined by their ability to self renew and generate mature cells of a particular tissue through differentiation [13]. CSC have been
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described from several human solid cancers including breast [15], brain [16, 17], colon [18, 19]
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head and neck [20] and pancreatic cancer [21]. Cancer stem cells have also been identified in gynecologic malignancies using extracellular markers including clusters of differentiation (CD)
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133 [22-26], CD44 [27] and CD117 [28] in ovarian and endometrial cancer.
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Nakamura et al. have shown that overexpression of CD133 in endometrial cancer patients is an independent predictor of decreased overall survival[24]. Their group also demonstrated
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that when patient derived tumor cells were isolated and treated with cisplatin or paclitaxel they were resistant to chemotherapy[24]. CD133+ cells also have differential response to estrogen
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treatment. Rutella et al. demonstrated that CD133+ endometrial cancer cells respond with increased growth after treatment with estradiol indicating that this is also a hormonally
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responsive sub-population [23].
The response of CD133 cells to hormonal treatment with progestins has not been explored. We explored the hypothesis that CD133+ endometrial cancer cells express ER and PR and that progestin therapy differentially regulates those cells. Specifically, by treating ER+/PR+ and ER-/PR- cell lines with progestins we anticipated that we would see decreases in cell number, increases in apoptosis and decreases in this unique CD133+ cell population. Secondly, in a clinical project, we hypothesized a similar decrease in CD133+ cells would occur in patients who responded to treatment with progestin therapy.
Materials and Methods Cell Lines and cell culture
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ACCEPTED MANUSCRIPT The Ishikawa (endometrial cancer), KLE (endometrial cancer), MCF7 (breast cancer), LNCaP (prostate cancer), and 2008 (ovarian cancer) cell lines were cultured at 37°C and 5%
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CO2 in RPMI 1640 (Invitrogen, Grand Island, NY 11875-093) with 10% fetal bovine serum
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(Phoenix Research Products, Asheville, NC A704410-7007) and 5% Pen Strep (Invitrogen, Grand Island, NY 15140-22). Cell lines were authenticated by polymorphic short tandem repeat
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analysis at the University of Colorado DNA Sequencing and Analysis Core as previously
Cambridge, MA) were used in western blots.
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Drugs and Drug Treatment
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described[29]. Purchased lysates from the PC3 prostate cancer cell line (ab3954, Abcam,
In experiments involving flow cytometry and apoptosis assays, cells were cultured in
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phenol red free RPMI 1640 (Invitrogen, Grand Island, NY 11835-055) with charcoal stripped fetal bovine serum (DCC) (Phoenix Research Products Asheville, NC, FBS-500DC) and 5% Pen
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Strep. Cells were plated, and then treated after 24 hours. Treatments included a control vehicle of ethanol (Sigma-Aldrich, St. Louis, MO, E7023) or 30.4 uM medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich, St. Louis, MO, M1629) dissolved in ethanol. Final concentrations of ethanol in media were 0.3%. MPA dosing was derived from previously published IC50 for the Ishikawa cell line [30]. Media was changed every 48 hours, and treatment concluded 7 days after plating. Flow Cytometry Analysis 0.5x106 cells were suspended in a 1:20 solution of MACS BSA Stock Solution and autoMACS ™ Rinsing Solution (Mitenyi Biotec, Auburn, CA, 130-091-376). 20L of fragment crystallizable (Fc) receptor blocking antibody (Mitenyi Biotec, Auburn, CA, 130-059-901) was added followed by either 10uL of CD133/2 (293C3)-APC antibodies (Mitenyi Biotec, Auburn,
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ACCEPTED MANUSCRIPT CA, 130-090-854) or Mouse IgG1 isotype control antibodies (Mitenyi Biotec, Auburn, CA, 130092-214) and incubated for 10 minutes at 4°C. Cells were then washed, and resuspended. Flow
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cytometry analysis was completed with CyAn ADP 9 color Analyzer (Beckman Coulter, Inc.,
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Brea, CA). Cloning Efficiency Assay
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4x107 cells were labeled with CD133/2 (293C3)-APC antibodies as detailed above.
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Sorting into CD133+ and CD133- populations was completed on a Moflo XDP 100 (Beckman Coulter, Inc., Brea, CA). 10,000 KLE, or 2,500 Ishikawa cells were plated in 6 well plates.
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Media was changed every 72 hours. At days 3, 6 and 9 cell counts and images were collected. Cell counts were completed with a ViCELL® Cell Counter (Beckman Coulter, Brea, CA) using
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trypan blue exclusion. Images were captured with an Olympus CKX41 microscope fitted with an Olympus DD73 camera using cellSens digital imaging software (Olympus, Center Valley,
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PA). Immunocytochemistry (ICC)
3x105 cells were plated in single chamber slides. After 48 hours, cells were fixed with 70% acetone and 30% cold methanol for 5 minutes at room temperature. Immunocytochemistry was conducted according to standard protocols. Antibodies used were as follows: ER (1:200, SP1 clone RM9101; LabVision/NeoMarkers), PR (1:500, PgR1294 clone M3568; Dako) and AR (1:600, rabbit monoclonal D6F11, Cell Signaling). Secondary antibody was Alexa Flour 555 (1:200, A21429, Life Technologies). Vector Shield (Vector Laboratories) was utilized for DAPI counter stain and mounting media. Immunofluorescence images were captured at room temperature on a Olympus CKX41 fluorescence microscope equipped with an Olympus DD73 camera (Olympus, Center Valley, PA) using cellSens digital imaging software (Olympus, Center
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ACCEPTED MANUSCRIPT Valley, PA) with a 40X objective. Androgen receptor localization studies utilized the above protocol, but prior to fixation, 10nM testosterone was added to media and cells were stimulated
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for 2 hours as previously described by Li et al [31].
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Preparation of Whole Cell Lysates, Nuclear Extracts and Western Blotting
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Protein was extracted, and concentration was quantitated by Bradford Assay. 100 µg of nuclear extract and 50g of whole cell lysate per lane were resolved on 7.5% SDS-PAGE gels
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(Gels: 456-1023EDU, Bio-Rad, Hercules, CA; Blotter: 170-4070 Bio-Rad, Hercules, CA,). Proteins were transferred to nitrocellulose (88013 Thermo Scientific, Rockford, IL), blocked
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with 5% milk in 1x TBST (T9039, Sigma-Aldrich, St. Louis, MO), then probed with the respective primary antibodies, washed then labeled with secondary antibodies (Anti-mouse:
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7076S, Anti-rabbit: 7074S, Cell Signaling Technology, Danvers, MA) and visualized using enhanced chemiluminescence (Supersignal West Pico 34080 and SuperSignal West Femto
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34095, Pierce, Rockford, IL). Film (34091, Pierce Rockford, IL) was exposed and developed. PC3, a prostate derived cell line, whole cell lysates were purchased (ab3954, Abcam, Cambridge, MA) and used as a negative control for the androgen receptor. Apoptosis Assay
2.5x103 cells were plated in 8 chamber slides (C7182-1CS, Sigma-Aldrich, St. Louis, MO). Cells were treated with progestins as described above for 7 days. Apotag® In situ Apoptosis Detection Kit (S7100, Millipore, Billerica, MA) was utilized for detection of terminal deoxynucleotidyl transferase found in apoptotic cells after treatment with progestins. Images were captured utilizing equipment described in ICC methods. Total number of cells and apoptotic cells were counted in 3 separate 20X fields then averaged in triplicate experiments. Patient Samples and Immunohistochemistry
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ACCEPTED MANUSCRIPT Institutional review board approval was obtained through the Colorado Multiple Institutional Review Board (COMIRB Protocol 13-1563). Women ≥18 years of age with a
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diagnosis of endometrial adenocarcinoma that had been treated with progestins were identified
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through a review of the University of Colorado Tumor Registry, Gynecologic Oncology Tumor Board and relevant ICD-9 codes. Clinical variables abstracted from the medical record included
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age, race, body mass index (BMI), medical comorbidities, FIGO tumor grade and stage,
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histologic cell type, indication for treatment, menopausal status, type of hormonal treatment, duration of hormonal treatment and length of surveillance in patients undergoing hysterectomy.
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Tissue samples were retrieved from the pathology department. Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained
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for Androgen Receptor (Cell Signaling, Danvers, MA; rabbit monoclonal D6F11; cat#: 5153; Lot: #2; 1:500), Estrogen Receptor (Thermo Scientific, Pittsburgh, PA; rabbit monoclonal SP1:
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cat#: MA139540; 1:40), Progesterone Receptor (DAKO, Carpinteria, CA; mouse monoclonal 1294; cat#M3568; 1:100) and CD133 (Miltenyi Biotec, Auburn, CA; mouse monoclonal cat#: 130-090-422; 1:15). All antibodies were diluted in TBST + 1% BSA w/v + 0.05% ProClin 950. Antigens to AR, ER and PR were revealed in pH 9.5 BORG solution (Biocare Medical, Concord, CA) for 5 minutes at 125°C (22 psi; Decloaking chamber, Biocare) with a 10 minute ambient cool down. Immunodetection was performed on the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ) at an operating temperature of 37°C. Primary antibodies were incubated for 32 minutes and detected with the UltraView universal polymer kit (Ventana). Antigens to CD133 required modest retrieval for 15 minutes at 110°C in Target Retrieval Solution (DAKO, diluted 1:50). All staining steps were performed at ambient temperature. Endogenous peroxidase and non-specific staining were blocked with 3% hydrogen peroxide (aq.
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ACCEPTED MANUSCRIPT v/v; 10 minutes) and 2.5% normal goat serum (from Mouse ImmPress kit; Vector; 20 minutes), respectively. Diluted primary antibody was applied and incubated for 60 minutes in a
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humidified chamber. Then the antibody was detected with a Mouse ImmPress polymer kit
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(Vector, cat#: MP-7402; 30 minutes). The complexes were visualized with ImmPACT DAB (Vector: cat#: SK-4105; 5 minutes). All sections were counterstained in Harris hematoxylin for
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xylene and coverglass mounted using synthetic resin.
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2 minutes, blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in
Hemotoxylin and eosin (H+E) stained slides were reviewed to identify tumor. Then the
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IHC slides were assessed at 40X by three independent reviewers (MG, KB, MP) utilizing two variables (intensity and percent positive) that were multiplied to create a histoscore (H-score).
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Intensity was graded on a 0-3 scale based on positive and negative controls from non-sample tissue (0=no stain, 1=weak staining, 2=moderate staining, 3=strong staining). Percent positive
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was scored from 0 to 100 percent. H-scores were averaged for each sample to create the composite H-score for ER, PR and AR. Interclass correlation, or overall agreement between the three observers was calculated and reported as Cronbach’s alpha. Interclass correlations were poor if values less than 0.40, fair for values between 0.40 and 0.59, good for values between 0.60 and 0.74, and excellent for values between 0.75 and 1.0. A gynecologic pathologist (MP) reviewed the H+E slides at the time of scoring to assess for progestin effect. Patients who had a clinical response with regression of FIGO grade, complete resolution of cancer, or noted treatment effect were considered as “responders”. Patients who had no noted treatment effect, persistent tumor or worsening tumor grade were categorized as “non-responders”. Statistical Analysis
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ACCEPTED MANUSCRIPT Statistical analysis of continuous variables with student’s t-test was performed using Graphpad Prism 6 for Mac OS X. Interclass correlations were conducted with IBM SPSS
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Results Steroid receptor profiles differ in Ishikawa and KLE cell lines
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statistics version 21. Statistical significance was determined by P values less than 0.05.
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To set up a model for ER+ and ER- endometrial cancer, hormone receptor profiles were
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analyzed for the Ishikawa and KLE cell lines using both Western Blot and ICC. In Western blot the MCF7 breast cancer cell line served as ER and PR positive controls. The 2008 ovarian
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cancer cell line was a negative control for ER and PR. PC3 (ab3954, Abcam, Cambridge, MA) and LNCaP prostate cancer cell lines served as the AR positive and negative controls
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respectively. In ICC, MCF7 served as positive controls for ER, PR and AR. Ishikawa whole
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cell lysates and ICC expressed ER, PR and AR. KLE cell lysates and ICC were ER and PR
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negative and expressed AR (Figure 1). ICC negative IgG controls provided for all images in supplemental Figure 1.
CD133+ cells are present in endometrial cancer cell lines and demonstrate differential growth The ER+/PR+ Ishikawa and the ER-/PR- KLE endometrial cancer cell lines were assayed for the presence of CD133 populations by flow cytometry. CD133+ cells were detected in both cell lines, but with a higher proportion in the ER+/PR+ Ishikawa cell line. Repeated experiments demonstrated 6.5% of ER-/PR- KLE and 80.7% of ER+/PR+ Ishikawa cells were positive for CD133 (Figure 2, panel A). A clonogenic assay was used to demonstrate the differential proliferative potential between CD133- and CD133+ populations. Day 1 cell counts were the same between groups. By day 9, significant differential growth of the CD133+ cells was confirmed in the KLE (7.7 x
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Progestin treatment differentially increases apoptosis in CD133+ cells Cell lines were treated with MPA for 6 days. Compared to ethanol treated controls, there
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was a significant reduction in viable cells in the MPA treated Ishikawa (100% vs. 86.4%,
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P=0.036) and KLE (100% vs. 45.6%, P=0.0002) cell lines (Figure 3A). Significant reductions in
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CD133+ populations were noted after treatment when compared to controls in Ishikawa (62.2% vs. 81.1%, P<0.0001; normalized to controls 79.4% vs. 100%) and KLE (3.0% vs. 6.4%
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P=0.0001; normalized to controls 46.8% vs. 100%) cell lines (Figure 3B). MPA treatment significantly increased the number of apoptotic cells in both cell lines compared to ethanol
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controls (Ishikawa 32.1% vs. 0.4%, P=0.01; KLE 28.4% vs. 0.7%, P=0.0006) (Figure 3C). Androgen Receptor present in CD133+ endometrial cancer cells
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Progestins such as MPA also activate androgen receptor (AR) [32] and AR has been identified previously in the Ishikawa cell line[33]. Because the MPA driven apoptosis of CD133+ cells was also detected in the ER-/PR- KLE cells, we postulated that these cells would contain the AR. After KLE and Ishikawa cells were sorted into CD133+ and CD133populations with flow cytometry, cells were stimulated with testosterone for two hours and fixed. ICC was then completed demonstrating that AR was present in both CD133+ and CD133populations of the Ishikawa and KLE cell lines (Figure 4). Progestin mediated decrease in CD133+ cells confirmed in clinical tissue samples Twelve patients were identified who were treated with progestins, and a total of 28 individual endometrial cancer samples were reviewed. Five patients only had a single specimen available for analysis and could not be compared over time. These patients began hormonal
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ACCEPTED MANUSCRIPT treatment at outside facilities and presented to our institution for hysterectomy. Their prior biopsies were not available for comparison.
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Demographics and clinicopathologic characteristics are detailed in table 1 of the
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remaining 7 patients. The majority of the population analyzed was post-menopausal, white, extremely obese (class III, BMI > 40), with multiple major medical co-morbidities and had well-
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differentiated endometrioid carcinomas. Documented indications for progestin treatment were
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related to increased surgical risk and concern for significant perioperative complications. All patients were treated with megestrol acetate (Megace), and 3 patients also had levonorgestrel
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IUDs placed as an additional therapeutic intervention.
The median length of hormonal treatment in the cohort was 21 months. Two patients
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(28.6%) had complete regression of cancer. One patient underwent radiation with curative intent. Four patients (57.1%) underwent definitive treatment with hysterectomy due to non-response to
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treatment or improvement in co-morbid conditions. In patients who underwent hysterectomy, median length of treatment with progestins prior to surgery was 8.5 months (range 1-21 months). The patients with hysterectomy have been followed for a median of 18.5 months after surgery. One post-hystectomy patient whose final pathology revealed a poorly differentiated mixed endometrial cancer that declined adjuvant chemotherapy and radiation had a pelvic recurrence 12 months after surgery. ER was expressed in 96.4% (n=27/28) of individual endometrial cancer specimens with a median H-score of 263 (95% CI 223.3-280.0). PR was expressed in 96.4% (n=27/28) samples, with a median H-score of 175.0 (95% CI 120.0-250.0). AR was expressed in 89.3% (n=25/28) samples, with a median H-score of 95.0 (95% CI 53.3-200.0). CD133 was expressed in 100% of samples (n=28), with a median H-score of 66.0 (95% CI 50.0-90.0) (Figure 5A). Interclass
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ACCEPTED MANUSCRIPT correlations average measures were calculated for ER (Cronbach’s alpha=0.961; 95% CI 0.930.98), PR (Cronbach’s alpha=0.965; 95% CI 0.94-0.98), AR (Cronbach’s alpha=0.853; 95% CI
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0.75-0.92) and CD133 (Cronbach’s alpha=0.846; 95% CI 0.72-0.92). Inter-rate reliability was
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excellent between 3 independent reviewers. For each patient, we assessed for changes in ER, PR and AR H-scores over time and no significant trends were identified.
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We further assessed the endometrial samples for changes in the proportion of CD133
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cells in response to progestins over time. Of the 7 patients with multiple samples available for comparison, there were 4 responders and 3 non-responders to progestins. In the responders,
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100% (n=4) showed a decrease in CD133 H-score between their first and last endometrial cancer samples (Figure 5B). In the three non-responders, changes in CD133 levels were mixed with no
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clear trends noted (Supplemental Figure 2). In responders and non-responders, samples were evaluated for loss or gain of ER/PR and AR expression and no significant change in H-scores
Discussion
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occurred during treatment.
Endometrial cancer is a hormonally responsive malignancy. The factors that play a role in response and progression are incompletely understood. CD133 is a cancer stem cell or tumorinitiating cell marker that has been defined in many cancers including gynecologic malignancies. We hypothesized that the CD133+ cell population in endometrial cancer was hormonally responsive and would differentially regulated by progestins. Our study demonstrates that CD133 cells are present in the ER+/PR+ Ishikawa and ER/PR- KLE endometrial cancer cell lines and patient endometrial cancer samples. When treated with progestins, CD133+ cells decreased in endometrial cancer cell lines as well as patient samples for patients who responded to progestin treatment. In the in vitro model, MPA
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ACCEPTED MANUSCRIPT treatment resulted in both decreased cell number and increased apoptosis in both ER+/PR+ and ER-/PR- cells.
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Our hypothesis and findings are supported by previous research that has demonstrated
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that CD133+ endometrial cancer cells are responsive to hormonal treatment with another steroid hormone, estrogen. After sorting patient derived endometrial cancer cells into CD133 positive
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and negative groups, Rutella et al. treated with estradiol for 72 hours and showed increased
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growth in only the CD133+ population [23]. Combining our results with these prior findings indicate that CD133+ endometrial cancer cells can be both positively and negatively growth
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regulated by hormonal treatment with estrogens and progestins respectively. Because progestin treatment effect was noted in both ER+/PR+ (Ishikawa) and ER-/PR-
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(KLE) cell lines it is likely that the decrease in proliferation, increase in apoptosis and decrease in CD133+ cells related to progestin treatment is multifactorial. In addition to direct interaction
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with the progesterone receptor, other mechanisms may involve non-classical signaling pathways utilizing the androgen receptor. In breast cancer, another hormonally responsive malignancy, Hackenburg et al. demonstrated in ER-/PR-/AR+ breast cancer and endometrial cancer cell lines that there is growth inhibition via AR with MPA[32]. To further support this possible interaction we demonstrated AR was not only present in the whole cell lysates of endometrial cancer cells, but was also present in sorted CD133+ sub-population. Furthermore, GOG 119, a phase II clinical trial that evaluated response to tamoxifen and intermittent MPA in patients with recurrent or advanced stage endometrial cancer, demonstrated highest response rates in patients with ER+ tumors, but response rates of 26% were also noted in patients with receptor negative tumors[34]. While AR was not assayed in this study, one may postulate that AR expression might account for this response. These data from other disease sites and the clinical trial
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ACCEPTED MANUSCRIPT information support the concept of complex hormonal interplay between progestin and multiple targets, both direct and indirect.
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Strengths of this study include the addition of a clinical component evaluating tumor
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specimens of patients treated with progestins at our institution. Their history, co-morbid conditions, treatment course and physician clinical decision-making was well documented.
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Patient follow-up since complete response or definitive treatment with surgery reaches a median
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of almost 2 years. Weaknesses of this study include the small sample size that limits statistical analysis and more definitive conclusions regarding the clinical utility of CD133 H-scores and its
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role in initial patient selection for progestin therapy, as well as its use for monitoring treatment. All patient samples in our study were ER+ so we were not able to parallel the in vitro ER+/PR+
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versus ER-/PR- model in the patient specimens. In future research, use of a larger endometrial cancer sequential biopsy set through a GOG ancillary study or a multi-institutional effort would
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be beneficial.
In conclusion, this work provides insight into the differential impact of progestin treatment on the hormonally responsive CD133+ endometrial cancer cells. We have shown that MPA treatment results in change in cell number through apoptosis and decrease in CD133+ cell number. This effect was noted in ER+/PR+ and ER-/PR- endometrial cancer cell lines and may be mediated by the androgen receptor. This exciting finding indicates that progestin therapy should be considered for treatment in both ER+ and ER- tumors, in patients who are not candidates for definitive surgery or who desire future fertility.
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Acknowledgements This study was supported by the Academic Enrichment Fund administered by the Department of Obstetrics and Gynecology at the University of Colorado School of Medicine. We thank Dr. Twila Jackson, PhD for generously providing the Ishikawa and KLE cell lines and Dr. Britta Jacobsen, PhD (Univ of Colorado School of Medicine and Anschutz Medical Campus) for the LNCaP cell line.
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Disclosure Statement: All authors declare that there are no financial disclosures, nor conflicts of interest.
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[21] Li C, Lee CJ, Simeone DM. Identification of human pancreatic cancer stem cells. Methods Mol Biol. 2009;568:161-73. [22] Friel AM, Zhang L, Curley MD, Therrien VA, Sergent PA, Belden SE, et al. Epigenetic regulation of CD133 and tumorigenicity of CD133 positive and negative endometrial cancer cells. Reprod Biol Endocrinol. 2010;8:147. [23] Rutella S, Bonanno G, Procoli A, Mariotti A, Corallo M, Prisco MG, et al. Cells with characteristics of cancer stem/progenitor cells express the CD133 antigen in human endometrial tumors. Clin Cancer Res. 2009;15:4299-311. [24] Nakamura M, Kyo S, Zhang B, Zhang X, Mizumoto Y, Takakura M, et al. Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer. Hum Pathol. 2010;41:1516-29. [25] Kryczek I, Liu S, Roh M, Vatan L, Szeliga W, Wei S, et al. Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. Int J Cancer. 2012;130:29-39. [26] Baba T, Convery PA, Matsumura N, Whitaker RS, Kondoh E, Perry T, et al. Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells. Oncogene. 2009;28:209-18. [27] Steffensen KD, Alvero AB, Yang Y, Waldstrom M, Hui P, Holmberg JC, et al. Prevalence of epithelial ovarian cancer stem cells correlates with recurrence in early-stage ovarian cancer. J Oncol. 2011;2011:620523. [28] Luo L, Zeng J, Liang B, Zhao Z, Sun L, Cao D, et al. Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome. Exp Mol Pathol. 2011;91:596-602. [29] Korch C, Spillman MA, Jackson TA, Jacobsen BM, Murphy SK, Lessey BA, et al. DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecologic Oncology. 2012;127:241-8. [30] Moe BT, Vereide AB, Orbo A, Jaeger R, Sager G. Levonorgestrel, medroxyprogesterone and progesterone cause a concentration-dependent reduction in endometrial cancer (Ishikawa) cell density, and high concentrations of progesterone and mifepristone act in synergy. Anticancer Res. 2009;29:1047-52. [31] Li L, Yang G, Ebara S, Satoh T, Nasu Y, Timme TL, et al. Caveolin-1 mediates testosterone-stimulated survival/clonal growth and promotes metastatic activities in prostate cancer cells. Cancer Res. 2001;61:4386-92. [32] Hackenberg R, Schulz KD. Androgen receptor mediated growth control of breast cancer and endometrial cancer modulated by antiandrogen- and androgen-like steroids. J Steroid Biochem Mol Biol. 1996;56:113-7. [33] Apparao KB, Lovely LP, Gui Y, Lininger RA, Lessey BA. Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome. Biol Reprod. 2002;66:297-304. [34] Singh M, Zaino RJ, Filiaci VJ, Leslie KK. Relationship of estrogen and progesterone receptors to clinical outcome in metastatic endometrial carcinoma: a Gynecologic Oncology Group Study. Gynecologic Oncology. 2007;106:325-33.
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Figure 1. Steroid receptor profiles in Ishikawa and KLE cell lines. (A) Immunocytochemistry demonstrating estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR). MCF7 cells serve as positive controls for ER, PR and AR. DAPI (blue) highlights nuclei. Nuclear hormone receptor detected by red stain. Size bar is 20 μM. (B) Western blots demonstrating ER, PR and AR in nuclear extracts. Molecular weights are displayed on the lateral portions of images. MCF7 cells serve as positive controls for ER and PR. PC3 cells serve as positive control for AR. 2008 cells are negative control for ER and PR. LNCAP cells are the negative control for AR.
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Figure 2. CD133 expression and clonogenic assay in endometrial cancer cell lines. (A) Fluorescence-activated cell sorting analysis of the frequency of CD133+ cells in Ishikawa and KLE cell lines. Results representative of repeated experiments of FACS for CD133/2 (293C3). (B) Clonogenic assay demonstrates the proliferative differences between CD133+ and CD133populations in Ishikawa (P<0.001) and KLE (P =0.001) cell lines. After sorting, 10,000 KLE, or 2,500 Ishikawa cells were plated in 6 well plates. Viable cells were counted after exclusion of trypan blue-positive dead cells. Cell counts and photos were completed on days 3, 6 and 9. Photos are representative of triplicate experiments. Size bar is 100 μM. Graph represents mean cell counts ± SEM.
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Figure 3. MPA decreases total cell number, percent of CD133+ cells, and increases apoptosis. Ishikawa and KLE cell lines treated with MPA or ethanol control for a total of 6 days. (A) Cell counts, completed with typan blue exclusion. Results displayed as percent of control with SEM, *P < 0.05. (B) Cells were analyzed for CD133/2-APC using flow cytometry. MPA significantly decreases CD133+ fraction of cells. Results displayed as percent of control ± SEM, *P < .05. (C) MPA treatment increases apoptosis and decreases total cell number. Total number of cells and apoptotic cells were counted in triplicate. Photos are of MPA treated cells and ethanol controls, DAPI (blue) highlights nuclei, red is a secondary antibody marking apoptotic cells. Size bar is 100 μM. Figure 4. Androgen receptor is present in CD133+ and CD133- cells. After being sorted into CD133+ and CD133- fractions with flow cytometry, cells were plated, stimulated with testosterone and fixed, then cells were examined for AR using ICC. Nuclear stain is marked with DAPI (blue) and nuclear hormone receptor is red. Size bar is 20 μM. Figure 5. Progestin treatment decreases CD133 expression in patient samples over time. (A) Sample CD133 IHC demonstrating +3 intensity and 85% positive staining at 200X magnification. (B) Graph shows the responders change in H-score (histoscore) during hormonal treatment. Y-axis is the H-score. X-axis is time, far right is most recent biopsy or hysterectomy, graph moves backwards in time to first biopsy. Each line represents a unique patient (P4, P8, P10, P12) in the study.
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ACCEPTED MANUSCRIPT Table 1 Demographic and Clinicopathologic Characteristics Median (range) or % (n)
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n=7 Demographic
58 (44-65)
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Age (years) Caucasian
85.7 (6)
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African America Clinicopathologic BMI (kg/m2)
50.3 (22.4-65.4)
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Co-morbidities Diabetes, type 2 Cardiac
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Pulmonary Orthopedic Psychiatric
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FIGO Grade 1 3
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14.2 (1)
Endometrioid cell type
57.1 (4) 85.7 (6) 57.1 (4) 57.1 (4) 71.4 (4) 14.2 (1) 85.7 (6) 14.2 (1) 0 (0) 100 (7)
Reason for medical treatment MMP
85.7 (6)
Patient request
14.2 (1)
Post-menopausal
85.7 (6)
Hormonal Therapy*
Megestrol acetate
100 (7)
Levonorgestrel IUD
42.9 (3)
Response Complete regression
28.6 (2)
Definitive hysterectomy
57.1 (4)
Follow up Duration of hormonal treatment (months) Length of surveillance surgical patients (months)
21 (1-59) 18.5 (1-26)
BMI, body mass index; VTE, venous thromboembolus; FIGO, International Federation of Gynecology and Obstetrics; MMP, multiple medical problems; IUD, intrauterine device, *some patients had more than one treatment
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Figure 5
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