doi:10.1111/jog.12758
J. Obstet. Gynaecol. Res. Vol. 41, No. 10: 1630–1637, October 2015
Epithelial ovarian cancer with CD117 phenotype is highly aggressive and resistant to chemotherapy Irena Conic1, Zorica Stanojevic1, Ljubinka Jankovic Velickovic2, Slavica Stojnev2, Ana Ristic Petrovic2, Miljan Krstic2, Marko Stanojevic3, Dragan Bogdanović4 and Vladisav Stefanovic4,5 1 Clinic of Oncology, Clinical Center, 2Center of Pathology, Clinical Center, 3Clinic of Gynecology, Clinical Center, and 5Faculty of Medicine, University of Nis, Niš, Serbia 4State University of Novi Pazar, Novi Pazar, Serbia
Abstract Aim: CD117 expression has a pathogenic role in many malignancies, including ovarian carcinoma. The aim of the present study was to examine the correlation of stemness-associated marker CD117 with the clinicopathologic features of epithelial ovarian cancer and patient survival. Material and Methods: The analysis included 240 primary ovarian carcinomas (OC) diagnosed during the period from 2005 to 2011 in the region of South Serbia. Age, pathohistological characteristics, presence and size of residual tumor, choice of therapy and response to the therapy were studied. Results: Residual tumors were more frequently present in the patients with positive CD117 expression (18.1% vs 8.0%; P < 0.05). Chemotherapy according to paclitaxel/carboplatin protocol was more frequent in the patients with positive CD117 expression (70.9% vs 54.2%; P < 0.05), while carboplatin monotherapy was more frequent in the patients with negative CD117 expression (18.0% vs 6.4%; P < 0.05). Median survival time in patients with CD117-positive mucinous and endometrioid OC was significantly shorter, at 20 and 26.8 months, respectively. Median survival in serous OC was not related to CD117 expression. Conclusion: Residual tumors and chemotherapy treatment were more frequent in patients with positive CD117 expression. The outcome was dependent on the type of OC; a worse outcome, including a shorter survival, was documented in the mucinous and endometrioid OC cases. Key words: CD117 expression, ovarian carcinoma, survival, treatment.
Introduction Epithelial ovarian cancer (EOC) is the fourth most common cause of cancer death in European women.1 Treatment includes maximal surgical debulking and platinum combination chemotherapy. Complete remission is common, but the majority of women develop a relapse of the disease. This is followed with multiple lines of chemotherapy, due to increased drug resistance associated with relapse. This emphasizes the need for more effective therapies in order to improve survival and quality of life of EOC patients.
EOC demonstrates significant tumor cell population heterogeneity, with multilineage clones expressing different capacities for growth, survival, metastasis and resistance to chemotherapy and radiation therapy. So far, many biomarkers expressed by EOC cells have been investigated in terms of their diagnostic and prognostic potential. In the last few years, a great improvement in understanding tumor biology has been achieved due to the discovery of cancer stem cells (CSC), which play the main role in cancer progression and its resistance to therapy. CSC could open the possibility of creating a
Received: August 21 2014. Accepted: April 8 2015. Reprint request to: Dr Irena Conic, Clinic of Oncology, Clinical Center Niš, Dr Zoran Djindjic Street 48, 18000 Niš, Serbia. Email: [email protected]
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Epithelial OC with CD1117 phenotype
new target for the purpose of decreasing resistance to chemotherapy and of improving the effects of therapy.2,3 CD117 (c-kit) is a surface marker for embryonic, hematopoietic and mesenchymal stem cells. CD117 is associated with undifferentiated cell state and capacity for renewal.4 CD117 expression was found in various tumors of mesenchymal and epithelial origin. Patients with high expression and/or mutation of CD117 had a significantly worse disease prognosis in different types of tumors.5–10 However, the role of CD117 in EOC has not been completely clarified. The recent discovery of CSC could reveal the role of CD117 in the development and growth of EOC. Identification and isolation of CSC have been reported in different kinds of cancer, including ovarian carcinoma (OC). Moserle et al.11 identified a subpopulation of OC cells with the side-population (SP) phenotype from a single tumor xenograft. These cells were highly cancerous in mice, compared to the tumor cell population that did not contain SP cells. Zhang et al.12 observed a subpopulation of primary human ovarian cancer cells that could form independent spheres and were capable of self-renewal in in vitro conditions that highly expressed CD44 and CD117. The cells with CD 44+/CD 117+ phenotype were extremely tumorigenic and were capable of developing a tumor in a xenograft experiment. CSC isolated from primary tumors have the ability to regenerate the tumor cell population and reconstitute the original tumor phenotype with as few as 100 cells. CSC from ovarian carcinomas were found to display the cell surface markers CD44 + CD117 + CD133+. In addition, CSC are also thought to account for chemotherapy resistance.13 Luo et al.,14 demonstrated that human OC cells with the CD117+ phenotype possess the unique properties of CSC. This includes self-renewal, differentiation, a high tumorigenic potential, and chemoresistance. Targeting CD117+ cancer cells may be effective therapy for treatment of OC. Bapat et al.15 isolated tumor cells from the ascites of patients with serous OC and obtained 19 spontaneously immortalized clones. Performing molecular examination of the clones, they identified expression of CD 44, CD 117 and a ligand for CD117. Only one of these clones expressed tumorigenesis in vivo, which indicated the presence of cells with potential for tumor initiation and multilineage differentiation in ovarian tumors accompanied by ascites. Zhang et al.12 analyzed tumor spheres originating from the cells isolated from the ascites of five patients with serous OC. After 10 serial passages in stem cell medium, it was demonstrated that the remaining sphere
© 2015 Japan Society of Obstetrics and Gynecology
cells showed high expression of CD44 and CD117. CD44+ and CD117+ spheroid cells were resistant to chemotherapy, and they were capable of starting carcinogenesis, leading to tumor formation in mice. Tumor metastases are considered the main cause of death in various malignant tumors. Evidence from previous studies showed that CSC can be generated in the processes associated with aberrant activation of epithelial–mesenchymal transition (EMT), which affects cell differentiation and metastatic tumor potential. An anti-EMT strategy would be a new therapeutic option for treating aggressive carcinomas.2,3 The aim of the present study was to examine the correlation of stemness-associated marker c-kit with clinicopathologic features of EOC and patient survival.
Methods The study included female patients with ovarian carcinoma diagnosed during the period from 2005 to 2011 in the region of South Serbia. The patients’ survival rate was observed until September 2013. The study included 240 cases of primary EOC, including: borderline tumors, OC limited to ovaries (stage I according to the official FIGO staging system16), OC disseminated in pelvic organs (FIGO stage II), OC with peritoneal metastases (FIGO stage III) and OC with remote metastases (FIGO stage IV). In four cases of radical hysterectomy with adnexectomy, FIGO stage could not be determined, because omentectomy was not performed. In the patients with EOC, clinical parameters were observed, as well as their pathological features. The analyzed clinical parameters included: age, FIGO stage, presence and size of residual tumor, kind of therapy and response to therapy. The analyzed pathological characteristics included: histological type of tumor, histological grade, and nuclear grade, according to official World Health Organization grading system.16 The data sources were histories of patients with OC treated at the Oncology Clinic of the Clinical Center Niš. Pathological and immunohistochemical analyses of OC samples were performed at the Pathology Institute of the Faculty of Medicine in Niš. Pathohistological analysis was performed on biopsy samples of OC fixed in 10% neutral formalin, processed in the automatic tissue processor and embedded in paraffin. After deparaffinization and processing in graded alcohols, the selected samples of 5-μm thick cancer tissue sections were stained using the classic histological hematoxylin–eosin method for determination of tumor
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differentiation and its pathological stage. The immunohistochemical analysis was performed using Polyclonal Rabbit Antihuman CD 117 (c-kit) antibody in dilution 1:300. Paraffin tissue sections were processed according to the manufacturer’s protocol (DAKO LSAB2R system HRP). In brief, tissue slices were deparaffinized, rehydrated and incubated in 30 mL/L hydrogen peroxide, to block endogenic peroxidase, and then, thoroughly rinsed with phosphate-buffered saline (PBS) (pH = 7.4). The next step was incubation with CD117 antibodies for 24 h in the humid chamber at 4°C. After rinsing with PBS, the tissue was incubated with secondary antibody (Biotin labeled goat antirabbit antibody) for 30 min. After repeated rinsing with PBS, incubation with tertiary antibody for 30 min followed. The visualization of marked antigens was performed using diaminobenzidinetetrahydrochloride chromogen. Counterstaining was performed with Mayer’s hematoxylin. Simultaneously with CD117 staining, selected cancer sections that were used as negative control were incubated with antibody diluents without addition of CD117 antibody, while section of healthy testicular tissue served as positive control (CD117-stained germ cells in seminal tubules). The analysis of immunohistochemically stained microscopic preparations of OC was performed using a light microscope Leica DM 1000. The obtained results were systematized and grouped, while the statistical significance was tested using the corresponding statistical tests, according to the tested parameters and sample size.
Scoring of immunohistochemical reaction Scoring of immunohistochemical CD117 reactivity was performed on the grounds of estimating the positive staining (membranous or membranous and cytoplasmic expression) in tumor cells: The intensity of the immunostaining was graded as negative (no staining), weak (1+), moderate golden-brown (2+), or strong brown coloration (3+) (modified from Zhang et al.12). The tumor was considered positive for CD117 expression if more than 25% of cancer cells were stained with moderate or strong intensity.
Statistical methods The quantitative statistical analysis was performed on the computer. Recording, ranging, grouping, as well as tabular and graphic presentation of data was performed using Excel 2003. The calculations were performed using R 2.12.0 (R Foundation for Statistical Computing).
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The mean values of numeric features between the two groups of patients were compared using the Student’s t-test or the Mann–Whitney U-test in cases where the value distribution did not satisfy the demands of a standard schedule. The numeric values among three and four groups of patients were compared by variance analysis using Tukey’s post-hoc test with Cramer correction for uneven group sizes. Comparison of the frequency of certain categories of attributive features among certain groups of patients was performed by χ 2-test or Fisher’s exact test in cases where an expected frequency was less than 5. Estimating the correlation between lethal outcome and all other investigated factors was performed by multivariant Cox regression analysis. Odds ratio was used to calculate the values of the rate of presence and absence of lethal outcomes and their 95% confidence interval. Applying the step-by-step backward method (Backward: Wald), all factors of unconfirmed statistical significance were excluded. The Kaplan–Meier estimator and log–rank test were used to test the level of expression of certain markers and survival rate. A permitted level of estimation error lower than 5% (P < 0.05) was used as the level of statistical importance in concluding.
Results The average age of patients with absent CD117 expression was 56.9 ± 13.2 years and these patients were much older than the patients with positive CD117 expression, whose average age was 52.6 ± 11.7 years (P < 0.05) (Table 1). Residual tumors were more frequently present in the patients with a positive CD117 expression (18.1% vs 8.0%; P < 0.05). Chemotherapy according to paclitaxel/carboplatin protocol was more frequent in the patients with a positive CD117 expression (70.9% vs 54.2%; P < 0.05), while carboplatin monotherapy was more frequent in the patients with a negative CD117 expression (18.0% vs 6.4%; P < 0.05). The disease progression was present in a significantly higher percentage of patients with a negative CD117 expression (19.7% vs 6.4%; P < 0.05), while partial remission was present in a significantly higher percentage of patients with a positive CD117 expression (35.4% vs 22.3%; P < 0.05). The differences concerning the values and frequency of other characteristics between the patients with different levels of CD117 expression were not statistically significant (Table 1).
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Epithelial OC with CD1117 phenotype
Table 1 Characteristics of patients, applied therapy and disease outcome compared to CD117 expression CD117 expression
Characteristics
Age Time to diagnosis Type of tumor Primary tumor Metastasis Residual tumor Surgical treatment Radical hysterectomy Unilateral adnexectomy with hysterectomy Bilateral adnexectomy Unilateral adnexectomy Exploratory laparotomy with biopsies Applied chemotherapy No chemotherapy Paclitaxel/carboplatin Etoposide/carboplatin Carboplatin Etoposide Etoposide/doxorubicin/carboplatin Number of chemotherapies Disease outcome Progression Stable disease Partial remission Complete remission Survival (months) Death
Significance
Negative (n = 174)
Positive (n = 66)
56.9 ± 13.2 4.50 ± 4.97
52.6 ± 11.7 3.80 ± 3.65
0.022 NS
144 (82.7%) 30 (17.2%) 14 (8.0%)
52 (78.7%) 14 (21.2%) 12 (18.1%)
NS 0.024
106 (63.0%) 2 (1.1%) 12 (7.1%) 18 (10.7%) 30 (17.8%)
34 (51.5%) 2 (3.0%) 6 (9.0%) 10 (15.1%) 14 (21.2%)
NS NS NS NS NS
10 (6.0%) 90 (54.2%) 32 (19.2%) 30 (18.0%) 2 (1.2%) 2 (1.2%) 5.02 ± 1.91
2 (3.2%) 44 (70.9%) 12 (19.3%) 4 (6.4%) 0 (0.0%) 0 (0.0%) 4.97 ± 1.75
NS 0.022 NS 0.028 NS NS NS
30 (19.7%) 4 (2.6%) 34 (22.3%) 84 (55.2%) 36.7 ± 23.8 78 (49.3%)
4 (6.4%) 2 (3.2%) 22 (35.4%) 34 (54.8%) 40.91 ± 24.6 32 (55.1%)
0.016 NS 0.048 NS NS NS
NS, not significant.
Positive expression of CD117 was demonstrated in a significant percentage of patients with serous OC (32.6%) compared with patients with mucinous OC (9.1%; P < 0.05) and clear cell OC (0.0%; P < 0.05) (Table 2). Expression of CD117 in serous OC is given in Figure 1 as cytoplasmic (Fig. 1a), and both cytoplasmic and membranous staining (Fig. 1b). In the group of patients with FIGO stage II disease, CD117 expression was not observed in any of the investigated tumors (11.7% vs 0.0%; P < 0,01) (Table 3). The differences in the frequency of other FIGO stages, as well as in histologic and nuclear grades between the patients
with different levels of CD117 expression, were not statistically significant. Kaplan–Meier survival curves for all investigated patients and for every different EOC type are presented in Figure 2. The median survival time in patients with EOC and negative CD117 expression was 47.2 months, while for those with positive CD117 expression it was 52.1 months. The Kaplan–Meier estimator and log–rank test showed non-significant difference between survival distributions of patients with positive and negative CD117 expression (P = 0.823) (Table 4). Median survival time in patients with serous ovarian cancer and
Table 2 CD117 expression related to the type of epithelial ovarian cancer CD117 expression Negative Positive
Type of epithelial ovarian cancer Serous
Mucinous
Endometrioid
Clear cell
120 (67.4%) 58 (32.6%)
20 (90.9%) 2 (9.1%)
24 (80.0%) 6 (20.0%)
10 (100.0%) 0 (0.0%)
Sum
Significance
174 (72.5%) 66 (27.5%)
A*, C*
*P < 0.05. A, serous vs mucinous; B, serous vs endometrioid; C, serous vs clear cell; D, mucinous vs endometrioid; E, mucinous vs clear cell; F, endometrioid vs clear cell.
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(a)
Table 3 Correlation of clinicopathologic characteristics and CD117 expression Characteristics
(b)
FIGO stage Borderline I II III IV Histologic grade 0 1 2 3 Nuclear grade 0 1 2 3
CD117 expression
Significance
Negative (n = 174)
Positive (n = 66)
12 (7.0%) 36 (21.1%) 20 (11.7%) 86 (50.5%) 16 (9.4%)
6 (9.0%) 12 (18.1%) 0 (0.0%) 40 (60.6%) 8 (12.1%)
NS NS 0.004 NS NS
12 (6.9%) 22 (12.7%) 96 (55.8%) 42 (24.4%)
6 (9.0%) 10 (15.1%) 38 (57.5%) 12 (18.1%)
NS NS NS NS
12 (6.9%) 26 (15.1%) 96 (55.8%) 38 (22.0%)
6 (9.0%) 8 (12.1%) 36 (54.5%) 16 (24.2%)
NS NS NS NS
NS, not significant.
Figure 1 Expression of CD117 in serous ovarian cancer. (a) Cytoplasmic expression of CD117 in serous ovarian carcinoma (OC) (×400). (b) Cytoplasmic expression of CD117 in cells of serous OC and in stroma (×400).
negative/positive CD117 expression was similar to the expression of EOC in general. Median survival time in patients with mucinous OC was 47.9 months for CD117-negative tumors, and 20.0 months for CD117positive tumors. A similar low survival of 26.8 months was found in CD117-positive endometrioid ovarian cancer. All 10 patients with clear cell ovarian cancer had negative CD117 expression. The median survival time of these patients was 38.1 months.
Discussion CD117 expression (c-kit) has a pathogenic role in many malignancies, including ovarian cancer.17,18 Recent research has indicated the relevance of response to imatinib (kinase inhibitor) in patients with active c-kit mutations. In the majority of cases, the role of c-kit in neoplasia is not quite clear.
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In this study, chemotherapy according to paclitaxel/ carboplatin protocol was more frequent in patients with a positive CD117 expression, while carboplatin monotherapy was more frequent in patients with a negative CD117 expression, which matched the disease stage. Residual tumors and chemotherapy treatment were more frequent in patients with positive CD117. In this study, the outcome was dependent on the type of ovarian carcinoma; a worse outcome, including a shorter survival, was documented in the mucinous and endometrioid ovarian cancer with a positive expression of CD117. Luo et al.14 observed a correlation between CD117 expression and the response to the applied chemotherapy in OC. The response to chemotherapy was correlated to many clinical factors, including the disease stage, residual tumor after surgical intervention and applied chemotherapy. All cases in the study were in an advanced stage (FIGO stage III). All patients underwent optimal cytoreduction (residual tumor smaller than 1 cm) and paclitaxel/platinum therapy. There was a significant correlation between CD117 expression and the response to secondary therapy. Compared to CD117– tumors, CD117+ tumors were less sensitive to chemotherapy, and they reoccurred in a shorter period of time after chemotherapy. However, this study was conducted on a small number of patients: only 25 cases of serous ovarian carcinoma. In the present study, patients with serous carcinoma constituted the majority of the group, which is in
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Epithelial OC with CD1117 phenotype
Figure 2 Kaplan–Meier curve of patients’ survival related to the (—) negative and ( ) positive expression of CD117. (a) All types of EOC. (b) Serous ovarian carcinoma (OC). (c) Mucinous OC. (d) Endometrioid OC. (e) Clear cell OC.
accordance with the relative frequency of malignant ovarian epithelial neoplasms.19 Patients with CD117 tumors from this group had a significantly higher chance of receiving paclitaxel/carboplatin protocol, which is
© 2015 Japan Society of Obstetrics and Gynecology
more aggressive therapy and superior to carboplatin monotherapy. This is associated with a higher stage of the disease in which serous carcinoma is detected in general. This may partially explain why CD117-positive
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Table 4 Survival in months of patients with ovarian carcinoma related to CD117 expression Type of carcinoma
CD117-negative
CD117-positive
Significance
All Serous Mucinous Endometrioid Clear cell
47.2 (42.2–52.2) 45.0 (39.0–50.0) 47.9 (32.6–63.1) 49.7 (38.5–60.8) 38.1 (24.4–51.8)
52.1 (42.9–61.3) 55.0 (45.2–64.8) 20.0 (20.0–20.0) 26.8 (15.3–38.4) —
0.823 0.539 0.330 0.205 —
Ranges in parentheses indicate 95% confidence intervals.
serous carcinoma patients in our study had longer survival compared to those with CD117-negative tumors. Although CD117 positivity was previously associated with a more aggressive phenotype and higher resistance to chemotherapy,14,15 our study suggested that CD117positive serous EOC was associated with better survival, higher rate of partial remissions and less disease progression. However, the difference in survival in the CD117positive and -negative groups was not statistically significant. Interestingly, all of the tumors classified as FIGO stage II were negative for CD117. This may be because serous EOC that were most prominently positive for CD117 were more often FIGO stage III or IV at diagnosis. In our study, EOC with mucinous, endometrioid and clear-cell histopathological patterns displayed CD117 expression less frequently than tumors with serous morphology. The correlation of these findings with previous studies was hindered by the paucity of studies investigating c-kit in non-serous EOC and by the very small number of non-serous EOC tumors samples tested. In a study by Ozer et al.,20 none of the investigated malignant mucinous EOC expressed c-kit. In the study by Medingen et al.,21 CD117 was expressed in the cytoplasm, as well as in the membrane in at least 10% of the cells. The tested tumors showed low CD117 expression, except sarcoma, kidney carcinoma, and melanoma, compared to gastrointestinal stromal tumour. Tumors with activated c-kit or plateled-derived growth factor receptor alpha mutation were potential targets for imatinib and other selective tyrosine kinase inhibitors. Medingen et al.,21 did not find a significant correlation between c-kit expression and disease prognosis, even though the tendency of worse prognosis in c-kit positive tumors was recorded in breast carcinoma and sarcoma. The available data on c-kit expression and prognosis are controversial. Due to the complexity of pathways included in each type of EOC, it is unreasonable to expect that a single marker could predict therapeutic response and outcome of the disease. Tumor screening for expression of CD117 still has no relevance in clinical
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practice, but it may add valuable information about tumor features and contribute to better understanding the complex molecular networks involved in EOC pathogenesis, which may lead to the development of specific and more effective therapy for this devastating disease.
Acknowledgments This study was supported in part by Grant No. 175092 from the Ministry of Education, Science and Technological Development of Serbia.
Disclosure No potential conflict of interest relevant to this article was reported.
References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2. Kusumbe AP, Bapat SA. Cancer stem cells and aneuploid populations within developing tumors are the major determinants of tumor dormancy. Cancer Res 2009; 69: 9245–9253. 3. Dong Y, Stephens C, Walpole C et al. Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment. PLoS One 2013; 8: e57056. 4. Palmqvist L, Glover CH, Hsu L et al. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells 2005; 23: 663–680. 5. Agarwal S, Kazi JU, Ronnstrand L. Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation. J Biol Chem 2013; 288: 22460–22468. 6. Won D, Chi HS, Shim H, Jang S, Park CK, Lee JH. The prognostic impact of c-KIT mutation in systemic mastocytosis associated with acute myeloid leukaemia patients. Leuk Res 2013; 37: 883–888. 7. Donnenberg AD, Zimmerlin L, Landreneau RJ, Luketich JD, Donnenberg VS. KIT (CD117) expression in a subset of
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non-small cell lung carcinoma (NSCLC) patients. PLoS One 2012; 7: e52885. Azevedo MF, Horvath A, Bornstein ER et al. Cyclic AMP and c-KIT signaling in familial testicular germ cell tumor predisposition. J Clin Endocrinol Metab 2013; 98: E1393–E1400. Fan H, Yuan Y, Wang J et al. CD117 expression in operable oesophageal squamous cell carcinomas predicts worse clinical outcome. Histopathology 2013; 62: 1028–1037. Dos Santos C, McDonald T, Ho YW et al. The Src and c-Kit kinase inhibitor dasatinib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents. Blood 2013; 122: 1900–1913. Moserle L, Indraccolo S, Ghisi M et al. The side population of ovarian cancer cells is a primary target of IFN-alpha antitumor effects. Cancer Res 2008; 68: 5658–5668. Zhang S, Balch C, Chan MW et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 2008; 68: 4311–4320. Baba T, Convery PA, Matsumura N et al. Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells. Oncogene 2009; 28: 209–218. Luo L, Zeng J, Liang B 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. Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of
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human epithelial ovarian cancer. Cancer Res 2005; 65: 3025–3029. Tavassoli FA, Devilee P (eds). World Health Organization Classification of Tumors. Pathology and Genetics: Tumours of the Breast and Female Genital Organs. Lyon: IARC Press, 2003. Mikami T, Nemoto Y, Numata Y et al. Small gastrointestinal stromal tumor in the stomach: Identification of precursor for clinical gastrointestinal stromal tumor using c-kit and αsmooth muscle actin expression. Hum Pathol 2013; 44: 2628–2635. Zhao F, Chen Y, Wu Q, Wang Z, Lu J. Prognostic value of CD117 in cancer: A meta-analysis. Int J Clin Exp Pathol 2014; 7: 1012–1021. Makwana HH, Maru AM, Lakum NR, Agnihotri AS, Trivedi NJ, Joschi JR. The relative frequency and histopathological pattern of ovarian masses – 11 year study at tertiary care centre. Int J Med Sci Public Health 2014; 3: 81–84. Ozer H, Yenicesu G, Arici S, Cetin M, Tuncer E, Cetin A. Immunohistochemistry with apoptotic-antiapoptotic proteins (p53, p21, bax, bcl-2), c-kit, telomerase, and metallothionein as a diagnostic aid in benign, borderline, and malignant serous and mucinous ovarian tumors. Diagn Pathol 2012; 7: 124. Medinger M, Kleinschmidt M, Mross K et al. c-kit (CD117) expression in human tumors and its prognostic value: an immunohistochemical analysis. Pathol Oncol Res 2010; 16: 295–301.
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J. Obstet. Gynaecol. Res. Vol. 41, No. 10: 1630–1637, October 2015
Epithelial ovarian cancer with CD117 phenotype is highly aggressive and resistant to chemotherapy Irena Conic1, Zorica Stanojevic1, Ljubinka Jankovic Velickovic2, Slavica Stojnev2, Ana Ristic Petrovic2, Miljan Krstic2, Marko Stanojevic3, Dragan Bogdanović4 and Vladisav Stefanovic4,5 1 Clinic of Oncology, Clinical Center, 2Center of Pathology, Clinical Center, 3Clinic of Gynecology, Clinical Center, and 5Faculty of Medicine, University of Nis, Niš, Serbia 4State University of Novi Pazar, Novi Pazar, Serbia
Abstract Aim: CD117 expression has a pathogenic role in many malignancies, including ovarian carcinoma. The aim of the present study was to examine the correlation of stemness-associated marker CD117 with the clinicopathologic features of epithelial ovarian cancer and patient survival. Material and Methods: The analysis included 240 primary ovarian carcinomas (OC) diagnosed during the period from 2005 to 2011 in the region of South Serbia. Age, pathohistological characteristics, presence and size of residual tumor, choice of therapy and response to the therapy were studied. Results: Residual tumors were more frequently present in the patients with positive CD117 expression (18.1% vs 8.0%; P < 0.05). Chemotherapy according to paclitaxel/carboplatin protocol was more frequent in the patients with positive CD117 expression (70.9% vs 54.2%; P < 0.05), while carboplatin monotherapy was more frequent in the patients with negative CD117 expression (18.0% vs 6.4%; P < 0.05). Median survival time in patients with CD117-positive mucinous and endometrioid OC was significantly shorter, at 20 and 26.8 months, respectively. Median survival in serous OC was not related to CD117 expression. Conclusion: Residual tumors and chemotherapy treatment were more frequent in patients with positive CD117 expression. The outcome was dependent on the type of OC; a worse outcome, including a shorter survival, was documented in the mucinous and endometrioid OC cases. Key words: CD117 expression, ovarian carcinoma, survival, treatment.
Introduction Epithelial ovarian cancer (EOC) is the fourth most common cause of cancer death in European women.1 Treatment includes maximal surgical debulking and platinum combination chemotherapy. Complete remission is common, but the majority of women develop a relapse of the disease. This is followed with multiple lines of chemotherapy, due to increased drug resistance associated with relapse. This emphasizes the need for more effective therapies in order to improve survival and quality of life of EOC patients.
EOC demonstrates significant tumor cell population heterogeneity, with multilineage clones expressing different capacities for growth, survival, metastasis and resistance to chemotherapy and radiation therapy. So far, many biomarkers expressed by EOC cells have been investigated in terms of their diagnostic and prognostic potential. In the last few years, a great improvement in understanding tumor biology has been achieved due to the discovery of cancer stem cells (CSC), which play the main role in cancer progression and its resistance to therapy. CSC could open the possibility of creating a
Received: August 21 2014. Accepted: April 8 2015. Reprint request to: Dr Irena Conic, Clinic of Oncology, Clinical Center Niš, Dr Zoran Djindjic Street 48, 18000 Niš, Serbia. Email: [email protected]
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Epithelial OC with CD1117 phenotype
new target for the purpose of decreasing resistance to chemotherapy and of improving the effects of therapy.2,3 CD117 (c-kit) is a surface marker for embryonic, hematopoietic and mesenchymal stem cells. CD117 is associated with undifferentiated cell state and capacity for renewal.4 CD117 expression was found in various tumors of mesenchymal and epithelial origin. Patients with high expression and/or mutation of CD117 had a significantly worse disease prognosis in different types of tumors.5–10 However, the role of CD117 in EOC has not been completely clarified. The recent discovery of CSC could reveal the role of CD117 in the development and growth of EOC. Identification and isolation of CSC have been reported in different kinds of cancer, including ovarian carcinoma (OC). Moserle et al.11 identified a subpopulation of OC cells with the side-population (SP) phenotype from a single tumor xenograft. These cells were highly cancerous in mice, compared to the tumor cell population that did not contain SP cells. Zhang et al.12 observed a subpopulation of primary human ovarian cancer cells that could form independent spheres and were capable of self-renewal in in vitro conditions that highly expressed CD44 and CD117. The cells with CD 44+/CD 117+ phenotype were extremely tumorigenic and were capable of developing a tumor in a xenograft experiment. CSC isolated from primary tumors have the ability to regenerate the tumor cell population and reconstitute the original tumor phenotype with as few as 100 cells. CSC from ovarian carcinomas were found to display the cell surface markers CD44 + CD117 + CD133+. In addition, CSC are also thought to account for chemotherapy resistance.13 Luo et al.,14 demonstrated that human OC cells with the CD117+ phenotype possess the unique properties of CSC. This includes self-renewal, differentiation, a high tumorigenic potential, and chemoresistance. Targeting CD117+ cancer cells may be effective therapy for treatment of OC. Bapat et al.15 isolated tumor cells from the ascites of patients with serous OC and obtained 19 spontaneously immortalized clones. Performing molecular examination of the clones, they identified expression of CD 44, CD 117 and a ligand for CD117. Only one of these clones expressed tumorigenesis in vivo, which indicated the presence of cells with potential for tumor initiation and multilineage differentiation in ovarian tumors accompanied by ascites. Zhang et al.12 analyzed tumor spheres originating from the cells isolated from the ascites of five patients with serous OC. After 10 serial passages in stem cell medium, it was demonstrated that the remaining sphere
© 2015 Japan Society of Obstetrics and Gynecology
cells showed high expression of CD44 and CD117. CD44+ and CD117+ spheroid cells were resistant to chemotherapy, and they were capable of starting carcinogenesis, leading to tumor formation in mice. Tumor metastases are considered the main cause of death in various malignant tumors. Evidence from previous studies showed that CSC can be generated in the processes associated with aberrant activation of epithelial–mesenchymal transition (EMT), which affects cell differentiation and metastatic tumor potential. An anti-EMT strategy would be a new therapeutic option for treating aggressive carcinomas.2,3 The aim of the present study was to examine the correlation of stemness-associated marker c-kit with clinicopathologic features of EOC and patient survival.
Methods The study included female patients with ovarian carcinoma diagnosed during the period from 2005 to 2011 in the region of South Serbia. The patients’ survival rate was observed until September 2013. The study included 240 cases of primary EOC, including: borderline tumors, OC limited to ovaries (stage I according to the official FIGO staging system16), OC disseminated in pelvic organs (FIGO stage II), OC with peritoneal metastases (FIGO stage III) and OC with remote metastases (FIGO stage IV). In four cases of radical hysterectomy with adnexectomy, FIGO stage could not be determined, because omentectomy was not performed. In the patients with EOC, clinical parameters were observed, as well as their pathological features. The analyzed clinical parameters included: age, FIGO stage, presence and size of residual tumor, kind of therapy and response to therapy. The analyzed pathological characteristics included: histological type of tumor, histological grade, and nuclear grade, according to official World Health Organization grading system.16 The data sources were histories of patients with OC treated at the Oncology Clinic of the Clinical Center Niš. Pathological and immunohistochemical analyses of OC samples were performed at the Pathology Institute of the Faculty of Medicine in Niš. Pathohistological analysis was performed on biopsy samples of OC fixed in 10% neutral formalin, processed in the automatic tissue processor and embedded in paraffin. After deparaffinization and processing in graded alcohols, the selected samples of 5-μm thick cancer tissue sections were stained using the classic histological hematoxylin–eosin method for determination of tumor
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differentiation and its pathological stage. The immunohistochemical analysis was performed using Polyclonal Rabbit Antihuman CD 117 (c-kit) antibody in dilution 1:300. Paraffin tissue sections were processed according to the manufacturer’s protocol (DAKO LSAB2R system HRP). In brief, tissue slices were deparaffinized, rehydrated and incubated in 30 mL/L hydrogen peroxide, to block endogenic peroxidase, and then, thoroughly rinsed with phosphate-buffered saline (PBS) (pH = 7.4). The next step was incubation with CD117 antibodies for 24 h in the humid chamber at 4°C. After rinsing with PBS, the tissue was incubated with secondary antibody (Biotin labeled goat antirabbit antibody) for 30 min. After repeated rinsing with PBS, incubation with tertiary antibody for 30 min followed. The visualization of marked antigens was performed using diaminobenzidinetetrahydrochloride chromogen. Counterstaining was performed with Mayer’s hematoxylin. Simultaneously with CD117 staining, selected cancer sections that were used as negative control were incubated with antibody diluents without addition of CD117 antibody, while section of healthy testicular tissue served as positive control (CD117-stained germ cells in seminal tubules). The analysis of immunohistochemically stained microscopic preparations of OC was performed using a light microscope Leica DM 1000. The obtained results were systematized and grouped, while the statistical significance was tested using the corresponding statistical tests, according to the tested parameters and sample size.
Scoring of immunohistochemical reaction Scoring of immunohistochemical CD117 reactivity was performed on the grounds of estimating the positive staining (membranous or membranous and cytoplasmic expression) in tumor cells: The intensity of the immunostaining was graded as negative (no staining), weak (1+), moderate golden-brown (2+), or strong brown coloration (3+) (modified from Zhang et al.12). The tumor was considered positive for CD117 expression if more than 25% of cancer cells were stained with moderate or strong intensity.
Statistical methods The quantitative statistical analysis was performed on the computer. Recording, ranging, grouping, as well as tabular and graphic presentation of data was performed using Excel 2003. The calculations were performed using R 2.12.0 (R Foundation for Statistical Computing).
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The mean values of numeric features between the two groups of patients were compared using the Student’s t-test or the Mann–Whitney U-test in cases where the value distribution did not satisfy the demands of a standard schedule. The numeric values among three and four groups of patients were compared by variance analysis using Tukey’s post-hoc test with Cramer correction for uneven group sizes. Comparison of the frequency of certain categories of attributive features among certain groups of patients was performed by χ 2-test or Fisher’s exact test in cases where an expected frequency was less than 5. Estimating the correlation between lethal outcome and all other investigated factors was performed by multivariant Cox regression analysis. Odds ratio was used to calculate the values of the rate of presence and absence of lethal outcomes and their 95% confidence interval. Applying the step-by-step backward method (Backward: Wald), all factors of unconfirmed statistical significance were excluded. The Kaplan–Meier estimator and log–rank test were used to test the level of expression of certain markers and survival rate. A permitted level of estimation error lower than 5% (P < 0.05) was used as the level of statistical importance in concluding.
Results The average age of patients with absent CD117 expression was 56.9 ± 13.2 years and these patients were much older than the patients with positive CD117 expression, whose average age was 52.6 ± 11.7 years (P < 0.05) (Table 1). Residual tumors were more frequently present in the patients with a positive CD117 expression (18.1% vs 8.0%; P < 0.05). Chemotherapy according to paclitaxel/carboplatin protocol was more frequent in the patients with a positive CD117 expression (70.9% vs 54.2%; P < 0.05), while carboplatin monotherapy was more frequent in the patients with a negative CD117 expression (18.0% vs 6.4%; P < 0.05). The disease progression was present in a significantly higher percentage of patients with a negative CD117 expression (19.7% vs 6.4%; P < 0.05), while partial remission was present in a significantly higher percentage of patients with a positive CD117 expression (35.4% vs 22.3%; P < 0.05). The differences concerning the values and frequency of other characteristics between the patients with different levels of CD117 expression were not statistically significant (Table 1).
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Epithelial OC with CD1117 phenotype
Table 1 Characteristics of patients, applied therapy and disease outcome compared to CD117 expression CD117 expression
Characteristics
Age Time to diagnosis Type of tumor Primary tumor Metastasis Residual tumor Surgical treatment Radical hysterectomy Unilateral adnexectomy with hysterectomy Bilateral adnexectomy Unilateral adnexectomy Exploratory laparotomy with biopsies Applied chemotherapy No chemotherapy Paclitaxel/carboplatin Etoposide/carboplatin Carboplatin Etoposide Etoposide/doxorubicin/carboplatin Number of chemotherapies Disease outcome Progression Stable disease Partial remission Complete remission Survival (months) Death
Significance
Negative (n = 174)
Positive (n = 66)
56.9 ± 13.2 4.50 ± 4.97
52.6 ± 11.7 3.80 ± 3.65
0.022 NS
144 (82.7%) 30 (17.2%) 14 (8.0%)
52 (78.7%) 14 (21.2%) 12 (18.1%)
NS 0.024
106 (63.0%) 2 (1.1%) 12 (7.1%) 18 (10.7%) 30 (17.8%)
34 (51.5%) 2 (3.0%) 6 (9.0%) 10 (15.1%) 14 (21.2%)
NS NS NS NS NS
10 (6.0%) 90 (54.2%) 32 (19.2%) 30 (18.0%) 2 (1.2%) 2 (1.2%) 5.02 ± 1.91
2 (3.2%) 44 (70.9%) 12 (19.3%) 4 (6.4%) 0 (0.0%) 0 (0.0%) 4.97 ± 1.75
NS 0.022 NS 0.028 NS NS NS
30 (19.7%) 4 (2.6%) 34 (22.3%) 84 (55.2%) 36.7 ± 23.8 78 (49.3%)
4 (6.4%) 2 (3.2%) 22 (35.4%) 34 (54.8%) 40.91 ± 24.6 32 (55.1%)
0.016 NS 0.048 NS NS NS
NS, not significant.
Positive expression of CD117 was demonstrated in a significant percentage of patients with serous OC (32.6%) compared with patients with mucinous OC (9.1%; P < 0.05) and clear cell OC (0.0%; P < 0.05) (Table 2). Expression of CD117 in serous OC is given in Figure 1 as cytoplasmic (Fig. 1a), and both cytoplasmic and membranous staining (Fig. 1b). In the group of patients with FIGO stage II disease, CD117 expression was not observed in any of the investigated tumors (11.7% vs 0.0%; P < 0,01) (Table 3). The differences in the frequency of other FIGO stages, as well as in histologic and nuclear grades between the patients
with different levels of CD117 expression, were not statistically significant. Kaplan–Meier survival curves for all investigated patients and for every different EOC type are presented in Figure 2. The median survival time in patients with EOC and negative CD117 expression was 47.2 months, while for those with positive CD117 expression it was 52.1 months. The Kaplan–Meier estimator and log–rank test showed non-significant difference between survival distributions of patients with positive and negative CD117 expression (P = 0.823) (Table 4). Median survival time in patients with serous ovarian cancer and
Table 2 CD117 expression related to the type of epithelial ovarian cancer CD117 expression Negative Positive
Type of epithelial ovarian cancer Serous
Mucinous
Endometrioid
Clear cell
120 (67.4%) 58 (32.6%)
20 (90.9%) 2 (9.1%)
24 (80.0%) 6 (20.0%)
10 (100.0%) 0 (0.0%)
Sum
Significance
174 (72.5%) 66 (27.5%)
A*, C*
*P < 0.05. A, serous vs mucinous; B, serous vs endometrioid; C, serous vs clear cell; D, mucinous vs endometrioid; E, mucinous vs clear cell; F, endometrioid vs clear cell.
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(a)
Table 3 Correlation of clinicopathologic characteristics and CD117 expression Characteristics
(b)
FIGO stage Borderline I II III IV Histologic grade 0 1 2 3 Nuclear grade 0 1 2 3
CD117 expression
Significance
Negative (n = 174)
Positive (n = 66)
12 (7.0%) 36 (21.1%) 20 (11.7%) 86 (50.5%) 16 (9.4%)
6 (9.0%) 12 (18.1%) 0 (0.0%) 40 (60.6%) 8 (12.1%)
NS NS 0.004 NS NS
12 (6.9%) 22 (12.7%) 96 (55.8%) 42 (24.4%)
6 (9.0%) 10 (15.1%) 38 (57.5%) 12 (18.1%)
NS NS NS NS
12 (6.9%) 26 (15.1%) 96 (55.8%) 38 (22.0%)
6 (9.0%) 8 (12.1%) 36 (54.5%) 16 (24.2%)
NS NS NS NS
NS, not significant.
Figure 1 Expression of CD117 in serous ovarian cancer. (a) Cytoplasmic expression of CD117 in serous ovarian carcinoma (OC) (×400). (b) Cytoplasmic expression of CD117 in cells of serous OC and in stroma (×400).
negative/positive CD117 expression was similar to the expression of EOC in general. Median survival time in patients with mucinous OC was 47.9 months for CD117-negative tumors, and 20.0 months for CD117positive tumors. A similar low survival of 26.8 months was found in CD117-positive endometrioid ovarian cancer. All 10 patients with clear cell ovarian cancer had negative CD117 expression. The median survival time of these patients was 38.1 months.
Discussion CD117 expression (c-kit) has a pathogenic role in many malignancies, including ovarian cancer.17,18 Recent research has indicated the relevance of response to imatinib (kinase inhibitor) in patients with active c-kit mutations. In the majority of cases, the role of c-kit in neoplasia is not quite clear.
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In this study, chemotherapy according to paclitaxel/ carboplatin protocol was more frequent in patients with a positive CD117 expression, while carboplatin monotherapy was more frequent in patients with a negative CD117 expression, which matched the disease stage. Residual tumors and chemotherapy treatment were more frequent in patients with positive CD117. In this study, the outcome was dependent on the type of ovarian carcinoma; a worse outcome, including a shorter survival, was documented in the mucinous and endometrioid ovarian cancer with a positive expression of CD117. Luo et al.14 observed a correlation between CD117 expression and the response to the applied chemotherapy in OC. The response to chemotherapy was correlated to many clinical factors, including the disease stage, residual tumor after surgical intervention and applied chemotherapy. All cases in the study were in an advanced stage (FIGO stage III). All patients underwent optimal cytoreduction (residual tumor smaller than 1 cm) and paclitaxel/platinum therapy. There was a significant correlation between CD117 expression and the response to secondary therapy. Compared to CD117– tumors, CD117+ tumors were less sensitive to chemotherapy, and they reoccurred in a shorter period of time after chemotherapy. However, this study was conducted on a small number of patients: only 25 cases of serous ovarian carcinoma. In the present study, patients with serous carcinoma constituted the majority of the group, which is in
© 2015 Japan Society of Obstetrics and Gynecology
Epithelial OC with CD1117 phenotype
Figure 2 Kaplan–Meier curve of patients’ survival related to the (—) negative and ( ) positive expression of CD117. (a) All types of EOC. (b) Serous ovarian carcinoma (OC). (c) Mucinous OC. (d) Endometrioid OC. (e) Clear cell OC.
accordance with the relative frequency of malignant ovarian epithelial neoplasms.19 Patients with CD117 tumors from this group had a significantly higher chance of receiving paclitaxel/carboplatin protocol, which is
© 2015 Japan Society of Obstetrics and Gynecology
more aggressive therapy and superior to carboplatin monotherapy. This is associated with a higher stage of the disease in which serous carcinoma is detected in general. This may partially explain why CD117-positive
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Table 4 Survival in months of patients with ovarian carcinoma related to CD117 expression Type of carcinoma
CD117-negative
CD117-positive
Significance
All Serous Mucinous Endometrioid Clear cell
47.2 (42.2–52.2) 45.0 (39.0–50.0) 47.9 (32.6–63.1) 49.7 (38.5–60.8) 38.1 (24.4–51.8)
52.1 (42.9–61.3) 55.0 (45.2–64.8) 20.0 (20.0–20.0) 26.8 (15.3–38.4) —
0.823 0.539 0.330 0.205 —
Ranges in parentheses indicate 95% confidence intervals.
serous carcinoma patients in our study had longer survival compared to those with CD117-negative tumors. Although CD117 positivity was previously associated with a more aggressive phenotype and higher resistance to chemotherapy,14,15 our study suggested that CD117positive serous EOC was associated with better survival, higher rate of partial remissions and less disease progression. However, the difference in survival in the CD117positive and -negative groups was not statistically significant. Interestingly, all of the tumors classified as FIGO stage II were negative for CD117. This may be because serous EOC that were most prominently positive for CD117 were more often FIGO stage III or IV at diagnosis. In our study, EOC with mucinous, endometrioid and clear-cell histopathological patterns displayed CD117 expression less frequently than tumors with serous morphology. The correlation of these findings with previous studies was hindered by the paucity of studies investigating c-kit in non-serous EOC and by the very small number of non-serous EOC tumors samples tested. In a study by Ozer et al.,20 none of the investigated malignant mucinous EOC expressed c-kit. In the study by Medingen et al.,21 CD117 was expressed in the cytoplasm, as well as in the membrane in at least 10% of the cells. The tested tumors showed low CD117 expression, except sarcoma, kidney carcinoma, and melanoma, compared to gastrointestinal stromal tumour. Tumors with activated c-kit or plateled-derived growth factor receptor alpha mutation were potential targets for imatinib and other selective tyrosine kinase inhibitors. Medingen et al.,21 did not find a significant correlation between c-kit expression and disease prognosis, even though the tendency of worse prognosis in c-kit positive tumors was recorded in breast carcinoma and sarcoma. The available data on c-kit expression and prognosis are controversial. Due to the complexity of pathways included in each type of EOC, it is unreasonable to expect that a single marker could predict therapeutic response and outcome of the disease. Tumor screening for expression of CD117 still has no relevance in clinical
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practice, but it may add valuable information about tumor features and contribute to better understanding the complex molecular networks involved in EOC pathogenesis, which may lead to the development of specific and more effective therapy for this devastating disease.
Acknowledgments This study was supported in part by Grant No. 175092 from the Ministry of Education, Science and Technological Development of Serbia.
Disclosure No potential conflict of interest relevant to this article was reported.
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