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Obstetrics & Gynecology 2000;96:65-69
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Serum Soluble Fas Levels in Ovarian Cancer

LUKAS HEFLER, MD, KLAUS MAYERHOFER, MD, ALESSANDRA NARDI, PhD, ALEXANDER REINTHALLER, MD, CHRISTIAN KAINZ, MD and CLEMENS TEMPFER, MD

From the Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas; and the Departments of Gynecology and Obstetrics and Medical Computer Sciences, University of Vienna Medical School, Vienna, Austria.

Address reprint requests to: Lukas Hefler, MD, Department of Obstetrics and Gynecology, Baylor College of Medicine, Smith Tower, 6550 Fannin, Suite 901, Houston, TX 77030, E-mail: lhefler{at}bcm.tmc.edu


   Abstract
Top
Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To determine the value of serum soluble Fas levels as a prognostic marker for survival of women with ovarian cancer and as a discriminator between benign and malignant adnexal masses.

Methods: Serum soluble Fas levels were measured with an enzyme-linked immunosorbent assay in 52 women with ovarian cancer, 30 women with benign ovarian cysts, and 35 healthy women.

Results: Median serum soluble Fas levels in women with ovarian cancer, women with benign ovarian cysts, and healthy women were 3.7 (range 1.6–14.5), 2.3 (range 1.3–4.1), and 1.5 ng/mL (range 0.1–5.6), respectively (P < .001). A univariate logistic regression model showed a significant influence of serum soluble Fas and CA 125 levels on the odds of presenting with ovarian cancer versus benign cysts (P < .001 and P = .001, respectively). In a multivariable logistic regression model for soluble Fas and CA 125, both markers showed a statistically significant influence on the odds of presenting with ovarian cancer versus benign cysts (P = .01 and P = .01, respectively). Increased pretreatment serum soluble Fas levels were associated with shortened disease-free and overall survival (P = .002 and P = .001, respectively). A multivariable Cox regression model identified serum soluble Fas levels as a significant prognostic factor for disease-free and overall survival, independent of tumor stage (P = .04 and P = .03, respectively).

Conclusion: Soluble Fas levels might be useful as a discriminator between benign ovarian cysts and ovarian cancer, adding to the information obtained with the use of the established tumor marker CA 125. Pretreatment serum soluble Fas levels also might be an independent prognostic factor for disease-free and overall survival.

Apoptosis includes condensation and segmentation of nuclei, condensation and fragmentation of cytoplasm, and fragmentation of chromosomal DNA into nucleosome units.1,2 After extensive investigation of apoptosis in malignant diseases, the belief is that tumor cells develop various mechanisms, such as aberration of cell surface molecules or tumor-derived soluble inhibitory factors, to subvert the immune system and subsequently induced apoptosis.3,4

Fas/CD95/APO-1, a type I transmembrane cell surface protein with a molecular weight of 48 kD, belongs to the tumor necrosis factor family.5 Fas and its ligand, FasL, have been described as trigger molecules of apoptosis.2 Fas is expressed in human organs such as the liver, thymus, heart, and ovaries. FasL expression has been described mainly on blood cells, such as activated T cells, B cells, and natural killer cells, but also on stromal cells of the eye and Sertoli cells of the testis.6 Binding of FasL to Fas induces trimerization of cell surface Fas, which recruits caspase-8, a member of the human protease superfamily, through a Fas-specific death domain and an adaptor. Oligomerization of caspase-8 triggers a cascade of proteases, resulting in proteolysis and degradation of chromosomal DNA.7,8 Another line of evidence indicates that Fas-driven apoptosis is mediated by the sphingomyelin-ceramide pathway. Binding of FasL to Fas causes hydrolysis of sphingomyelin to ceramide, resulting in induction of a ceramide-activated protein kinase, which leads to subsequent cell death.1,9

Soluble Fas is produced by alternative splicing of Fas messenger RNA encoding a soluble form of the Fas protein that lacks the transmembrane domain.10 Soluble Fas binds to and neutralizes FasL, thus functionally antagonizing Fas/FasL-driven apoptosis.10,11 The effect of serum soluble Fas levels has been investigated in various human malignancies, including non-Hodgkin lymphoma, myeloid leukemia, and colon, breast, bladder, and renal cancer.11–15

Ghahremani et al9 showed that anti-Fas antibodies induce apoptosis in ovarian cancer cell lines, suggesting that Fas is expressed on ovarian cancer cells and that the Fas-FasL mechanism is involved in the natural history of ovarian cancer. Wakahara et al16 showed that anti-Fas antibodies increase the potential of chemotherapeutic agents to cause apoptosis in ovarian cancer cell lines. Baldwin et al,17 using immunohistochemistry and Western blot analysis, found that Fas expression of ovarian cancer cells was not restricted to the cell surface but was detectable also in cytoplasm. Preliminary data suggest that serum soluble Fas levels are significantly increased in women with ovarian cancer compared with healthy controls.

The aim of our study was to evaluate serum soluble Fas levels in women with ovarian cancer, women with benign ovarian cysts, and healthy women. We investigated whether serum soluble Fas levels could serve as a discriminator between ovarian cancer and benign ovarian cysts and thus add to the information obtained with the use of the established tumor marker CA 125. We also evaluated the prognostic potential of pretreatment serum soluble Fas levels regarding disease-free and overall survival.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
This retrospective study included serologic examinations of 52 consecutive women with International Federation of Gynecology and Obstetrics stage I (n = 8), II (n = 9), or III (n = 35) ovarian cancer who were treated between December 1992 and March 1999 at the Department of Gynecology and Obstetrics, University of Vienna Medical School. Median age at the time of diagnosis of ovarian cancer was 57 years (range 29–87). Histologically, 28 tumors were graded as serous adenocarcinomas, 12 as mucinous adenocarcinomas, four as undifferentiated carcinomas, three as clear-cell carcinomas, and five as endometrioid ovarian cancers. Eleven tumors were graded as well differentiated, 14 as moderately differentiated, and 27 as poorly differentiated. The median duration of follow-up was 8 months (range 0.5–67). Twenty-three of 52 women had recurrent ovarian cancer and ten died from cancer during the observation period.

We also investigated serum soluble Fas levels in 30 women with benign ovarian cysts and 35 healthy women. Median ages of women with benign cysts and healthy women were 50 (range 24–79) and 39 years (range 23–58), respectively. Women with ovarian cancer were significantly older than women with benign cysts and healthy women (P < .001). Serum samples from women with benign cysts were collected consecutively between 1993 and 1999. Serum samples from apparently healthy women were collected in 1996. Serum CA 125 levels were evaluated in all women with ovarian cancer or benign cysts. Women with borderline ovarian cancer were not included in our study. A power analysis showed that with an estimated standard deviation of serum soluble Fas levels of 1.5 ng/mL, a sample of 50 subjects was needed to detect a difference in soluble Fas levels of 0.86 ng/mL among women with ovarian cancer, women with benign cysts, and women who were healthy with a power of 85%.

Women with ovarian cancer underwent hysterectomies, bilateral salpingo-oophorectomies, pelvic and para-aortic lymphadenectomies, and omentectomies. Optimal debulking and suboptimal debulking were achieved in 38 and 14 women, respectively. All patients with tumor stages Ic–III disease and those with clear-cell carcinoma received platinum-based, first-line chemotherapy with carboplatin and cyclophosphamide from 1992 to 1997 and carboplatin and paclitaxel from 1997 to 1999. Histologic grading was performed using International Union Against Cancer criteria. Women underwent vaginal-rectal palpation, abdominal ultrasound examination, and evaluation of serum levels of tumor marker every 3 months. In cases of clinically doubtful findings or increased levels of tumor marker, computed tomography was done.

Blood was drawn 24–48 hours before surgery by peripheral venous puncture and was centrifuged immediately at 3000 rpm for 15 minutes. Only serum samples drawn before therapy were evaluated. Serum was kept at -80C until examination. For measuring serum soluble Fas levels, a commercially available enzyme-linked immunosorbent assay (ELISA) (Medical & Biological Laboratories Co., Nagoya, Japan) was used. All serum soluble Fas analyses were done at the same time, in the same batch, and in duplicate, according to manufacturer instructions. The sensitivity of the test was 0.5 ng/mL; intra- and interassay variability were less than 7.12% and less than 7.32%, respectively. Serum CA 125 levels were measured using a commercially available enzyme immunoassay (Enzymun-Test, CA 125 II; Boehringer Mannheim, Mannheim, Germany), as described.18

Because of skewed distribution of soluble Fas serum values, median and ranges of serum soluble Fas levels are given. Unpaired groups were compared using the Mann-Whitney U test. Survival probabilities were calculated using the Kaplan-Meier product-limit method. Survival times of women who were disease free or still alive were censored with the last follow-up date. Differences between groups were tested using the log-rank test. Univariate and multivariate logistic regression models were used to analyze the influence of serum soluble Fas and CA 125 levels on the probability of ovarian cancer versus benign cysts. Sensitivity and specificity were calculated for each possible threshold value of estimated probability for ovarian cancer versus benign cysts. Receiver operating characteristic (ROC) curves were constructed based on those values to determine the relationship between soluble Fas, CA 125, and the combination of both markers and ovarian cancer. Ninety-five percent confidence intervals for odds ratios were calculated using the profile likelihood method. Odds ratios refer to doubling of serum soluble Fas and CA 125 levels. A multivariable Cox proportional hazards regression model was used to assess the independence of different prognostic factors. P <= .05 was considered statistically significant. We used the SAS statistical software system (SAS Institute Inc., Cary, NC) to do the calculations.

In a search of PubMed, using "ovarian cancer," "ovarian cyst," "Fas," and "serum" as search terms, we identified publications on serum soluble Fas levels in women with ovarian cancer and those with benign ovarian cysts. The period covered by the search was 1961 through October 1999.


   Results
Top
Abstract
 Materials and Methods
 Results
Discussion
 References
 
Median serum soluble Fas levels in women with ovarian cancer, women with benign ovarian cysts, and healthy women were 3.7 (range 1.6–14.5), 2.3 (range 1.3–4.1), and 1.5 ng/mL (range 0.1–5.6), respectively (Mann-Whitney U test, P < .001). Table 1Go includes serum soluble Fas levels in women with ovarian cancer, broken down by tumor stage, lymph node involvement, histologic grade, and age at diagnosis. A box plot of serum soluble Fas levels in women with ovarian cancer, women with benign ovarian cysts, and healthy women is shown in Figure 1Go. Median serum CA 125 levels in women with ovarian cancer and those with benign ovarian cysts were 343 (range 13.7–33200) and 16.6 U/mL (range 3.1–378), respectively (Mann-Whitney U test, P < .001). Univariate logistic regression models showed that serum soluble Fas and CA 125 levels had significant influences on the odds of presenting with ovarian cancer versus benign cysts (P < .001 and P = .001, respectively). At 3.69 ng/mL, soluble Fas achieved a sensitivity of 53% and a specificity of 95%. At 409 U/mL, CA 125 achieved a sensitivity of 71% and a specificity of 95%.


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  		Table 1. Serum Soluble Fas Levels and Pathologic Characteristics in Ovarian Cancer
 


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  		Figure 1. Box plot of serum soluble Fas (sFas) levels (ng/mL) in women with ovarian cancer (n = 52), women with benign ovarian cysts (n = 30), and healthy women (n = 35). Horizontal lines in the boxes represent the first, second (the median), and third quartiles; whiskers extend from the box to a distance of 1.5 interquartile ranges. Open squares represent the mean, and x and dashes represent values outside a distance of 1.5 interquartile ranges from the box.

 
In a multivariable regression model considering serum soluble Fas and CA 125 levels simultaneously, both soluble Fas and CA 125 showed a significant influence on the odds of presenting with ovarian cancer versus benign ovarian cysts (P = .01 and P = .01, respectively). Soluble Fas and CA 125 together achieved a sensitivity of 81% and a specificity of 95%. Shown in Figure 2Go are ROC curves of soluble Fas, CA 125, and their simultaneous consideration.



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  		Figure 2. Receiver operating characteristic curves of serum soluble Fas levels (thin dashed line) and serum CA 125 levels (thick dashed line) in patients with ovarian cancer (n = 52) and those with benign ovarian cysts (n = 30). The thick line represents a simultaneous consideration of both variables.

 
Serum soluble Fas levels lack a clearly defined cutoff value. Our chosen cutoff value of 3.7 ng/mL was the median serum level in the ovarian cancer patients. Increased pretreatment serum soluble Fas levels were associated with shortened disease-free and overall survival (log-rank test, P = .002; and log-rank test, P = .001, respectively) (Figures 3Go and 4Go). A multivariable Cox regression model showed that serum soluble Fas levels are a prognostic factor for disease-free and overall survival, independent of tumor stage (P = .04 and P = .03, respectively).



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  		Figure 3. Kaplan-Meier analysis of 5-year disease-free survival of patients with serum soluble Fas (sFas) levels less than 3.7 ng/mL and patients with levels greater than or equal to 3.7 ng/mL.

 


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  		Figure 4. Kaplan-Meier analysis of 5-year overall survival of patients with serum soluble Fas (sFas) levels less than 3.7 ng/mL and patients with levels greater than or equal to 3.7 ng/mL.

 

   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first study to show that serum soluble Fas levels are significantly increased in women with ovarian cancer compared with those who have benign ovarian cysts. A multivariable regression model showed that serum soluble Fas levels might be a powerful discriminator between benign and malignant adnexal masses. Measuring of soluble Fas levels added to the information obtained with the use of the established tumor marker CA 125. Increased pretreatment serum soluble Fas levels were associated with shortened disease-free and overall survival.

Serum soluble Fas levels have been investigated in various malignancies, including breast, colon, bladder, and hepatocellular cancer.11,14,19,20 Serum soluble Fas levels are significantly higher in women with malignant disease compared with healthy controls.11,14,15 Our results accord with preliminary data showing that serum soluble Fas levels are significantly increased in women with ovarian cancer compared with healthy women.17 However, serum soluble Fas levels in our series were markedly higher than in the study by Baldwin et al,17 perhaps because of a difference in the number of patients evaluated, the distribution of disease stage, or patient ages or because of the use of two different ELISAs. Our results indicate that soluble Fas production and secretion into the circulation, as reflected by serum soluble Fas levels, are involved in the natural history of ovarian cancer. The precise cellular origin of increased serum soluble Fas levels remains unclear. Increased soluble Fas levels in culture supernatants of breast and colon cancer cells lead to the assumption that soluble Fas is derived from tumor cells.14 It also has been speculated that soluble Fas is produced by activated peripheral blood lymphocytes and stromal tissue surrounding the tumor, in response to immune activation.14

We found that serum soluble Fas levels were significantly increased in women with ovarian cancer compared with those with benign ovarian cysts. The higher the serum soluble Fas levels were, the higher were the odds of presenting with ovarian cancer. A multivariable logistic regression model showed that measuring serum soluble Fas levels added diagnostic information beyond that obtained with the use of the established tumor marker CA 125, which suggests that serum soluble Fas levels might be useful in efforts to find adequate combined screening systems (eg, by combining the use of CA 125 with transvaginal ultrasound examination).

The relationship between soluble Fas and pathologic characteristics has been investigated in various malignancies. Midis et al14 reported an association between increased serum soluble Fas levels and a metastatic phenotype and tumor stage in patients with breast or colon cancer. Mizutani et al11 showed that serum soluble Fas levels in bladder cancer did not correlate with tumor stage, histologic grade, or patient age at diagnosis. In ovarian cancer, we found lower serum soluble Fas levels in women with stage I or II tumors than in women with stage III tumors, whereas serum soluble Fas levels were not associated with lymph node involvement, histologic grade, or patient age at diagnosis. Those findings indicate that serum soluble Fas levels in ovarian cancer are reflective of tumor bulk but not tumor differentiation and lymphatic spread. Serum soluble Fas levels were significantly lower in younger women with ovarian cancer. Patients with ovarian cancer who had higher serum soluble Fas levels were significantly older than healthy women and those with benign cysts. We cannot rule out that patient age might influence serum soluble Fas levels.

The Fas-FasL system is involved in apoptosis, and the belief is that failure of that defense mechanism, possibly mediated by soluble Fas, is involved in ovarian carcinogenesis.16,17,21 Our results accord with those experimental data showing that serum soluble Fas levels are indicative of the patient prognosis. Our findings support the assumption that soluble Fas binds to and neutralizes FasL before transformation and activation of the Fas/FasL complex occur. Loss of cell-bound Fas function due to increased availability of soluble Fas might be important in the pathogenesis and progression of malignant disease.


   Footnotes
 
Supported in part by the Erwin-Schroedinger-Auslandsstipendium (J 1830 MED to LH) and the Ludwig Boltzmann Foundation, Institute for Gynecologic Oncology.

PII S0029-7844(99)00840-1

Received November 2, 1999. Received in revised form January 18, 2000. Accepted February 1, 2000.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Nagata S, Goldstein P. The Fas death factor. Science 1995;267:1449–56.[Abstract/Free Full Text]

2. Rowe PM. Glimmers of clinical relevance for Fas. Lancet 1996;347: 1398.[Medline]

3. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–62.[Abstract/Free Full Text]

4. Carson DA, Ribeiro JM. Apoptosis and disease. Lancet 1993;341: 1251–4.[Medline]

5. Suda T, Takahashi T, Goldstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993;75:1169–78.[Medline]

6. Gratas C, Tohma Y, Barnas C, Taniere P, Hainaut P, Ohgaki H. Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer. Cancer Res 1998; 58:2057–62.[Abstract/Free Full Text]

7. Nagata S. Apoptosis by death factor. Cell 1997;88:355–65.[Medline]

8. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998;281:1305–8.[Abstract/Free Full Text]

9. Ghahremani M, Foghi A, Dorrington JH. Activation of Fas ligand/receptor system kills ovarian cancer cell lines by an apoptotic mechanism. Gynecol Oncol 1998;70:275–81.[Medline]

10. Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 1994;263:1759–62.[Abstract/Free Full Text]

11. Mizutani Y, Yoshida O, Bonavida B. Prognostic significance of soluble Fas in the serum of patients with bladder cancer. J Urol 1998;160:571–6.[Medline]

12. Yufu Y, Choi I, Hirase N, Tokoro A, Noguchi Y, Goto T, et al. Soluble Fas in the serum of patients with non-Hodgkin’s lymphoma: Higher concentrations in angioimmunoblastic T-cell lymphoma. Am J Hematol 1998;58:34–6.

13. Munker R, Midis G, Owen-Schaub L, Andreff M. Soluble FAS (CD95) is not elevated in the serum of patients with myeloid leukemias, myeloproliferative and myelodysplastic syndromes. Leukemia 1996;10:1531–3.[Medline]

14. Midis GP, Shen Y, Owen-Schaub LB. Elevated soluble Fas (sFas) levels in nonhematopoietic human malignancy. Cancer Res 1996; 56:3870–4.[Abstract/Free Full Text]

15. Kimura M, Tomita Y, Imai T, Saito T, Katagiri A, Tanikawa T, et al. Significance of serum-soluble CD95 (Fas/APO-1) on prognosis in renal cell cancer patients. Br J Cancer 1999;80:1648–51.[Medline]

16. Wakahara Y, Nawa A, Okamoto T, Hayakawa A, Kikkawa F, Suganama N, et al. Combination effect of anti-Fas antibody and chemotherapeutic drugs in ovarian cancer cells in vitro. Oncology 1997;54:48–54.[Medline]

17. Baldwin RL, Tran H, Karlan BY. Primary ovarian cancer cultures are resistant to Fas-mediated apoptosis. Gynecol Oncol 1999;74: 265–71.[Medline]

18. Kenemans P, Bon GG, Kessler AC, Verstraeten AA, van Kamp GJ. Multicenter technical and clinical evaluation of a fully automated enzyme immunoassay for CA 125. Clin Chem 1992;38:1466–71.[Abstract/Free Full Text]

19. Gutierrez LS, Eliza M, Niven-Fairchild T, Naftolin F, Mor G. The Fas/Fas-ligand system: A mechanism for immune evasion in human breast carcinomas. Breast Cancer Res Treat 1999;54:245–53.[Medline]

20. Jodo S, Kobayashi S, Nakajima Y, Matsunaga T, Nakayama N, Ogura N, et al. Elevated serum levels of soluble Fas/APO-1 (CD95) in patients with hepatocellular carcinoma. Clin Exp Immunol 1998;112:166–71.[Medline]

21. Ghahremani M, Foghi A, Dorrington JH. Etiology of ovarian cancer: A proposed mechanism. Med Hypotheses 1999;52:23–6.[Medline]





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