Use of the concomitant serum dosage of CA 125, CA 19-9 and interleukin-6 to detect the presence of endometriosis. Results from a series of reproductive age women undergoing laparoscopic surgery for benign gynaecological conditions

E. Somigliana1,4, P. Viganò2, A.S. Tirelli3, I. Felicetta3, E. Torresani3, M. Vignali1 and A.M. Di Blasio2

1 Department of Obstetrics and Gynecology, Clinica ‘L.Mangiagalli’, University of Milano, 2 Istituto Auxologico Italiano and 3 Laboratorio di Chimica Clinica Istituti Clinici di Perfezionamento Milano, MilanoItaly

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Clinica ‘L.Mangiagalli’, Via Commenda 12, Milano, 20122, Italy. Email: dadosomigliana{at}yahoo.it


    Abstract
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 Materials and methods
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BACKGROUND: Recent studies have proposed the measurement of CA 19-9 and IL-6 as an alternative to CA 125 as markers for endometriosis. This study was performed in order to verify the clinical value of serum CA 125, CA 19-9 and IL-6 levels, either by themselves or combined, in the detection of the disease. METHODS: In a prospective cohort study, serum concentrations of CA 125, CA 19-9 and IL-6 were measured in a consecutive series of 80 women of reproductive age who underwent laparoscopy for benign gynaecological pathologies. RESULTS: Endometriosis was documented in 45 women (stage I–II in 14 cases and stage III–IV in 31 cases). Patients with endometriosis had significantly higher levels of CA 125 than controls [23.4 IU/ml (13.3–37.6) versus 11.4 IU/ml (9.1–18.5), P<0.001)]. Conversely, women with and without the disease were shown to have similar levels of both IL-6 pg/ml [0.6 (undetectable–1.4) versus 1.0 pg/ml (0.4–1.9), P=0.09] and CA 19-9 [9.8 IU/ml (4.5–20.8) versus 7.4 IU/ml (2.8–11.5), P=0.11]. The area under the receiver operating characteristics curve resulted in a statistically significant difference from the null hypothesis only for CA 125 (P<0.001). Sensitivity and specificity of CA 125 were 27 and 97% respectively and were higher than those related to CA 19-9 and IL-6. Concomitant use of the three dosages led to a sensitivity and a specificity of 42 and 71% respectively. CONCLUSIONS: The concomitant dosage of CA 125, CA 19-9 and IL-6 does not add significant information in respect to the CA 125 test alone in diagnosing either early or advanced stages of endometriosis.

Key words: diagnosis/endometriosis/CA 125/CA 19-9/IL-6


    Introduction
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 Abstract
 Introduction
 Materials and methods
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In recent years, many efforts have been made in trying to identify an easy way to diagnose endometriosis (Brosens et al., 2003Go). Laparoscopic surgery is, so far, considered indispensable in assessing the presence, severity and recurrence of the disease. Indeed, ultrasonography is solely effective in detecting ovarian endometriomas whereas physical examination may be helpful only in the diagnosis of deeply infiltrative endometriosis of the cul-de-sac and rectovaginal septum (Spaczynski and Duleba, 2003Go). Therefore, the endoscopic examination, albeit invasive, expensive and potentially risky, still represents an integral part of the diagnostic course (Spaczynski and Duleba, 2003Go).

The current task in the field of endometriosis diagnosis is to identify a serum marker of the disease. The role of the serum CA 125 assay for the diagnosis of the disease is indeed limited (Mol et al., 1998Go; Somigliana et al., 2002Go). This tumour-associated antigen has been suggested to be more useful in excluding cancer, in following the effects of medical treatments and in detecting recurrences (Moloney et al., 1989Go; Bast et al., 1998Go; Chen et al., 1998Go; Mol et al., 1998Go).

However, recently, two other molecules have been proposed as valuable tools in predicting the presence of endometriosis. CA 19-9 is a glycoprotein whose concentrations are elevated in patients with malignant and benign ovarian tumours (Harada et al., 2002Go). Serum levels of the antigen CA 19-9 have been shown to increase in accordance with the advancement of the clinical stage of endometriosis and no significant difference was found between CA 19-9 and CA 125 as positive and negative predictive values for this disorder (Harada et al., 2002Go). Moreover, on the basis of the increasing significance of the pleiotropic cytokine interleukin (IL)-6 in the pathophysiology of endometriosis (Witz et al., 2000Go), the serum measurement of the protein has been proposed in order to discriminate between women with and without the disease (Bedaiwy et al., 2002Go) and to identify specific forms of the disease (Iwabe et al., 2003Go). Sources of IL-6 in women affected may be represented by both eutopic and ectopic endometrium and peritoneal macrophages (Rier et al., 1994Go; Tseng et al., 1996Go; Tsudo et al., 2000Go; Witz et al., 2000Go).

Based on these observations, we have conducted a prospective evaluation of pre-operative sera from a consecutive series of women undergoing laparoscopic surgery for benign gynaecological conditions to determine the serum levels of CA 125, CA-19-9 and IL-6. The specific aim of the study was to verify the clinical usefulness of these markers in the diagnosis of endometriosis, either by themselves or when combined.


    Materials and methods
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 Materials and methods
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Between October 2002 and January 2003, all women who were referred to the Gynecological Surgical Service of the II Department of Obstetrics and Gynecology were evaluated prior to surgery for eligibility. Inclusion criteria were as follows: (i) a gynaecological indication for surgery, (ii) to have undergone laparoscopic surgery, (iii) age between 18 and 45 years. Exclusion criteria were: (i) a suspected or ascertained diagnosis of malignancy, (ii) pregnancy, (iii) menopausal age, (iv) refusal to participate in the study. The protocol of the study was approved by the local Ethics Committee and all recruited patients gave written informed consent.

Overall, 80 consecutive patients satisfied both our inclusion and exclusion criteria. Forty-five of them were found to have endometriosis. Stages according to the revised American Society for Reproductive Medicine (ASRM) classification (1997)Go were as follows: 12 were stage I, 2 stage II, 19 stage III and 12 stage IV. Concomitant presence of other gynaecological benign pathologies were observed in 14 of these 45 patients and were as follows: ovarian dermoids (two cases), sero-mucinous ovarian cysts (five cases), paraovarian cysts (three cases) and myomas (four cases). Endometriosis could not be detected in 35 women. These cases were used as controls. The main diagnoses of this group were as follows: ovarian dermoids (12 cases), pelvic inflammatory disease (six cases), sero-mucinous ovarian cysts (seven cases), paraovarian cysts (three cases), myomas (two cases) and normal pelvis in patients with infertility and/or pelvic pain (five cases).

Peripheral venous blood samples were obtained from the patients immediately before laparoscopy and successively centrifuged at 700 g at 4°C for 10 min. The resulting sera were then frozen at –20°C until assayed. The quantitative detection of CA 125 and CA 19-9 was performed using a commercially available chemiluminescent immunometric assay provided by Roche Diagnostics GmbH (Germany) to be analysed with the Elecsys Analyzer. The concentrations of CA 125 and CA 19-9 were expressed as IU/ml. Sensitivity for both CA 125 and CA 19-9 was <0.6 IU/ml. The quantitative detection of IL-6 levels was performed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit provided by R&D Systems, Inc. (USA) with an intra-assay coefficient of variation (CV) of 2.5%, an inter-assay CV of 4.5% and a sensitivity of <0.7 pg/ml. The concentration of IL-6 was expressed as pg/ml.

Since we have failed to observe previously reported differences between endometriosis and controls in regard of the serum levels of IL-6 (Bedaiwy et al., 2002Go), we have hypothesized that this result could be ascribed to the assay used. Therefore, dosages of this cytokine were repeated using another system. Specifically, the quantitative measurement of IL-6 was also performed by a sequential immunometric assay to be used with the Immulite analyzer (Diagnostic Products Corporation, Medical Systems, Italy) with an intra-assay CV of 4%, an inter-assay CV of 7% and a sensitivity of 5 pg/ml.

Data were analysed using {chi}2-test, Fisher's exact test, Student's t-test and non-parametric Wilcoxon test as appropriate (SPSS/Windows, USA). Serum levels of CA 125, IL-6 and CA 19-9 are expressed as median and interquartile range (IQR). Sensitivity, specificity, positive predictive value and negative predictive values were calculated using two different cut-off values. First, cut-off values were based on levels of the three different molecules observed in the control group of our series. Specifically, these levels (c) were calculated as follows:

Second, we have used the most commonly employed cut-off limit according to the literature. Specifically, the cut-off levels used for CA 125, IL-6 and CA 19-9 were 35 IU/ml, 2 pg/ml and 37 IU/ml respectively (Mol et al., 1998Go; Bedaiwy et al., 2002Go; Harada et al., 2002Go).


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Baseline characteristics of patients with and without endometriosis are shown in Table I. Both groups were similar in regard of these variables. The phase of the cycle did not appear to be a relevant factor for any of the molecules tested (P=0.21, P=0.57, P=0.30 for CA 125, IL-6 and CA 19-9 respectively).


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Table I. Characteristics of the patients

 
Patients with endometriosis had significantly higher levels of CA-125 [23.4 U/ml (13.3–37.6) versus 11.4 U/ml (9.1–18.5), P<0.001)]. Conversely, women with and without the disease were shown to have similar levels of both IL-6 [0.6 pg/ml (undetectable–1.4) versus 1.0 pg/ml (0.4–1.9), P=0.09] and CA 19-9 [9.8 IU/ml (4.5–20.8) versus 7.4 IU/ml (2.8–11.5), P=0.11]. These results are illustrated in Figure 1. Levels of these three molecules were also analysed according to the different forms and severity of the disease. Results of this analysis are shown in Table II.



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Figure 1. Serum levels of CA 125 (upper panel), IL-6 (middle panel) and CA 19-9 (lower panel) in patients with endometriosis (right) and controls (left). The statistical comparison was performed using the non-parametric Wilcoxon test for unpaired data. A statistically significant difference was observed only for CA 125 dosage (P<0.001).

 

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Table II. Serum levels of CA 125, IL-6 and CA 19-9 according to the presence of the different forms of endometrosis

 
We have used receiver operating characteristic (ROC) curves to evaluate the performance of the various serum dosages to screening (Figure 2). The area under the curve (AUC) was statistically significantly different from the null hypothesis (AUC of 0.5) only for CA 125 (P<0.001). Based on the levels of the molecules in the control group, the cut-off levels for CA 125, IL-6 and CA 19-9 were 31.0 IU/ml, 3.9 pg/ml and 36.4 IU/ml respectively. Sensitivity, specificity, positive predictive value and negative predictive value of the three molecules using both these cut-off levels and those more frequently employed in the literature are shown in Table III. The possibility of using a combination of all three variables was also evaluated. Specifically, we have tested two possibilities: ‘all tests positive’ and ‘at least one of them positive’. No patients were found to have all three tests positive; results from this analysis were thus not reported. The performance of the ‘at least one of them positive’ approach is shown in Table III.



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Figure 2. Receiver operating characteristics curve for identification of endometriosis for CA 125 (upper line), CA 19-9 (middle line) and IL-6 (lower line)

 

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Table III. Performance of serum concentration of CA 125, IL-6 and CA 19-9 in identifying the presence of endometrosis

 
This study refers to the results of IL-6 detection as performed by the ELISA assay. Dosage of this cytokine was repeated using the Immunolite immunometric system (Diagnostic Products Corporation, Medical Systems, Italy). The above statistical analysis was repeated using results from this second assay. This IL-6 assay was even more inadequate in detecting the presence of endometriosis (data not shown).


    Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IL-6 and CA 19-9 have been recently suggested to be of help in the diagnosis of endometriosis (Bedaiwy et al., 2002Go; Harada et al., 2002Go; Iwabe et al., 2003Go). However, the real usefulness of these proteins as serum markers is still to be established.

IL-6 is a pleiotropic cytokine that is produced by a variety of cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells and keratinocytes (Van Snick, 1990Go). It is also produced by both eutopic and ectopic endometrium (Akoum et al., 1996Go; Tseng et al., 1996Go; Bergqvist et al., 2001Go; Tsudo et al., 2000Go; Witz et al., 2000Go). The cytokine is important in mediating acute phase reaction, inflammation and angiogenesis during folliculogenesis and formation of decidua (Biffl et al., 1996Go; Witz et al., 2000Go). Recently, Bedaiwy et al. (2002)Go have emphasized the diagnostic value of serum IL-6 in detecting endometriosis providing a sensitivity of 90% and a specificity of 67% with a serum threshold of 2 pg/ml. However, due to the limited casistics, data from this specific study had to be verified. Moreover, Iwabe et al. (2003)Go have found higher serum concentrations of IL-6 in patients with endometriomas that decrease after laparoscopic surgery or treatment with a GnRH agonist.

CA 19-9 was initially detected as an antigen related to colorectal carcinoma and can serve as a marker of potential metastasis and malignancy (Gadducci et al., 1992Go). Endometriotic tissue also expresses CA 19-9 (Harada et al., 2002Go). There are a limited number of reports on the significance of serum CA 19-9 levels on the diagnosis of endometriosis (Panidis et al., 1988Go; Imai et al., 1998Go; Matalliotakis et al., 1998Go, 2000Go). However, a recent study reported that, while the sensitivity of the CA 19-9 test in detecting endometriosis was significantly lower than that of the CA 125 test (34 versus 49%), specificity was 100% for both the assays and positive and negative predictive values were not significantly different between the two (Harada et al., 2002Go). Moreover, all the patients with elevated CA 19-9 levels were at stages III and IV endometriosis, suggesting that this assay would be particularly suitable to detect more severe forms of the disease (Harada et al., 2002Go).

The results of the present study would indicate that neither IL-6 nor CA 19-9 can be used to discriminate between patients with or without endometriosis, even in combination with CA 125. Discrepancies among these studies are difficult to explain and may be due to differences in study design or patients considered. The presence of patients with non-endometriotic ovarian cysts and/or with pelvic inflammatory disease may have reduced the specificity of CA 125, CA 19-9 and IL-6 in our series. Indeed, these pathologies have been reported to be associated with increased serum levels of these molecules (Gadducci et al., 1992Go; Biffl et al., 1996Go; Bast et al., 1998Go). On the other hand, it has to be considered that the present study was specifically designed to assess the ability of CA 125, CA 19-9 and IL-6 to detect endometriosis among a non-selected population of patients undergoing laparoscopic surgery for gynaecological indications. Thus, we have estimated that recruiting controls excluding those with gynaecological conditions known to be associated with increased levels of one or more of these molecules would have led to an overestimate of the effectiveness of these molecules in terms of specificity and sensitivity.

Another way to explain discrepancies with previous studies is related to the assays employed. A role for this bias in the evaluation of CA 19-9 and CA 125 is unlikely since the currently available methods have been highly established for diagnostic purposes for many years. Conversely, techniques used for evaluation of IL-6 levels are less standardized. However, the specific assay used should not have played a crucial role since we have observed similar results using two different systems (an ELISA and an automatic immunometric assay). Of note, the ELISA that we have employed is exactly the same test used in the study by Bedaiwy et al. (2002)Go but was not a highly sensitive kit such as that of Iwabe et al. (2003)Go. The use of this high sensitivity assay could have been more appropriate. In particular, it might be hypothesized that the calculated cut-off value may be influenced by the use of a different assay. However, there are at least two reasons to think that this effect would not be relevant in our series: (i) the ROC curve of serum levels of IL-6 (which is independent from a single cut-off value) was highly inadequate, (ii) the low performance of this dosage in detecting the disease was confirmed also using previously determined cut-off values. In line with our results, in a study that has evaluated serum levels of IL-6 in a group of patients with malignant ovarian tumours when compared to patients with benign tumours, the cytokine could not be detected in women with endometriosis (Schroder et al., 1994Go).

Calculation of the cut-off levels in our series might also be a matter of discussion. Indeed, the formula used would have been more appropriate for variables with a normal distribution. Since it does not appear that CA 125, IL-6 or CA 19-9 have a normal distribution, using the 95th percentile of the distribution would have been more appropriate. However, in this regard it must be noted that an extremely large sample size is mandatory to reliably assess a 95th percentile. The 35 control patients enrolled in our study are insufficient. Nevertheless, the analysis of sensitivity, specificity, positive predictive value and negative predictive value was also repeated using the 95th percentile of the control group, leading to similar results (data not shown). Finally, in order to validate our data, the statistical analysis has been repeated using the threshold values most frequently used in the literature.

In conclusion, based on the results presented herein, we believe that the concomitant concentration of CA 125, CA 19-9 and IL-6 does not add significant information in respect of the concentration of CA 125 alone for the diagnosis of endometriosis. Nevertheless, we cannot exclude that assays able to measure lower amounts of IL-6 in the serum could provide a better performance. Moreover, larger series are required to evaluate whether the concentration of this cytokine may have some value in detecting specific forms of endometriosis. However, the most important message that derives from the present study is the necessity, when investigating the usefulness of a novel marker, to compare its clinical value to that of CA 125. The performance of serum CA 125 measurement in the detection of any type of endometriosis is low (Mol et al., 1998Go) but, awaiting other reliable serum indicators, it represents and remains the reference test.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 26, 2004; accepted on April 22, 2004.





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