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Plasma Bikunin As a Favorable Prognostic Factor in Ovarian Cancer

From the NetForce Co Ltd; Computer Technology Integration Co Ltd, Nakamura, Nagoya, Aichi; Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka; Department of Knowledge-Based Information Engineering, Toyohashi University of Technology, Toyohashi, Japan

Address reprint requests to Hiroshi Kobayashi, MD, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka, 431-3192, Japan; e-mail: hirokoba{at}hama-med.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Bikunin is a multifunctional glycoprotein, which mediates suppression of tumor cell invasion and metastasis. The measurement of bikunin levels in the tissue of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the evaluation of prognosis. The high bikunin expression in ovarian cancer tissue would enable the use of soluble bikunin protein present in the circulation of ovarian cancer patients as a biomarker of disease.

PATIENTS AND METHODS: We developed a double-antibody immunoassay for bikunin and detected its presence in normal human circulation. We quantified, by enzyme-linked immunosorbent assay and/or immunoblot assay bikunin in sera from 200 healthy women (controls), 200 patients with benign gynecologic diseases, and 327 patients with ovarian cancer before surgical removal of the tumor.

RESULTS: When the values of bikunin corresponding to the median were used as the cutoff value (11.5 µg/mL), low plasma bikunin was strongly associated with late-stage, suboptimal debulking with large residual tumor (> 2 cm) and low response to chemotherapy. The median survival time of the patients with a high bikunin level was more than 60 months as compared with 26 months among those with low bikunin level (P = .002). This difference corresponded to a 2.2-fold increased risk of dying for the lower plasma bikunin patients (hazard ratio, 0.45; P = .023) and remained significant in multivariate analysis (hazard ratio, 0.63; P = .041).

CONCLUSION: Preoperative plasma bikunin concentration is a strong and independent favorable prognostic marker for ovarian cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Bikunin is a Kunitz-type protease inhibitor and a heavily glycosylated protein.¹ It has an inhibitory function for trypsin, chymotrypsin, human leukocyte elastase, and plasmin. In our previous experiments, several proteins, including urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) known to be involved in invasion and metastasis, were significantly suppressed by exogenously added bikunin.^2-5 Further, transfection of human ovarian cancer HRA cells with bikunin gene reduced the expression of uPA and uPAR and diminished cellular invasiveness.⁶ Current investigations have focused on the understanding of the molecular mechanism(s) by which bikunin downregulates uPA and uPAR expression both in vitro and in vivo and controls invasiveness and tumor growth in highly invasive cancer cells.^2-6

We showed that there is a direct correlation between bikunin overexpression and the reduced metastatic potential of primary tumor biopsies.⁷ We have shown that bikunin is part of the negative invasive program, as evaluated by their invasion and uPA/uPAR synthesis.^8-11 In the previous study, we examined the levels of bikunin mRNA using a reverse transcription (RT) polymerase chain reaction (PCR) in ovarian cancer tissues.⁷ The major finding of our study was the striking correlation between low bikunin mRNA expression and poor prognosis in patients with ovarian cancer.⁷ The link between reduced bikunin mRNA expression and poor prognosis was independent of age at surgery, surgical stage, tumor size, tumor histology, degree of differentiation, and plasma CA125 level.⁷ When the Cox proportional hazards model was constructed for the entire series, low bikunin mRNA expression remained an independent predictive factor of death.⁷

Plasma concentrations of bikunin have not been investigated in the same patients. To our knowledge, there are no data available on the prognostic influence of circulating plasma bikunin in ovarian cancers. In this study, we determined the plasma bikunin levels in a series of ovarian cancer patients and correlated the results with conventional clinicopathologic characteristics. Most importantly, this large study allowed the prospective collection of plasma samples from a reasonably homogeneous group of ovarian cancer patients treated on the same clinical trial.

	PATIENTS AND METHODS

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Patient Specimens
The Shizuoka Cohort Study on Ovarian Cancer Screening (SCSOCS) started in September 1985 as a part of the Cancer Registration System established in January 1982. The SCSOCS trial was carried out at 212 hospitals in Shizuoka prefecture in Japan. The participants were living in 35 townships. From September 1985 to December 2002, a total of 71,683 women between the ages of 20 and 85 years participated in the SCSOCS. Each woman who had signs or symptoms leading to the diagnosis of pelvic masses such as lower abdominal discomfort or pain visited a hospital for gynecologic examination, including cancer screening. Each woman who enrolled received pelvic examinations and transvaginal (occasionally transabdominal) ultrasound performed by gynecologists. Peripheral venous blood samples were collected in sterile test tubes before any cancer therapy was given, centrifuged at 3,000 x g for 10 minutes, and then stored at –70°C. Plasma CA125 determination was performed. Furthermore, residual plasma samples were stored at –70°C. The plasma samples had been freeze-thawed twice before the bikunin assay. Investigators responsible for determining the CA125 levels were aware of the participants' clinical status.

The report of a diagnosis of disease (including cancer) in a participant was checked against medical records from the hospital where the disease had been diagnosed. The validity of benign and malignant ovarian masses in the SCSOCS has been found to be in accordance with registration at local hospitals. Index cancers were defined as primary epithelial carcinomas of the ovary and fallopian tube. The medical records, ovarian ultrasonograph, other radiologic studies, and pathology studies were reviewed by the investigators.

In cases of death, the cause was investigated by a review of hospital records and autopsy reports. The status of patients lost to follow-up was checked at intervals with the Death Registry in Shizuoka so that no instances of death would be missed. Incident ovarian cancer cases occurring in the entire cohort were identified by record linkage to the Shizuoka Cancer Registry (SCR). This register covers the entire Shizuoka population, including diagnoses of ovarian cancer and time of death. New cases of cancer were generally registered immediately after diagnosis. Entries within this registry have proven to be accurate in 96% of cases. No cohort members were lost to follow-up.

In December 2002, the SCSOCS and SCR were searched to determine both malignant and nonmalignant diagnoses. These registries included entries of all in-patients and all cancer diagnoses made throughout Shizuoka prefecture. Excluded from the study were patients in whom cancer had been diagnosed at any time before registration according to information from the patient's history and from medical records available to the investigator. In addition, 3,035 patients in whom benign gynecologic disease including uterine leiomyoma (n = 2,026) and adenomyosis (n = 1,009) had been diagnosed at this screening were excluded from the study and independently analyzed. To obtain information on vital status of registered cases, the SCR has used the following three steps: (1) collation with annual cancer death file, (2) collation with annual death certificate file in Shizuoka, and (3) confirmation of the cases' living status by referring to registers in the local municipality offices of inhabitants. Study participants underwent a maximum of nine ultrasound scans or eight CA125 screens (one prevalence and others incidence). For analysis, each scan or screen episode was classified, on the basis of last scan or screen result, into normal or abnormal.

The study was approved by the regional ethics committee of the Hamamatsu University School of Medicine and by the Shizuoka Data Inspection Board.

Ovarian Cancer Patients and Control Subjects
Plasma samples from the 429 patients enrolled onto the SCSOCS were collected. The number 429 includes all women diagnosed with ovarian cancer during this study. Aliquots of specimens were frozen after collection, shipped on dry ice by overnight mail to a central repository, and stored at –70°C until the time of assay. Aliquots were removed for triplicate enzyme-linked immunosorbent assay (ELISA) assays. Plasma CA125 antigen was first measured from the sera, and plasma bikunin, from the stored frozen samples.

From this series, we selected 327 patients with histologically diagnosed and typed ovarian cancer in the analysis. The baseline characteristics of the 327 ovarian cancer patients in this study are presented in Table 1. These patients were selected from a consecutive series of 429 women diagnosed and treated for ovarian malignancy at Hamamatsu University Hospital and the SCSOCS, Japan, between 1985 and 2002 by excluding cases with a malignancy not of the histologically epithelial type (n = 30), patients who were not operated on (n = 52), and those with insufficient material (n = 11). Patients who died because of any postoperative complications were excluded from the survival analyses (n = 9). Tumor staging and histologic typing of the tumors were done according to the International Federation of Gynecology and Obstetrics (FIGO) classification. The median age was 67 years (range, 31 to 86 years). One hundred sixty patients died during the follow-up.

Tissue Lysates
Tissue lysates from human ovarian cancer were prepared using radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 50 mmol/L Tris [pH 7.5]) containing a mixture of protease inhibitors (aprotinin, bestatin, leupeptin, and pepstatin). The homogenate was centrifuged at 14,000 x g at 4°C for 10 minutes, and lysates were stored at –70°C until use. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA).

Site-Directed Antibody Production
Sequence-specific antibodies were raised by previously described procedures¹² against the following bikunin peptides: the N-terminal segment of bikunin (peptide 1; ¹AVLPQEEEGSGGGQ¹⁴), the central segment of bikunin (peptide 2; ⁶⁰MGNGNNFVTEKE⁷¹), and the C-terminal segment of bikunin (peptide 3; ¹¹⁶QGNGNKFYSEKEC¹²⁸). Each of these antibodies was reactive against their cognate peptide (shown by ELISA; data not shown).

Solid-Phase Binding Assay
Two µg of each bikunin, deglycosylated bikunin, or bovine plasma albumin (BSA) in 100 µL of phosphate-buffered saline (PBS) were dried in a 96-well microtiter plate (Corning, Cambridge, MA) overnight at 37°C. The wells were rinsed with PBS three times and were blocked with PBS 100 µL per well, containing 1 mg/mL BSA for 1 hour at room temperature. After rinsing three times with PBS, known concentrations of each antibody (antibikunin peptide 1, antibikunin peptide 2, antibikunin peptide 3, or anti–inter--trypsin inhibitor [ITI] Dako antibody; Copenhagen, Denmark) in PBS containing 0.1 mg/mL BSA were incubated in triplicate (100 µL per well) for 1 hour at room temperature. After rinsing the wells with PBS three times, 100 µL goat antirabbit IgG conjugated to horseradish peroxidase in PBS containing 0.1 mg/mL BSA (1:800) was added for 1 hour at room temperature. The binding of antibody was detected using the colorimetric substrate tetramethyl benzidine (Sigma-Aldrich Japan, Tokyo) as described by the manufacturer.

Western Blot Analysis
Bikunin was immunopurified from human plasma pretreated with or without chondroitinase ABC using peptide 1 antibody immobilized on a Sepharose 4B gel column. Isolated bikunin antigen was separated on SDS–polyacrylamide-gel electrophoresis under nonreducing conditions and was electrophoretically transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore Co, Bedford, MA). The membrane was blocked in PBS containing 2% BSA, and incubated in a solution containing anti-ITI Dako antibody. Antibodies bound on the membrane were visualized by antirabbit IgG labeled with peroxidase and 4-chloro-1-naphtol substrates. Equal loading of protein was confirmed by antiactin antibodies (Santa Cruz). The molecular masses on the membrane were determined by prestained markers (Bio-Rad).

Plasma Bikunin Analysis
To measure plasma bikunin level, deglycosylated bikunin concentrations were determined using human bikunin ELISA that we performed. The system uses a solid-phase antibikunin peptide 1 antibody raised against human bikunin peptide 1 and a biotinylated anti-ITI Dako antibody. The detection steps include streptoavidin-horseradish peroxidase and tetramethylbenzidine as chromogens. This two-step ELISA was performed to measure the concentration of bikunin in plasma. For each analysis, 10 µL plasma samples were pretreated with chondroitinase ABC (5 µL, 50 U/mL) at 37°C for 30 minutes. 50 µL of 1:10 diluted samples were used. Standards and specimens were added into the wells and incubated for 1 hour at room temperature. After incubation, the wells were washed three times with PBS, and the solution containing Dako antibody coupled with peroxidases was added. One hour later, the wells were washed with PBS, and tetramethyl benzidine liquid substrates were added. The enzyme reaction was terminated after 10 minutes of incubation by the addition of H₂SO₄, and the absorbance at 450 nm was measured by a Bio-Rad microplate reader. All analyses and calibrations were carried out in triplicate. The calibrations on each microtiter plate included purified human bikunin or deglycosylated bikunin standards. Optical densities were determined using a microtiter plate reader (Bio-Rad multireader) at 450 nm. The blank was subtracted from the triplicate readings for each standard and sample. Concentrations are reported as ng/mL. No loss of bikunin immunoreactivity was observed when five samples were subjected to either three or five freeze-thaw cycles. No significant difference in plasma bikunin concentrations was found when cancer patient plasma samples stored at –70°C for 1 to 5 years (n = 30) and samples stored for 10 to 15 years (n = 30) were compared, suggesting that there is no major loss of bikunin immunoreactivity during long-term storage as compared with shorter storage.

Receiver Operator Characteristic Curve Analysis
Receiver operating characteristic (ROC) curves were used to examine the performance characteristics of the bikunin over their entire range of values. The area under the curve (AUC) was used as an index of global test performance, with an AUC of 0.5 indicating no discrimination ability. For each set of patients, ROC curves were generated for the parameters of bikunin.

Statistical Analysis
Pretreatment plasma bikunin levels were used to dichotomize patients into low- and high-level groups based on the median values of the plasma bikunin measurements. The Kaplan-Meier product-limit method¹³ was used to estimate the survival duration in the low- and high-level bikunin patient groups. The log-rank statistical method was used to test for differences in the distribution of the survival times between the two baseline bikunin groups. Univariate analyses were carried out for pretreatment bikunin. Analyses were performed using bikunin levels as categoric variables with median cutoff points. In addition, the proportional hazards regression model¹⁴ was used to assess the prognostic importance of pretreatment plasma bikunin levels, and to adjust for potentially confounding pretreatment variables reported in other series to be of prognostic significance. Statistical analyses were done using a StatView computer program (HULINKS, Tokyo). All P values are two-tailed.

	RESULTS

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Specificity of Polyclonal Antibodies to Bikunin Peptides: ELISA for Bikunin
Three of the site-directed polyclonal antibodies to bikunin peptides we established were selected to be reacted with bikunin and its deglycosylated form. Of these antibodies, the peptide 1 antibody strongly reacts with bikunin (Fig 1A) and deglycosylated bikunin (Fig 1B) by a solid-phase binding assay.

View larger version (27K): [in this window] [in a new window]   Fig 1. Reactivity of the three established polyclonal antibodies to the bikunin peptides. Relative binding of the antibodies to bikunin (A) or deglycosylated bikunin (B) absorbed on the 96-well microplates was determined by measuring the absorbance obtained in the solid-phase immunoassay. (C) Western blot analysis for plasma pretreated with or without chondroitinase ABC. The data are representative of three separate experiments. Dako, Copenhagen, Denmark.

Plasma Bikunin Concentration in Noncancer and Ovarian Cancer Patients
The bikunin protein levels were measured in the plasma of 327 ovarian cancer patients, 200 noncancer patients, and 200 healthy volunteers by a specific ELISA (Fig 2). Plasma bikunin concentration is similar in normal controls and patients with benign gynecologic diseases (means and medians were 6.1 and 6.2, and 6.3 and 6.5 µg/mL for the two groups, respectively). The levels measured in ovarian cancer patients were significantly higher than those found in the controls (P < .001). The median and mean bikunin plasma levels were 11.5 and 12.1 µg/mL, respectively. Thus, plasma bikunin concentration is only elevated in ovarian cancer patients. ROC curve for bikunin was also constructed (Fig 3). The AUCs were 0.81 (95% CI, 0.74 to 0.86) for bikunin. ROC curve indicates that the cutoff of bikunin is 11.45 µg/mL. Taken together, the upper limit of normal for bikunin is considered to be 11.5 µg/mL.

View larger version (11K): [in this window] [in a new window]   Fig 2. Distribution of plasma bikunin in healthy controls, patients with benign gynecologic diseases, and patients with ovarian cancer. The boxplots display the 10th, 25th, 50th (bold horizontal line is the median), 75th, and 90th percentiles. P values calculated by the Mann-Whitney test were found to be less than .001 between bikunin concentrations in patients versus controls, less than .001 between cancer versus benign, and .93 between healthy versus benign. (*) P < .001.

View larger version (34K): [in this window] [in a new window]   Fig 3. Receiver operating characteristic curve for bikunin. We arrived at the optimum cut point of 11.45 µg/mL by selecting the point on the ROC curve that maximized both sensitivity and specificity.

View larger version (49K): [in this window] [in a new window]   Fig 4. Comparison of soluble bikunin proteins in the chondroitinase ABC-treated plasma of noncancer patients (A) and ovarian cancer patients (B), as well as in ovarian cancer tissue extracts pretreated with chondroitinase ABC (C). Bikunin-related proteins present in the chondroitinase ABC-treated plasma of five noncancer patients (A), five ovarian cancer patients (B), and in five ovarian cancer tissue extracts pretreated with chondroitinase ABC (C) were immunoblotted with antibikunin peptide 1 antibody (A and B) or antiactin antibody (C), respectively. The data are representative of two separate experiments.

Association of Bikunin Levels With Survival: Univariate Analysis
The strength of the associations between each individual predictor and progression-free survival (PFS) or overall survival (OS) is shown in the univariate analysis in Table 2. Stage of disease, histologic grade, residual tumor size, histologic types and CA125 level showed a strong association with cancer relapse and death. In univariate analysis for bikunin, the following parameter showed statistically significant associations with survival: pretreatment plasma bikunin using the median level as cut point (Table 2 and Fig 5A). Although bikunin levels seem to retain independent prognostic significance on multivariate analysis, the strong association with stage is of some concern. In that regard, separate survival curves were shown for patients with stage I/II (Fig 5B) versus stage III/IV disease (Fig 5C). A pretreatment plasma bikunin level greater than 11.5 µg/mL was associated with improved survival, with a hazard ratio of 0.45, P < .001. With log-rank testing, this difference corresponded to a median survival of 26 months for patients with preoperative plasma bikunin 11.5 µg/mL compared with a median survival of more than 60 months for patients with preoperative plasma bikunin more than 11.5 µg/mL. High bikunin level was found to be a more significant predictor of patients with stage I/II, compared with those with stage III/IV (P = .014 versus P = .043, respectively). High bikunin level was also found to be a significant predictor of PFS (hazard ratio, 0.41; P < .001, respectively).

View larger version (18K): [in this window] [in a new window]   Fig 5. Kaplan-Meier survival curves for patients with high- and low-level bikunin. Overall survival (OS) curves for all patients (n = 327; A), patients with stage I/II (n = 147; B), and patients with stage III/IV (n = 180; C), respectively.

	DISCUSSION

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In this study, plasma specimens were prospectively collected with the intent of determining bikunin levels and assessing its prognostic value of ovarian cancer in this large trial. Here, we show that plasma bikunin is a novel biomarker for ovarian cancer and is an independent powerful predictor of patient outcomes. This difference held true in a multivariate analysis as well. Patients with preoperative bikunin concentration more than 11.5 µg/mL have a significantly improved prognosis compared with patients with low preoperative bikunin. Patients with 11.5 µg/mL at pretreatment had a 2.2-fold increase in the hazard ratio (risk of dying) compared with those with bikunin level greater than 11.5 µg/mL. Median survivals for these two groups were 26 months and longer than 60 months, respectively (P = .002). High bikunin level was found to be a more significant predictor of patients with stage I/II compared with those with stage III/IV. The effects were largely independent of cancer histologic type and grading. This is the first demonstration in any cancer that plasma bikunin levels are predictive of survival.

In a pilot study, we found no significant association between the bikunin tumor tissue expression and the plasma levels (data not shown), but this finding should be interpreted with caution because we had adequate tissue samples only from 10 patients available for bikunin immunostaining. In the present series, plasma bikunin levels greater than 11.5 µg/mL were found in as many as 57% of the patients with stage I or II disease at presentation as compared with 40% of those with stage III to IV disease (P = .039; Table 1). High levels of circulating bikunin may in part reflect the presence of a small tumor mass. Why are bikunin levels in plasma elevated in early-stage ovarian cancer? Furthermore, what stimulates bikunin production? The findings of the previous studies suggested that the levels of plasma bikunin changed in patients with liver dysfunction and were closely related to the abnormalities of coagulofibrinolysis, including prothrombin time. At this time, we have no data showing whether plasma bikunin levels show positive correlations with the levels of prothrombin time, hepaplastin test, antithrombin III, 2-plasmin inhibitor, plasminogen, or fibrinogen. We speculate that, in the early stage, ovarian cancer cell–mediated humoral factor(s) may stimulate bikunin from hepatocytes and that endogenous bikunin may efficiently prevent invasion and metastasis, especially in the early stage. Further studies are needed to clarify whether plasma bikunin levels may be related to the liver function as well as the origin of elevated bikunin in plasma.

The basic release of bikunin is thought to have its origin predominantly in hepatocytes.^15,16 Furthermore, RT-PCR analysis indicated that other human organs, including pancreas and kidney express bikunin.¹⁹ Elevated bikunin tissue levels have been observed for several tumor types using an immunoblot (data not shown). The high expression of bikunin by ovarian cancer tumors incited us to investigate the prognostic value of bikunin levels in the circulation of ovarian cancer patients. We examined whether ovarian cancer–specific bikunin might be present in the plasma of ovarian cancer patients and not in the plasma of noncancer controls. However, comparison of the spectrum of bikunin proteins present in the circulation of ovarian cancer and noncancer patients did not reveal any changes related to the presence of a tumor (Fig 4).

A significant difference between the bikunin plasma levels of ovarian cancer patients and noncancer patients could be observed. Our recent unpublished data demonstrated that tumor-infiltrating macrophages in ovarian cancer tissues do express bikunin. Taken together, these data suggest that the amount of soluble bikunin proteins released from the tumor of ovarian cancer patients is enough to significantly exceed the basic bikunin plasma level. However, we did not determine whether surgical removal of the tumor did not result in a reduction of the bikunin plasma level.

In our previous study examining the prognostic value of bikunin analysis in ovarian tumor extracts, we verified the overexpression of bikunin in tumor tissues by RT-PCR and additionally provided evidence that intratumor bikunin protein concentration is also a strong predictor of improved prognosis in both univariate and multivariate analyses.⁷ The findings of the previous study are therefore in general agreement with the results of our study (ie, that higher plasma bikunin levels correlate with improved survival). This trend would suggest that an increase in plasma bikunin level might portend a better clinical outcome. There are no data on bikunin expression in human ovarian cancer or ovarian cancer cell lines tested, but lung cancer cells express bikunin in moderate to high levels in the majority of the cancer cells.²⁰ The origin of serum bikunin and the mechanism by which serum levels are elevated, especially in the early stage, now need to be further evaluated in ovarian cancer.

Serine proteases have been shown to have prognostic value in ovarian cancer.²¹ It has been known for years that many other proteolytic enzymes have prognostic value in many different cancers.^22-25 The biologic mechanisms of proteolytic enzyme involvement in cancer prognosis include their ability to degrade extracellular matrix, thus facilitating invasion and metastasis.²⁶ It seems likely that bikunin is a protease inhibitor that can suppress tumor cell invasion and metastasis. This may be the reason why the high plasma bikunin level predicts an improved survival.

Established adverse prognostic factors in ovarian cancer consist of advanced stage and grading at diagnosis and a poor performance status, but many other factors have been investigated with variable success.²⁷ Of the established prognostic factors, a high plasma bikunin level was significantly associated with an improved prognosis. This plasma factor is quick to measure, is relatively inexpensive, and might be incorporated into a panel of prognostic factors in ovarian cancer. However, there seems to be substantial overlap in bikunin levels among the three groups. It is thus unclear how checking bikunin levels would have any practical clinical utility at present. Confirmatory studies in other series of patients with other malignancies need to be carried out before recommending its use in clinical decision making.

Prognostic markers are needed to design more rigorous strategies for treating advanced ovarian cancer. The Kaplan-Meier curves of Figure 5 suggest that plasma bikunin analysis can assist physicians in selecting therapeutic options, for the following reason: virtually all patients with high presurgical bikunin level tend not to relapse within 1 year, and most are alive. Therefore, presurgically low bikunin level ( 11.5 µg/mL) is most frequently associated with patients who are destined to relapse and die. These patients should be good candidates for clinical trials evaluating alternative treatments.

In summary, the data suggest that plasma bikunin levels may be able to identify a subset of ovarian cancer patients who have a worse or improved outcome. Plasma bikunin is a powerful, favorable prognostic factor in ovarian cancer. Therapies targeted at this glycoprotein are under development,²⁸ and in the future, prospective trials could be designed to enrich for patients who might benefit from such therapies.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
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Submitted March 1, 2004; accepted November 7, 2004.

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