Circulating Haptoglobin Is an Independent Prognostic Factorin the Sera of Patients with Epithelial Ovarian Cancer*
Changqing Zhao*, Loganath Annamalai*, Changfa Guo y, Narasimhan Kothandaraman z,Stephen Chee Liang Koh*, Huoming Zhang*, Arijit Biswas* and Mahesh Choolani*
Departments of *Obstetrics and Gynecology, and ySurgery, Yong Loo Lin School of Medicine, National Universityof Singapore, Singapore, Singapore; zDepartment of Obstetrics and Gynecology, National University Hospital,Singapore, Singapore
Abstract
OBJECTIVE: This study was conducted to evaluate the
prognosticsignificanceofhaptoglobin levels in theover-
all survival of patients presenting with various stages of
epithelial ovarian cancer. MATERIALS AND METHODS:
We employed an in-house sandwich enzyme-linked
immunosorbent assay method to determine the con-
centrations of preoperative haptoglobin and C-reactive
protein (CRP) in sera samples obtained from 66 malig-
nant tumors, 60 benign tumors, and 10 normal healthy
women. RESULTS: Levels of serum haptoglobin signifi-
cantly correlated with tumor type (P < .001) and Interna-
tional Federation of Gynecology and Obstetrics stage
(P < .05). A significant correlation was observed be-
tween clinical stage and patient survival (r = 5.99, P =
.026). Our data also indicated that elevated serum
haptoglobin levels were associated with poor outcome
for overall survival using both univariate and multi-
variate analyses (P = .048 and P = .036 respectively).
Using Pearson’s correlation, we have noted that serum
CRP concentrations significantly correlated with hapto-
globin levels (r2 = 0.22, P < .001). Immunohistochemical
findings and Western blot analyses were compatible
with sera levels of haptoglobin in which a higher in-
tensity of staining occurred in late-stage epithelial
ovarian cancers. CONCLUSION: This study provides
evidence that preoperative serum levels of haptoglobin
could serve as an independent prognostic factor in
patients presenting with epithelial ovarian cancer.
Neoplasia (2007) 9, 1–7
Keywords: Haptoglobin, epithelial ovarian cancer, sandwich ELISAmethod, CRP levels, prognostic biomarker.
Introduction
Epithelial ovarian cancer is the most lethal of female genital
tract cancers. Owing to a paucity of symptoms and their
insidious onset, most patients presented with advanced
stages of the disease, with the 5-year survival rate being a
mere 30% because of failure in early diagnosis. The prog-
nosis of ovarian cancer is largely dependent on stage and
histologic grade on presentation. The 5-year survival rate for
stage I epithelial ovarian cancer was reported to be approxi-
mately 90% [1]. Previous studies have also indicated that
patients with well-differentiated tumors based on histologic
grade tend to have a survival rate better than those with poorly
differentiated tumors [2]. The current management of patients
with ovarian cancer involves optimal cytoreductive surgery
followed by chemotherapy. Despite an initial favorable reac-
tion to therapy, most patients, however, appear to have a re-
lapse within 5 years after the management of tumors. Although
CA-125 is, by far, themost useful biomarker for ovarian cancer, it
is only limited to the evaluation of therapeutic effectiveness
in patients diagnosed with ovarian cancer and in the detection
of recurrent disease during posttreatment surveillance [3].
Therefore, it is imperative to identify alternative prognostic
markers for improvement in patient survival, to formulate ade-
quate treatment, and to monitor this ominous disease.
The basic haptoglobin molecule is a tetrameric protein with
two a/b dimers. b-Chains are identical in all haptoglobin types,
and polymorphisms of haptoglobin arise from differences in a-
chains [4]. In a manner similar to other acute-phase proteins,
haptoglobin also originates mainly from the liver, and elevation
of this peptide could be observed in infections, inflammations,
and various malignant diseases, including lung and bladder
cancers [5,6], leukemia [7], breast cancer [8], malignant lym-
phoma [9], urogenital tumors [10], and esophageal squamous
cell carcinoma [11]. Elevation of haptoglobin in the sera and
ascitic fluid of ovarian cancer has been reported in previous
studies using enzyme-linked immunosorbent assay (ELISA)
[12,13]. In addition, recent studies using proteomics-based
methods have shown that haptoglobin levels were elevated in
Address all correspondence to: Dr. Mahesh Choolani, Department of Obstetrics and
Gynecology, Yong Loo Lin School of Medicine, National University Hospital, 5 Lower Kent
Ridge Road, Singapore 119074, Singapore. E-mail: [email protected]
*This article refers to supplementary material, which is designated by ‘‘W’’ (i.e., Figure W1)
and is available online at www.bcdecker.com.
Received 28 September 2006; Revised 16 November 2006; Accepted 16 November 2006.
Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00
DOI 10.1593/neo.06619
Neoplasia . Vol. 9, No. 1, January 2007, pp. 1 – 7 1
www.neoplasia.com
BRIEF ARTICLE
the sera of patients with ovarian cancer [14,15]. Using con-
ventional two-dimensional gel electrophoresis, it was noted
that a decreased intensity of staining for this protein was
observed in samples from patients undergoing chemo-
therapy [16]. This change also correlated well with the pro-
file of CA-125 levels before and after chemotherapy [16]. The
evaluation of haptoglobin on survival rates and the out-
come of patients with ovarian cancer have not been exam-
ined as yet. In this study, we employed the in-house ELISA
method to measure serum haptoglobin levels in the sera
of patients presenting with epithelial ovarian cancer. Our
data indicate that haptoglobin levels could provide a use-
ful measure in the prognostic evaluation of ovarian cancer.
Materials and Methods
Patient and Clinical Samples
Women aged 20 to 72 years with malignant (n = 66) and
benign (n = 60) epithelial ovarian tumors were recruited for
open surgical or laparoscopic treatment at the Department
of Obstetrics and Gynecology, National University Hospital
(Singapore, Singapore), from 1998 to 2006. The histologic
type of ovarian cancer was classified as defined by Inter-
national Federation of Gynecology and Obstetrics (FIGO)
staging [17]. Blood and tissue samples from cysts of ovarian
carcinomas and benign ovarian cysts were collected during
surgery without intraoperative spillage. Thirteen patients di-
agnosed with ovarian cancer were treated with combination
therapy consisting of carboplatin (AUC 5)/Taxol (175 mg/m2
body weight) following surgery. Blood were drawn before
and after six cycles of chemotherapy. Venous blood samples
were collected in 8-ml Vacuette serum tubes containing clot-
activating factor (Greiner Bio-One, Kremsmuenster, Aus-
tria). All samples were transported on ice to the laboratory,
centrifuged immediately at 4jC for 10 minutes at 1500g, and
stored at �80jC until analysis, according to the method
described previously [18]. Tissue samples were snap-frozen
in liquid nitrogen and stored in a freezer at�80jC. Ten normal
healthy controls without any evidence of cancer or meta-
bolic disease were also included in this study. The sample
collection for this study was approved by the Institution
Review Board of the National University Hospital, and in-
formed consent was obtained from each patient. All patients
will be observed every 3 months for the first 2 years, then for
6 months for up to 5 years, and annually thereafter. For
Western blot and immunohistochemistry analyses, tumor
tissue sections that comprised > 60% of cancer cells in the
field were carefully selected by a histopathologist.
Quantitative Analysis by Sandwich ELISA Method
Total haptoglobin levels were quantified using the sand-
wich ELISA technique with two polyclonal antibodies against
this protein. Goat anti-human haptoglobin antibody (Bio-
design, Saco, ME) in an appropriate dilution (1:200) with
0.5% bovine serum albumin (BSA) in PBST buffer [0.05%
Tween-20 in phosphate-buffered saline (PBS) buffer] was
coated onto PVC plates (NUNC, Roskilde, Denmark) over-
night at 4jC. After blocking with 200 ml of 3% nonfat dry milk
in PBST for 2 hours at room temperature, individual serum
samples were mixed with 0.5% BSA in PBST buffer (1:100).
A diluted mixture of 200 ml was added and incubated for
2 hours at room temperature. Purified human haptoglobin
(Sigma-Aldrich, St. Louis, MO) was used as standard at
concentrations ranging between 10 ng/ml and 10 mg/ml.
The plate was then incubated with rabbit anti-human hapto-
globin antibody (Dakocytomation, Glostrup, Denmark) di-
luted in 0.5% BSA in PBST (1:400) at room temperature for
2 hours, followed by the addition of biotin-labeled anti-rabbit
secondary antibody (1:400; Vector Laboratories, Burlingame,
CA) and alkaline phosphatase streptavidin (1:400; Vector
Laboratories). p-Nitrophenylphosphate (Vector Laboratories)
was applied at 5 mM in 100 mM sodium bicarbonate solution
at 37jC for about 1 hour. To determine the concentration of
haptoglobin, the plate was read at 405 nm using an ELISA
reader (Tecan, Salzburg, Austria). Between each stage of
the assay, the plate was washed four times by immersion in
PBST buffer followed by aspiration.
Serum concentrations of human C-reactive protein (CRP)
were determined by a specific ELISA kit (Chemicon Inter-
national, Temecula, CA) according to the manufacturer’s
instructions.
Western Blot Analysis
Ten samples of late-stage ovarian cancer and benign
ovarian tumors were selected for further Western blot and
immunohistochemical analyses. All tissues used were lysed
using Tris buffer (pH 8.0; 50 mM Tris, 150 mM NaCl, and
5 mM EDTA with protease inhibitor cocktail; Roche Diagnos-
tics, Mannheim, Germany). To performWestern blot analysis
for haptoglobin, 10 mg of protein from each sample was
separated by 12% sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. The bands were then electrically
transferred onto 0.45-mm nitrocellulose membranes using
Semidry Transblot Cell (Bio-Rad Laboratories, Hercules,
CA). Membranes were soaked in 5% nonfat dry milk in TBST
(20 mM Tris base, 500 mM NaCl, and 0.05% Tween 20,
pH 7.5) for 1 hour at room temperature and incubated
overnight at 4jC with rabbit anti-human haptoglobin poly-
clonal antibody (1:5000; Dakocytomation). They were
washed thrice with TBST and incubated with horseradish
peroxidase–labeled anti-rabbit secondary antibody (Pierce
Biotechnology, Rockford, IL) diluted in 5% nonfat dry milk
in TBST (1:1000) for 1 hour at room temperature. The mem-
branes were then washed and developed with chemilumi-
nescent substrates (Pierce Biotechnology).
Semiquantitative Reverse Transcription–Polymerase
Chain Reaction (RT-PCR)
In addition, semiquantitative RT-PCR was employed to
confirm mRNA expression for haptoglobin in three ovarian
epithelial cancer cell lines TOV-112D, Caov-3, and SKOV-3
obtained from the American Type Culture Collection (Manas-
sas, VA). These cells were cultured at 37jC in 5% CO2 in
RPMI 1640medium (Invitrogen Corp., Carlsbad, CA) supple-
mented with 100 U/ml penicillin, 100 mg/ml streptomycin
2 Haptoglobin: Prognostic Factor in Ovarian Cancer Zhao et al.
Neoplasia . Vol. 9, No. 1, 2007
(Sigma-Aldrich), and 10% fetal calf serum (Hyclone, Logan,
UT). The cells were grown to 90% confluency and harvested
by treatment with 0.25% trypsin and 0.02% EDTA (GIBCO
Invitrogen, Carlsbad, CA).
Total RNA from cultured ovarian cancer cells (5� 106) was
extracted using an RNeasy Mini Kit (Qiagen GmbH, Hilden,
Germany). Specific mRNA for haptoglobin was analyzed by
RT-PCR, which was carried out using a one-step RT-PCR Kit
(Qiagen GmbH) in accordance with the manufacturer’s
instructions. Optimal PCR conditions for haptoglobin were
determined in preliminary experiments so that the number of
cycles selected was within the linear range of amplification
[19]. The following specific primers were used for the am-
plification of a 197-bp region of human haptoglobin cDNA
fragment: 5V-CTG TGC TGG CAT GTC TAAG-3V (5V up-
stream) and 5V-CAG CTATGG TCT TCT GAAC-3V (3V down-
stream) [20]. Amplification consisted of 40 cycles of 94jCfor 45 seconds, 55jC for 45 seconds, and 72jC for 60 sec-
onds. Finally, after an additional extension step at 72jC for
10 minutes, PCR products were analyzed by electrophoresis
on 2% agarose gels stained with ethidium bromide and
visualized under ultraviolet radiation. GAPDH was used as
positive control.
Immunohistochemistry
Rabbit anti-human haptoglobin polyclonal antibody
(1:5000; Dakocytomation) was used in standard indirect
immunoperoxidase procedures, as reported previously [21].
Statistical Analysis
Statistical significance was evaluated using ANOVA for
comparison of the means of haptoglobin between normal
healthy controls, benign ovarian tumors, and malignant ovar-
ian tumors. Pearson’s chi-square test was used to compare
demographic and clinical data. Overall cancer-specific sur-
vival was defined from the date of blood sampling to the
date of death due to cancer. Kaplan-Meier method was used
to estimate the probability of survival, and differences in sur-
vival rates were assessed by log-rank test. Multivariate
analysis of prognostic factors was performed using the Cox
regression model. All statistical analyses were performed
using SPSS 13.0 (SPSS, Inc., Chicago, IL). Results were
considered statistically significant at P < .05.
Results and Discussion
Tetrameric protein haptoglobin has three phenotypes
(Hp 1-1, Hp 2-1, and Hp 2-2) [22]. All phenotypes are com-
posed of different combinations of b, a1, and a2 subunits.
Results from Western blot analysis showed that the two
antibodies used for the in-house sandwich ELISA method
possessed the capacity of binding both a and b subunits
(Figure W1). Based on this finding, the ELISA method used
in this study measured the total serum haptoglobin concen-
trations. Three samples of different concentrations were
tested eight times on the same day (intra-assay) and were
repeated on five consecutive days (interassay). The coeffi-
cients of variance were 3.8% and 9.2% for intra-assay and
interassay for haptoglobin, respectively. The sensitivity of
this test is 1 mg/ml. We observed that serum haptoglobin con-
centrations were significantly higher in late-stage ovarian
cancer patients (4.6 ± 0.6 mg/ml) than in benign ovarian
tumors and healthy controls (3.12 ± 0.5 and 1.5 ± 0.5 mg/ml,
respectively; P < .05 and P < .01). Moreover, a significant
difference was observed in haptoglobin levels between
early-stage cancer (3.6 ± 0.5 mg/ml) and healthy controls
(P < .01). This observation appears to be in agreement with
previous reports [23,24].
Because CRP is a serological marker of inflammation that
can be used to investigate the association between inflam-
mation and risk of cancer, the levels of this acute-phase
reactant were measured in our patients. CRP concentrations
were observed to be significantly elevated in both early-
stage and late-stage ovarian cancers (13.9 ± 3.0 and 12.7 ±
2.7 mg/ml, respectively) when compared with normal healthy
women and benign tumors (1.28 ± 0.4 and 2.67 ± 0.9 mg/ml;
P < .01). The distribution of haptoglobin and CRP levels in
benign, early, and late epithelial ovarian cancers, as well as in
normal women, is presented in a boxplot, which is bounded
by 75th and 25th percentiles (Figure 1). A significant ele-
vation in serum haptoglobin and CRP levels was observed
in the sera of early-stage and late-stage epithelial ovarian
cancer when compared with those from normal healthy
women and benign tumors, as illustrated in Figure 1.
The association of haptoglobin levels with clinicopatho-
logical variables, including age, FIGO stage, tumor type,
grade, and pathology, is given in Table 1. In this study, we
have used a cutoff value of 4 mg/ml for haptoglobin concen-
tration because this value corresponds to better diagnostic
accuracy in our series of patients. Hence, using the cutoff
value of 4 mg/ml, we were able to minimize false-positive and
false-negative rates in differentiating normal and benign
cases from malignant ovarian tumors. As indicated in the
receiver operating characteristic (ROC) curve (Figure W2),
by adding a series of sensitivity and specificity at different
cutoff coordinate points, we found that at the 4.0-mg/ml
point, the largest value of the sum of sensitivity and specificity
could be achieved. Accordingly, following this criterion, the
ovarian cancer patients were divided into two groups based
on haptoglobin concentrations. Tumor staging/grading ac-
cording to FIGO classification is known to be the most
important prognostic factor for ovarian cancer. In this study,
we noted a significant association between the serum hap-
toglobin level and FIGO stage on diagnosis (P < .05; Table 1).
However, no significant association was observed between
the serum haptoglobin level and age, histology, or tumor
grade on diagnosis (P > .05).
In addition, tumor haptoglobin expression levels were
also examined for overall survival rates using Kaplan-Meier
analysis with log-rank statistics for determining significance.
The mean follow-up time for the entire cohort of 66 patients
was 37.7 months (range, 4–100 months). Using the 4-mg/ml
haptoglobin concentration as a cutoff point, as mentioned
earlier, the median and mean survival times of patients
with enhanced serum haptoglobin levels were observed
to be significantly less than those with low serum levels for
Haptoglobin: Prognostic Factor in Ovarian Cancer Zhao et al. 3
Neoplasia . Vol. 9, No. 1, 2007
this acute-phase reactant protein (38 vs 79, and 46.7 vs
84.7 months; P = .048). Kaplan-Meier survival curves gener-
ated for high versus low concentrations of haptoglobin are
illustrated in Figure 2. It is noteworthy that significantly
elevated haptoglobin concentrations were associated with
poor outcome for survival in women with epithelial ovarian
cancer (Figure 2; P = .048).
Using the Cox proportional hazards model multivariate
survival analysis of 66 patients, we observed enhanced
haptoglobin levels [relative risk = 5.054; 95% confidence
interval (95% CI) = 1.11–22.98] and tumor stage on diag-
nosis (relative risk = 5.99; 95% CI = 1.24–29.0), which ap-
peared to be significant predictor variables of overall survival
in these patients. In contrast, however, age, tumor type, his-
tology, and grade on diagnosis were not significantly corre-
lated with survival rates, as illustrated in Table 2.
Western blot analysis and immunohistochemistry tech-
niques were performed to demonstrate tumor tissue expres-
sion of haptoglobin. Results of Western blot analysis indicated
that the intensity of bands corresponding to haptoglobin in
tissue extracts was higher in advanced ovarian cancer than
in benign ovarian tumors (Figure 3). Data from RT-PCR have
convincingly demonstrated that there occurs a strong ex-
pression of haptoglobin mRNA in ovarian epithelial cancer
cells in culture (Figure 4). Moreover, immunohistochemical
analysis also indicated intense staining of haptoglobin in the
cytoplasm of ovarian cancer cells obtained from late-stage
ovarian cancers when compared with those from benign
ovarian tumors (Figure 5). This elevation in haptoglobin there-
fore provides strong evidence that malignant ovarian tumor
tissues possess the capacity to synthesize this protein.
Table 1. Association of Serum Haptoglobin Levels with Clinicopathological
Variables.
Variable n Haptoglobin Concentration P *
Low (<4 mg/ml) High (>4 mg/ml)
Age (years)
< 52 31 13 18 .691
z 52 35 13 22
Tumor type
Benign 60 41 19 < .001
Malignant 66 26 40
FIGO stage
Early (I/II) 19 11 8 < .05
Late (III/IV) 47 15 32
Histology
Serous 27 11 16 .254
Mucinous 20 8 12
Endometrioid 11 5 6
Clear cell 6 0 6
No record 2
Differentiation
Well 24 8 16 .918
Moderate 14 5 9
Poor 23 9 14
No record 5
*Pearson’s chi-square test was used to compare demographic and clinical data.
Figure 2. Overall survival of 66 patients with epithelial ovarian cancer in
relation to haptoglobin level; a cutoff value of 4 mg/ml was used to divide the
patients into two groups. Patients with higher haptoglobin concentrations had
a significantly worse survival probability.
Figure 1. Serum haptoglobin and CRP concentrations obtained from 66 pa-
tients with epithelial ovarian cancer, 60 benign ovarian tumors, and 10 healthy
controls. Serum levels of haptoglobin and CRP were significantly higher in pa-
tients with early-stage and/or late-stage ovarian cancer than in control subjects.
4 Haptoglobin: Prognostic Factor in Ovarian Cancer Zhao et al.
Neoplasia . Vol. 9, No. 1, 2007
Coupled to our data on survival rates, a prominent role for
haptoglobin in predicting the survival outcome of patients with
ovarian cancer is indicated (Figure 2).
It is envisaged that inflammation could serve as a potential
contributory factor in the development of a variety of cancers.
CRP is a serological marker of inflammation that could be
used to investigate the association between inflammation
and risk of cancer [25]. It is noteworthy that, in a manner
similar to that of haptoglobin, serum CRP levels were also
significantly elevated in early-stage and late-stage ovarian
cancers. Moreover, a significant correlation existed between
CRP and haptoglobin levels (r2 = 0.22, P < .001; Figure W3),
indicating that inflammation occurs in the early and late
stages of ovarian cancer development. Based on this ob-
servation, it seems plausible to suggest that CRP could serve
as an adjunct to haptoglobin in the evaluation of ovarian
cancers. Because haptoglobin is among the most abundant
glycoproteins secreted by the liver and because circulating
hepatic haptoglobin accounts for the major elevation of
this protein in the serum of ovarian cancer patients [16], it is
reasonable to suggest that enhanced hepatic synthesis of
this protein occurs due to an acute-phase response in these
ovarian cancer patients.
It has been previously reported that abnormally elevated
glycosylated haptoglobin was detected in the blood of women
presenting with ovarian and breast cancers [26]. The oligo-
saccharide involved in this binding was identified as the
fucose-specific lectin Lotus tetragonolobus, which was ele-
vated seven-fold in the sera of patients with ovarian tumors
[27]. The serum concentration of fucosylated haptoglobin
closely correlated with tumor burden and levels of a-3-fucosyl
transferase—an enzyme that adds fucose toN-acetylglucos-
amine on glycoproteins [28,29]. Similar changes in oligo-
saccharide structures have been reported in other types of
malignant transformations, including pancreatic cancers
[30]. The origin of fucosylated haptoglobin is, however, un-
known at present, but it could be due to either aberrant
glycosylation by malignant ovarian cancer cells or a factor
produced by cancer cells that could enhance its liver synthe-
sis in afflicted women. Disturbances in fucose metabolism
with respect to haptoglobin binding may be implicated in the
pathophysiology of ovarian cancer.
Haptoglobin levels in the sera of 13 patients who were
subjected to chemotherapy were also measured to deter-
mine whether chemotherapeutic treatment had any effect
on the expression of this acute-phase reactant protein. Its
concentration was significantly reduced after six cycles of
chemotherapeutic treatment (2.2 ± 0.8 mg/ml) when com-
pared with those before treatment (5.2 ± 0.7 mg/ml; P <
.001). This result is in accordance with a previous study
[16] indicating a role for haptoglobin in monitoring patients
undergoing chemotherapy.
Recent investigations revealed that haptoglobin is a nat-
ural inhibitor of collagen degradation and is locally expressed
by fibroblasts in arterial walls [31]. This glycoprotein there-
fore plays an important role in cell migration and arterial
restructuring. Moreover, collagen turnover is an important
feature in many physiological processes such as growth
Table 2. Multivariate Survival Analysis By Cox Regression.
Variables Hazard Ratio (95% CI) P
Age 1.032 (0.357–2.981) .954
FIGO stage 5.99 (1.237–29.005) .026
Histology 0.839 (0.522–1.346) .466
Grade 1.162 (0.457–3.035) .896
Haptoglobin 5.054 (1.111–22.983) .036
Figure 3. Western blot analysis from ovarian tumor tissue samples. Ten micrograms of samples from benign (A) and late-stage ovarian cancer (B) was selected.
Expression of haptoglobin in cancer was significantly higher than that in benign ovarian tumor. Actin was used as loading control.
Figure 4. Analysis of ovarian cancer cell lines for haptoglobin mRNA ex-
pression by RT-PCR. A 197-bp fragment was amplified by RT-PCR from
samples of total RNA extracted from the cultured ovarian cancer cell lines TOV-
112D, Caov-3, and SKOV-3. RT control was performed without Sensiscript
reverse transcriptase, and PCR control was conducted without RNA template.
Haptoglobin: Prognostic Factor in Ovarian Cancer Zhao et al. 5
Neoplasia . Vol. 9, No. 1, 2007
and wound healing, and enhanced collagen degradation is
causally related to severe tissue destruction andmalfunction,
which are often encountered in pathological processes such
as arthritis and cancer [32]. The importance of haptoglobin
in extracellular matrix degradation and cell migration sug-
gests a role for this polypeptide in cancer and also provides
evidence supporting haptoglobin as a molecular target for
therapy in epithelial ovarian cancer.
Our retrospective analysis therefore provides evidence
that circulating haptoglobin levels could be a useful indepen-
dent prognostic indicator in patients with epithelial ovarian
cancer. Overall, our data suggest that lower haptoglobin
levels in preoperative sera are associated with better survival
outcome. More studies are, however, warranted to confirm
these findings.
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Haptoglobin: Prognostic Factor in Ovarian Cancer Zhao et al. 7
Neoplasia . Vol. 9, No. 1, 2007
Figure W1. Western blot analysis showing the binding capacity of antibodies used in ELISA (A, rabbit anti-human haptoglobin polyclonal antibody; B, goat anti-
human haptoglobin antibody). Haptoglobin standard (5 �g; Lanes 1 and 6) and 10 �g of protein from ovarian cancer (Lanes 2, 3, 7, and 8) and benign ovarian
tumors (Lanes 4, 5, 9, and 10) were loaded into each well.
Figure W2. ROC curves for serum haptoglobin. Using different cutoff
coordinate points, a series of sensitivity and specificity would be generated. It
is noted that at the 4.0-mg/ml point, the greatest value of the sum of sen-
sitivity and specificity could be achieved.
Figure W3. Single regression analysis between haptoglobin and CRP.
Haptoglobin was significantly correlated to CRP (P < .001). The central
straight line corresponds to the best-fit linear regression line. The two curved
lines surrounding the best-fit line define the 95% CI of regression line. The
graphs also show prediction interval (the curves defining the prediction
interval are further from the regression line than from confidence lines).
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