Download Title: The use of Osteopontin as an Adjunct to CA125, for the

Title:
The use of Osteopontin as an Adjunct to CA125, for the detection of ovarian carcinoma in
patients with a pelvic mass, in Epithelial Ovarian Cancer and Healthy Women
Running Title: Osteopontin for detection of ovarian carcinoma
Malihe Hasanzadeh1, Hossein Ayatollahi2, Laya Shirinzadeh3, Soodabeh Shahidsales 4
1- Associate Professor of Gynecology, Women Health Research center, Ghaem
Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran
2- Assistant Professor of Pathology, Ghaem Hospital, MUMS, Mashhad, Iran
3- Gynecologist, MUMS, Mashhad, Iran
4- Assistant Professor of Radiation oncology , Cancer Research Center, Omid
Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad,
Iran
Corresponding Author: Soodabeh Shahidsales
Assistant professor of Radiation oncology, Cancer Research Center, MUMS, Mashhad, Iran,
Tel:+98-511-8461518
Email: [email protected]
1
Abstract
Objective: We have evaluated the diagnostic value of osteopontine serum level as in patients
with epithelial ovarian tumors.
Materials and Methods: Twenty five patients with ovarian cancer and 25 patients with pelvic
masses and 25 healthy women were enrolled in this study. Blood samples were taken from all
patients before surgery. Serum level of osteopontine was measured by Elisa method using human
OPN Enzyme Immuno assay kits. Finally osteopontine levels before surgery were compared
between groups.
Results: The mean age in patients with ovarian cancer, benign tumor and normal groups were
35.1, 32.4 and 31.6 years old respectively. Clinical symptom in nearly half of patients referred
with pathology of ovaries whether benign or malignant was abdominal pain. The average level of
CA125 and osteopontine in ovarian cancer patients was 1818.4 and 9.6 respectively. The average
level of CA125 and Osteopontine in epithelial ovarian tumors was with significantly higher than
the other groups. Significant linear relationship was detected between CA125 and Osteopontine.
Conclusion: Our study results supports that commitment use of osteopontine with CA125 could
be useful tool for early detection of ovarian cancer and due to their significant increase in the
among ovarian cancer patients.
Keywords: Ovarian cancer - Tumor marker - Osteopontine - CA125
2
Introduction
Ovarian cancer as common disease [1] will be diagnosed in women, with 15,280 deaths annually
[2]. The majority of these women will present with an adnexal mass, with or without evidence of
metastatic disease, yet only a small percentage of these masses represent the ovarian malignancy.
Ovarian carcinomas, tumors of the surface epithelium, are the most common form of ovarian
cancer [3].
Receiving surgical intervention for an ovarian cyst or pelvic mass is appropriate for the majority
of patients; however, for patients with a malignancy, optimal management of their disease would
be in a tertiary care center with multidisciplinary teams specializing in ovarian cancer [4–6].
Different screening strategies have been promoting for its detection and increasing the survival
rate at an early stage in a larger fraction of cases including with ultrasonography [7–9], serum
markers such as CA125 [10–12] or a combination of the two modalities where a rising serum
marker would trigger transvaginal sonography [13,10-12]. Among these methods, CA125 is
currently the only tumor marker to have a well-defined and validated role in the monitoring of
ovarian cancer (14).
Its decreasing levels are associated with response to therapy and its increasing levels with tumor
progression (13, 15). Although most of patients by normal CA125 (<35 units/ml) and no clinical
evidence of disease at the completion of primary chemotherapy are still have microscopic
residual cancer (16).
Recently, osteopontin (OPN) as secreted into glycoprotein form in human body fluids was shown
to exhibit 184-fold over-expression by comparing pooled ovarian cancer cell lines to healthy
women ovarian surface epithelium (17). Plasma levels of this protein have been shown to
3
correlate with disease status in several types of carcinoma (18, 19). The average levels of OPN
are significantly higher in epithelial ovarian cancer patients compared with healthy controls (20).
The efficacy of this putative biomarker has not been directly compared with CA125 in
monitoring patients with ovarian cancer and pelvic masses. The purpose of this study was to test
the hypothesis that OPN is a clinically useful adjunct to CA125 in detecting ovarian cancer in
three groups’ healthy women, patients with pelvic masses tumors and ovarian epithelial cancer.
4
Methods and Material
Study Participants
Human serum samples serum samples from 25 patients diagnosed with ovarian cancer as well as
25 women with pelvic masses (including benign cyst, leiomyoma and eccysis) and 25 healthy
women that referred for check up were provided by the Gynecological Oncology Department of
our Hospital. The studied inclusion criteria were epithelial ovarian cancer, not renal failure, age
between 15- 83 years old. The patients with non epithelial ovarian cancer, renal failure and not in
the considered age range were excluded. The written informed consent was obtained for each
subject. The study procedure was approved by Mashhad University of Medical sciences Ethics
committee. All the patients were promoted for the cytoreductive surgery of ovarian cancer and
considered as International Federation of Obstetricians and Gynaecologists (FIGO) Staging
System for Ovarian Cancer. All plasma samples were centrifuged at 2000 g at 4°C for 15 min.
The separated plasma was removed, aliquoted, and frozen at - 80°C for future analysis. OPN
serum level was assay according to Enzyme-Linked Immunosorbent Assay (ELZA) technique in
two studied groups.
Enzyme-Linked Immunosorbent Assay
Levels of OPN were measured in plasma samples with a solid-phase sandwich ELISA using a
commercially available kit (Code 17158, Immuno-Biological Laboratories, Quantikine ELISA
Kits, USA). Briefly, microplates were first precoated with antihuman OPN rabbit IgG [100 µl of
20 µg/ml in 0.1 M carbonate buffer (pH, 9.5)] and blocked with 1% BSA and 0.05% Tween 20.
Plasma and standard OPN samples were diluted with 1% BSA and 0.05% Tween 20 in PBS and
incubated for 1 h at 37°C. After seven washes with 0.05% Tween 20 in phosphate buffer,
horseradish peroxidase-labeled conjugated antihuman OPN (10A16) mouse monoclonal antibody
5
(100 µl of 2 ng/ml) was added and incubated for 30 min at 4°C. After nine washes, 100 µl of
tetramethyl benzidine buffer was added, and the signal was allowed to develop for 30 min at
room temperature. The reaction was stopped with 100 µl of 1 N sulfuric acid. The absorbance at
450 nm was measured by an automatic ELISA reader (Bio-Rad, Hercules, CA). The applied
cutoff value for CA125 was 35 units/ml as recommended by Bast et al. (21). A partial response
was defined by a decrease of at least 50% in the sum of the largest dimensions of tumors as
measured by computed tomographic scanning. A smaller decrease or any increase in tumor size
during primary chemotherapy was defined as a nonresponder. The duration of overall survival
was the interval between initiation of treatment (surgery or chemotherapy) and death.
Statistical Analysis
Basic descriptive statistical analysis and tables as represent by mean± standard deviation were
created using the SPSS version 16 (SPSS Inc., Chicago, IL). Nonparametric test Wilcoxon’s
signed rank-sum test and rock curve were used to perform paired comparisons between
pretreatment and postoperative OPN and CA125 measurements. Also χ2 or the Fisher’s exact test
was applied for dichotomous variables between studied groups. P value less than 0.05 was
considered as significant level for all statistical analysis.
6
Results
The study participants divided into three groups with 25 individuals into each group; patients
with ovarian epithelial cancer (Group I), pelvic masses or benign ovarian pathology forms
(Group II) and healthy women (Group III). Age of studied individuals were not significant
between the studied groups (P=0.17). As patients in group I, II and III had 35.1±14.48,
32.4±11.27 and 31.6±7.85 years old respectively. Pregnancy status was evaluated between three
studied groups (Table 1). However there was not any significant statistical difference between
the studied groups in aspect of pregnancy status but the number of parity, gravidity and abortion
in healthy women were lower than the remained studied groups (P>0.05). Table 2 presents that
there was significant difference between studied patients complaints frequency (P=0.031). As in
healthy group complaint about painful masses was lower than group I and II and they referred
due to vaginal secretion to clinic. Also there was significantly difference between consumption
of oral contraceptive pills and ovarian cancer (P<0.001), as the patients in Group I used oral
contraceptive pills more than the remained two groups. Also the difference between consumption
of hormone replacement therapy (HRT) within there studied groups were not meaningful
(P>0.05). Pathology evaluation of epithelial tumors in patients with no benign pathology
revealed the 15 cases (60%) with serous adenocarcinoma and 10 cases (40%) with mucinous
adenocarcinoma. Tumor stage of patients in group I presented in Figure 1. Most of the studied
patients were in the stage I according to FIGO staging.
OPN and CA125 levels of three studied groups are presented in Table 3. This table showed that
there was significant direct relation between CA 125 levels between three studied groups
(P=0.001). According to considered cutoff value 35 units/ml for CA125 as positive results, the
ROC curve (Figure 2) showed that the relation between CA125 and OPN was significant. The
7
analysis of multiple biomarkers in this study demonstrated that the addition of CA125
significantly elevates the sensitivity and specificity.
8
Discussion
The levels of several novel tumor markers including osteopontin, CA72-4, EGFR, ERBB2 (Her2), activin, and inhibin are elevated in patients with ovarian cancer [22–24]. Increased expression
levels of OPN along with elevated plasma levels of this marker have been reported in patients
with ovarian cancer. However, to obtain a detection sensitivity of 80%, the specificity declined
to 80% [20].
OPN may be a clinically useful adjunct to CA125 for the detecting ovarian cancer. After primary
surgery and platinum-based chemotherapy, most patients with advanced disease achieve a
complete clinical remission. Although most of these women (75–80%) will relapse and neither
consolidation therapy nor maintenance therapy has been shown for promoting their survival
duration.
CA125 is the most widely used serum biomarker among patients with ovarian cancer. Its utility
in determining response to treatment or as a marker for the detection of recurrent disease is well
established. The mean age of our studied individuals with ovarian cancer and benign tumors
were 35.1 and 31.6 years old respectively. Also most of tumors were in the stage I. In a similar
study, More et al [25] found that the mean age of studied patients with ovarian cancer and benign
tumors were 65 and 50 years old respectively. And most of the studied cases (38 cases from 48
individuals) were in the stage III. Lower age mean and lower tumor stage in our study
comparison of the previous study might be raveled two points including early incidence of
ovarian cancer and rapid diagnosis of these tumors in our society [26].
Evaluation of CA 125 and OPN status in patients with ovarian cancer then in patients with
pelvic masses or benign tumors were significantly higher than healthy individuals. These
findings also confirmed by previous studies [27-28]. However expression of biomarkers in tissue
may not reflect levels of antigen in serum as previous study [29] showed that in some cases, such
9
as CA125, serum assays may have greater sensitivity than would be predicted from tissue levels,
but the opposite trend might be observed. Although we had not assay the expression of these
tumor markers (CA 125 and OPN) in tissue but we found significantly direct relation between
changes in OPN and CA 125 status as specificity of these two tumors markers were increase
toward increasing their sensitivity.
Conclusion
The analysis of these two biomarkers in this study demonstrated that these two serum markers
set that is part of a treatment algorithm that may include pelvic imaging, similar to the risk of
malignancy index, would be useful for the triage of patients with pelvic masses and predicting
the presence of ovarian cancer in patients with an adnexal mass.
10
Acknowledgment
This study was supported by Mashhad University of Medical Sciences Grant under the medical
gynecologist student thesis.
11
References
1- Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CACancer J Clin
2004;54:8– 29.
2- Shih Ie M, Kurman RJ. Ovarian tumorigenesis: a proposed model based on
morphological and molecular genetic analysis. Am J Pathol 2004;164 (5):1511–8.
3- Carney ME, Lancaster JM, Ford C, Tsodikov A,Wiggins CL.Apopulationbased study of
patterns of care for ovarian cancer: who is seen by a gynecologic oncologist and who is
not? Gynecol Oncol 2002;84(1):36–42.
4- Paulsen T, Kjaerheim K, Kaern J, Tretli S, Trope C. Improved short-term survival for
advanced ovarian, tubal, and peritoneal cancer patients operated at teaching hospitals. Int
J Gynecol Cancer 2006;16(Suppl 1):11–7.
5- Engelen MJ, Kos HE, Willemse PH, Aalders JG, de Vries EG, Schaapveld M, et al.
Surgery by consultant gynecologic oncologists improves survival in patients with ovarian
carcinoma. Cancer 2006;106(3):589–98.
6- Giede KC, Kieser K, Dodge J, Rosen B. Who should operate on patients with ovarian
cancer? An evidence-based review. Gynecol Oncol 2005;99 (2):447–61.
7- Bourne TH, Campbell S, Reynolds KM, Whitehead Mi, Hampson J, Royston P, et al.
Screening for early familial ovarian cancer with transvaginal ultrasonography and colour
blood flow imaging. Br Med J 1993;306:1025 –9.
8- Van Nagell JR, Depriest PD, Reedy MB, Gallion HH, Ueland FR, Pavlik EJ, et al. The
efficacy of transvaginal sonographic screening in asymptomatic women at risk for
ovarian cancer. Gynecol Oncol 2000;77:350– 6.
12
9- Sato S, Yokoyama Y, Sakamota T, Futagami M, Saito Y. Usefulness of mass screening
for ovarian carcinoma using transvaginal ultrasonography. Cancer 2000;89:585– 8.
10- Einhorn N, Sjovall K, Knapp RC, Hall P, Scully RE, Bast Jr RC, et al. Prospective
evaluation of serum CA125 levels for early detection of ovarian cancer. Obstet Gynecol
1992;80:14–8.
11- Jacobs IJ, Skates S, Davies AP, Woolas RP, Jeyerajah A, Weidemann P, et al. Risk of
diagnosis of ovarian cancer after raised serum CA125 concentration: a prospective cohort
study. Br Med J 1996;313:1355– 8.
12- Jacobs I, Stabile I, Bridges J, Kemsley P, Reynolds C, Grudzinkas J, et al. Multimodal
approach to screening for ovarian cancer. Lancet 1999:268– 71.
13- Bast Jr RC, Siegal FP, Runowicz C, Klug TL, Zurawski Jr VR, Schonholz D, et al.
Elevation of serum CA125 prior to diagnosis of an epithelial ovarian carcinoma. Gynecol
Oncol 1985;22:115– 20.
14- Meyer T, Rustin GJ. Role of tumour markers in monitoring epithelial ovarian cancer. Br J
Cancer 2000;82:1535–8.
15- Rustin GJ, Nelstrop AE, Bentzen SM, Piccart MJ, Bertelsen K. Use of tumour markers in
monitoring the course of ovarian cancer. Ann Oncol 1999;10:21–7.
16- Wong C, Dai ZM, Lele SB, Natarajan N. Comparison of CA125 after three courses of
chemotherapy and results of second-look surgery. Eur J Gynaecol Oncol 2000;21:70–3.
17- Wong KK, Cheng RS, Mok SC. Identification of differentially expressed genes from
ovarian cancer cells by MICROMAX cDNA microarray system. Biotechniques
2001;30:670–5.
13
18- Hotte SJ, Winquist EW, Stitt L, Wilson SM, Chambers AF. Plasma osteopontin:
associations with survival and metastasis to bone in men with hormone-refractory
prostate carcinoma. Cancer (Phila) 2002;95: 506–12.
19- Le Q, Sutphin PD, Raychaudhuri S, et al. Identification of osteopontin as a prognostic
plasma marker for head and neck squamous cell carcinomas. Clin Cancer Res 2003;9:59–
67.
20- Kim JH, Skates SJ, Uede T, et al. Osteopontin as a potential diagnostic biomarker for
ovarian cancer. J Am Med Assoc 2002;287:1671–9.
21- Bast RC Jr, Klug TL, St John E, et al. A radioimmunoassay using a monoclonal antibody
to monitor the course of epithelial ovarian cancer, N Engl J Med 1983;309:883–7.
22- Lu KH, Patterson AP, Wang L, Marquez RT, Atkinson EN, Baggerly KA, et al. Selection
of potential markers for epithelial ovarian cancer with gene expression arrays and
recursive descent partition analysis. Clin Cancer Res 2004;10(10):3291–300.
23- Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the
HER-2/neu
proto-oncogene
in
human
breast
and
ovarian
cancer.
Science
1989;244(4905):707–12.
24- Robertson DM, Stephenson T, Pruysers E, Burger HG, McCloud P, Tsigos A, et al.
Inhibins/activins as diagnostic markers for ovarian cancer. Mol Cell Endocrinol
2002;191(1):97–103.
25- Mor G, Visintin I, Lai Y, Zhao H, Schwartz P, Rutherford T, Yue L, Bray-Ward P, Ward
DC. Serum protein markers for early detection of ovarian cancer. Proc Natl Acad Sci U
S A. 2005; 24;102(21):7677-82.
14
26- Ghaemmagame F, Hasanzadeh M. Evaluation of epidemiology of ovarian cancer in ValiAsr Hospital. Iranian J Gynecol Oncol. 2008;1(1):5-11.
27- Nakae M, Iwamoto I, Fujino T, Maehata Y, Togami S, Yoshinaga M, Douchi T.
Preoperative plasma osteopontin level as a biomarker complementary to carbohydrate
antigen 125 in predicting ovarian cancer. J Obstet Gynaecol Res. 2006;32(3):309-14.
28- Schorge JO, Drake RD, Lee H, Skates SJ, Rajanbabu R, Miller DS, Kim JH, Cramer
DW, Berkowitz RS, Mok SC. Osteopontin as an adjunct to CA125 in detecting recurrent
ovarian cancer. Clin Cancer Res. 2004:15;10(10):3474-8.
29- Skates SJ, Horick N, Yu Y, Xu F-J, Berchuck A, Havrilesky L, et al. Pre-operative
sensitivity and specificity for early stage ovarian cancer when combining CA125, CA
15.3, CA 72.4 and M-CSF using mixtures of multivariate normal distributions. J Clin
Oncol 2004;22:4059– 4066.
15