Download the place of biochemical testing

The Scientific Basis for Detecting Early Stage Epithelial Ovarian Carcinoma
D.A. Fishman, L. Cohen, N. Maihle* A. Baron* and M. Sharon Stack
National Ovarian Cancer Early Detection Program, Northwestern University Medical
School, Northwestern Memorial Hospital, Chicago, IL, U.S.A.
*Mayo Clinic, Rochester, MN, U.S.A.
In the United States ovarian carcinoma is the leading cause of death from gynecologic
malignancies and is the fifth most common female cancer. The incidence of ovarian
cancer has been steadily increasing over the past 10 years (overall lifetime risk 1.8%),
with the mean age of occurrence at 60 years.1 Annually approximately 23,300 women
will be newly diagnosed with ovarian cancer and 13,900 will die from this disease. 1
Despite significant improvement in surgical technology, critical care, and new
chemotherapeutic regimens, the overall 5-year survival for women with stage III/IV
epithelial ovarian carcinoma has remained constant (12%) over the past 30 years.
However, those women opportune enough to be diagnosed with disease confined to the
ovary (stage IA-IB) often require less morbid surgical intervention, may not require
adjuvant chemotherapy, and most importantly have an overall 5-year survival
approximating 90%.2 Therefore, short of an effective ovarian cancer specific therapy,
the early detection of early stage epithelial ovarian cancer (EOC) is essential in
decreasing the morbidity and mortality associated with the disease.
Who Are the Women at Increased Risk?
Ovarian cancer most commonly occurs in a sporadic fashion without any
antecedent history of disease in the family. Epidemiologic factors associated with
ovarian cancer include nulliparity, a personal or family history of colon or breast cancer,
an affected first-degree relative with ovarian cancer or a family history of a recognized
inherited malignancy syndrome, as well as a history of prolonged use of fertility drugs. 2,
3-5
However, nulliparity and fertility drug use are contested as risk factors because those
nulliparous women who used oral contraceptive pills actually have a decreased risk as
compared to those with unintended infertility.
6-7
Similarly the use of infertility
medications to achieve fertility does not appear to increase the risk for epithelial ovarian
cancer.
8
Approximately 5% of epithelial ovarian cancers are attributable to the
inheritance of highly penetrant mutations in the breast/ovarian cancer susceptibility
genes BRCA-1 and BRCA-2. 3-8
Ovarian cancer is a component of the autosomal dominant hereditary breastovarian cancer syndrome, and may be due to a mutation in either the BRCA1 or BRCA2
genes. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2
(6174delT) are common in the Ashkenazi Jewish population. Each mutation is
associated with an increased risk of ovarian cancer and it is expected that a significant
proportion of Jewish women with ovarian cancer will carry one of these mutations. 5
Individuals with a family history suggestive of an inherited malignancy syndrome
should be considered for genetic counseling and testing. Additionally the following
individuals should be offered formal genetic evaluation and testing as deemed
appropriate: women with a diagnosis of breast or epithelial ovarian cancer before age
50, women with a significant family history of breast cancer, especially premenopausal,
or ovarian cancer (one or more affected first-degree relatives), women with a blood
relative with a known BRCA-1 or BRCA-2 mutation, and Ashkenazi women who have
ovarian or premenopausal breast cancer or a family history of one or both diseases.
Since Narod et al, as well as other authors, reported that approximately 40% of the
Jewish women with epithelial ovarian cancer have been found to have a BRCA-1 or
2
BRCA-2 mutation, it is now our clinical practice to offer all Jewish women with ovarian
cancer genetic testing.
5
The benefits of genetic testing for the BRCA mutations include
identification of those individuals at increased risk for the development of breast or
ovarian cancer, individualizing surveillance measures that may enhance the early
detection of cancer, offering prophylactic surgery (mastectomy and/or bilateral salpingooophorectomy {BSO}) as well as knowledge of the potential for passing the mutation to
future generations. Genetic testing for mutations in these genes also has potential risks
such as adverse psychological effects, disruption of family dynamics, insurance or
employer discrimination.
Prior to initiation of genetic testing it is imperative to assess who is appropriate
for such testing, provide expert genetic counseling regarding the implications of genetic
testing, and obtain consent from the individual. The American Society of Clinical
Oncology (ASCO) as well as the ethical, legal, and social issues branch of the Human
Genome Project emphasize the importance of expert counseling for genetic testing.
Genetic counseling is the process of translating medical and scientific knowledge into
practical, understandable information for the patient. Unfortunately, there are no
accepted guidelines on genetic counseling for cancer and the quality of counseling
provided to patients can be quite variable. The optimal clinical management of
individuals who test positive for BRCA mutations is evolving. Therefore, ovarian cancer
risk assessment and testing are most effective when performed within the context of a
multidisciplinary team approach. The American College of Obstetricians and
Gynecologists Committee Opinion states “women with a documented familial history of
an inherited malignancy syndrome that increases their risk for the development of
3
ovarian cancer who do not wish to retain fertility may be offered a prophylactic BSO
after age 35.”3,9 It is our practice to offer, those select women who have completed
childbearing, and have received formal counseling, laparoscopic surgery to remove the
fallopian tubes and ovaries (BSO) only. Unfortunately, approximately 1% to 11% of
women continue to develop primary peritoneal carcinoma, a distinct pathologic entity
from epithelial ovarian carcinoma, after prophylactic BSO.
Women with a BRCA mutation not desirous of prophylactic surgery may be at a
significantly increased risk for the development of ovarian cancer (up to 40%) as well as
breast cancer (up to 60%), by age 70 years and therefore require more intensive clinical
surveillance.2-9 Oral contraceptive pills (OCPs) have been shown to decrease the risk by
approximately 11% per year of use with a maximum decrease approaching 70%. 7 It is
believed that OCPs decrease the risk secondary to a reduction in the number of
ovulatory cycles a woman experiences in her lifetime, yet the degree of protection is
significantly greater than the relative reduction in lifetime ovulations especially since
approximately 30% of women continue to ovulate despite OCP use. One means for the
enhanced protection has been attributed to the progestin effect on the surface
epithelium to induce apoptosis.10-12
Women at significantly increased risk for the development of ovarian cancer
require more intensive clinical surveillance and should consider participation in an IRB
approved research program. The National Ovarian Cancer Early Detection Program
(NOCEDP) as part of the National Cancer Institute’s Early Detection Research Network
(NCI-EDRN) is committed to the development of an effective means for the accurate
detection of early stage epithelial ovarian cancer.9,14-16 Only asymptomatic women with
4
normal gynecologic examinations deemed at increased risk for ovarian cancer are
eligible to participate in our IRB approved program. Eligibility includes those women with
at least one affected first degree relative with ovarian cancer; a personal history of
breast, ovarian, or colon cancer; one or more affected first and/or second degree
relatives with breast and/or ovarian cancer; inheritance of a BRCA mutation from an
affected family member; or membership within a recognized cancer syndrome such as
HNPCC. All women are seen every 6 months for formal genetic counseling, pelvic
examination by a board certified gynecologic oncologist, ultrasound examination by an
expert sonologist, and have blood drawn for investigational biomarker analyses.
Diagnostic Imaging: The Use of Ultrasound for Early Detection
Ultrasound has proven utility in detecting ovarian cancer in asymptomatic women with
advanced stage disease. However, its value for the detection of early stage disease has
yet to be determined. Multiple studies have reported the utility and limitations of
ultrasound for identifying Stage I epithelial ovarian carcinoma in asymptomatic women.
As expected due to the low annual prevalence of ovarian cancer within the general
population, a large number of women must be screened to identify a single ovarian
cancer. Van Nagell et al. reported they performed 57,214 scans on a general population
consisting of postmenopausal women and women over age 25 with a family history of
ovarian cancer.17 They identified 11 epithelial ovarian carcinomas, three granulosa cell
tumors, and three borderline ovarian tumors. A total of 11 Stage I tumors were
identified, five of which were epithelial ovarian carcinoma (EOC), three granulosa cell
tumors, and three borderline tumors. Of the Stage I tumors, three were abnormally
enlarged as appreciated on clinical examination (two borderline, one EOC) yet only 1 of
5
15 malignancies (excluding borderline tumors and palpable tumors) developed in
women under the age of 50 years. If one excludes granulosa cell and borderline tumors,
that are usually clinically detected confined to the ovary (Stage I) due to unknown
inherent biological properties, the sensitivity for EOC is approximately 31%. It is also
recognized that a negative ultrasound examination while clinically reassuring is
imperfect as women continued to develop advanced stage ovarian cancer within 6 to 12
months of a normal scan. Therefore a major limitation of transvaginal architectural
screening is that ovarian cancers can arise from normal sized, structurally normal
appearing ovaries despite advanced diagnostic imaging technology.
Recent technological advances such as 3-D volume acquisition and 3-D power
Doppler may have clinical utility in the early identification of abnormal vascularity and
ovarian architecture.14,15,18-20 3-D volume acquisition allows for careful evaluation of the
internal surfaces of cyst walls for intracystic papillations otherwise not appreciated by 2D technology. While the addition of 3-D power Doppler provides a new tool for
measuring the quality of ovarian vascularity, its clinical value for the early detection of
ovarian carcinoma has yet to be determined. In our diagnostic paradigm, gray-scale
ultrasound is used as a primary evaluation, however, many masses are not sufficiently
characteristic in echo-pattern to predict histology with certainty, which is similar to other
reports.14-15 Our practice is to describe a mass as cystic, multiloculated, complex, or
solid. If the echo-features are highly suggestive of a functional cyst, hemorrhagic cyst,
endometrioma, cystic teratoma, cystadenoma, or hydrosalpinx, it is noted on the report.
Previous experience in our program suggests that endometriomas and cystic teratomas
can often be correctly identified by their characteristic echo-patterns.14-15 We observed
6
that 80% of endometriomas and 70% of cystic teratomas were correctly identified by
ultrasound. However only 28% of cystadenomas and adenofibromas were sufficiently
characteristic in echo-pattern for a correct histologic ultrasound prediction. Many
cystadenomas and cystadenofibromas referred to our program were architecturally
unusual in that they presented with a highly complex echo pattern. It is also important to
note that there can be overlap between the echo-features of certain benign histologies
and frank malignancies. For example, we have found that the gray-scale patterns of
granulosa cell tumors can resemble those of endometriomas, yet adenofibromas, cystic
teratomas, LMP tumors, and papillary serous ovarian carcinoma can contain areas of
dense echogenicity with posterior shadowing.
The addition of Doppler examination is helpful in this regard due to the absence
of vascular flow within the central regions of endometriotic cysts and the echogenic
portions of most cystic teratomas. It is not unexpected that 2D TVS identified 100% of
the malignant adnexal masses, because they were enlarged and complex in echoarchitecture. The published literature has found that 2D TVS is 85% to 100% sensitive
for identifying adnexal masses as malignant.14-15,18-20 Although 3D TVS with rendering
improves visualization of the internal capsule wall and intracystic papillations, it is the
addition of PD3D that we found most helpful. 3D TVS did not change the morphologic
score (viz, cystic, multiloculated, complex, or solid), compared to the 2D TVS, however
the rendering of the internal aspect of cystic masses can yielded high detail of internal
excrescences previously identified on 2D TVS (Figures 1 and 2). It is more useful,
however, in ruling out excrescences rather than in their identification. In our experience
three-dimensional power Doppler imaging better defines the morphologic and vascular
7
characteristics of ovarian lesions resulting in a significant improvement in specificity
(54% to 75%) for ovarian cancer detection.15 This improved diagnostic accuracy may
promote improved patient care by separating complex benign masses from ovarian
cancer, therefore facilitating appropriate physician referral.
We continue to evaluate the utility of new ultrasound technologies in evaluating
asymptomatic women deemed at significantly increased risk for the development of
ovarian carcinoma. Premenopausal scans are routinely performed transvaginally and
postmenopausal scans either transvaginally or transabdominally. Doppler and 3-D
studies are performed only if an adnexal mass is identified. Masses are graded as
cystic, multiloculated, complex, or solid. An overall impression of malignancy risk is
assigned to adnexal masses based on morphologic appearance and the presence or
absence of central vascularity. Simple cysts and premenopausal hemorrhagic-like cysts
are rescanned at 6- to 8-week intervals. Since several recent publications have
confirmed a less than 1% malignancy rate in simple menopausal cystic masses
measuring less than 5 cm, it is our practice to conservatively follow these lesions unless
architectural changes are demonstrated.14-15,18-20
Since 1996, within the NOCEDP, over 4000 women have been evaluated by
approximately 11,500 scans. The average age of the women was 46 years.
Visualization of both ovaries was noted in 98% of premenopausal and in 94% of
postmenopausal women. Recall rates at less than the routine 6-month interval were
0.4% in the premenopausal and 0.3% in postmenopausal women. Approximately 100
women with persistent adnexal masses were identified. Fourteen women with simple
cysts greater than 5 cm were treated with percutaneous drainage with benign cytology.
8
Thirty women received operative intervention with benign ovarian pathologies that were
scored at low risk for malignancy on ultrasound interpretation. Thirty-six menopausal
women with stable simple cysts, hydrosalpinges, or fluid-filled adhesive disease have
been followed expectantly. A total of 47 invasive surgical procedures were performed
for 38 benign ovarian tumors and 9 gynecologic malignancies. All cancers were
detected in asymptomatic women who had previous normal US and physical
examination 12 and 6 months prior to diagnosis. The detected malignancies were
fallopian tube carcinoma (2) stage IIIC, grade 3; primary peritoneal carcinoma (3) Stage
IIIA; epithelial ovarian carcinoma (2) Stage IIIA and IIIB grade 3; and uterine carcinoma
(2) Stage IA grade 1. Twenty-seven primary and 7 recurrent breast cancers were also
detected. Approximately 110 women with a genetic predisposition (BRCA+) for ovarian
cancer have had prophylactic bilateral salpingo-oophorectomies (BSO) with 30%
demonstrating atypical hyperplasia, one woman was surprisingly found to have Stage III
B primary peritoneal carcinoma, and one woman was found to have as expected a
Stage 1A borderline tumor (mucinous-type). Our ongoing study suggests the limited
value of diagnostic ultrasound as an independent primary screening tool for the
detection of early stage EOC in asymptomatic high-risk women.
The Scientific Basis for Early Detection
An understanding of the biochemical and molecular biology of ovarian
carcinogenesis, invasion, and metastasis is critical if we are to identify asymptomatic
women at increased risk for ovarian cancer. This knowledge will enable the
development of accurate serum/plasma assays for the early detection of asymptomatic
early stage EOC, chemoprevention programs, and ovarian cancer specific therapies
9
that shift the current paradigm from nonspecific antiproliferative therapy to
chemotherapies that prevent invasion and metastatic dissemination.
The common epithelial ovarian cancers, which represent approximately 90% of
all ovarian cancers, arise from the 2-cell epithelial covering of the external surface of the
ovary. The neoplastic transition of normal ovarian surface epithelium requires a complex
cascade of interrelated genetic, molecular, and biochemical events. Ovarian cancers
accumulate genetic aberrations that affect cell cycle control, apoptosis, adhesion,
angiogenesis, transmembrane signaling, DNA repair, and genomic stability. Specific
genetic
aberrations
found
in
ovarian
cancers
include
amplification
and/or
overexpression of ErbB2,21 and PIK3CA.22 Some of these genes have been identified in
ovarian cancers using comparative genomic hybridization (CGH) and analysis of allelic
imbalance. Those regions of recurrent abnormality may encode genes that contribute to
ovarian carcinoma progression when differentially expressed as a result of abnormal
copy number or mutation.21-25
The processes of cellular adhesion, migration, extracellular matrix degradation,
directed invasion into host parenchyma, proliferation, and neovascularization are
influenced by numerous regulatory molecules found within the tumor microenvironment,
which include epidermal growth factor (EGF) and receptors (EGFR/ErbB), urinary-type
plasminogen (uPA) and receptor (uPAR), matrix metalloproteinases (MMP-2, MMP-9),
vascular endothelial growth factor (VEGF), cytokines (such as IL-8), and biologically
active lysophospholipids such as lysophosphatidic acid (LPA).9,13,26-36 Many of these
factors regulate the expression of each other and initiate a cascade of extracellular and
intracellular signaling that stimulate hematogenous, lymphatic, and intraperitoneal
10
metastatic dissemination. Specifically, LPA, EGFR, VEGF, IL-8 can act either in a
paracrine and autocrine mode, affect gene and protein expression, and upregulate
proteinase (uPA, MMP) expression and activation.
Serum/Plasma Tumor Markers
CA125
The ability to detect early stage epithelial ovarian cancer by a simple blood test
has yet to be achieved despite tremendous clinical and scientific effort. The most
studied serum ovarian tumor marker is CA125, an ovarian cancer cell surfaceassociated high molecular weight glycoprotein that exists in multiple forms ranging from
220 to greater than 1000 kDa, and is expressed in 80% of nonmucinous epithelial
ovarian cancers.2,23 Overall, more than 80% of women with advanced ovarian cancer
will have an elevated CA125 level (greater than 35 u/mL), yet the test is not useful in the
detection of early stage disease.2,23,37 The NIH Consensus Statement specified that
CA125 should not be used as a screening test in the general or high-risk population
since an elevated value accurately detects malignancy in less than 3% of women. 38-39
CA125 in conjunction with ultrasound is under clinical evaluation in the United
States and England as a method of reducing mortality through early detection.
40-42
These longitudinal studies will evaluate the ability of both US and CA125 to detect early
ovarian cancers in the general population by comparing the results of screening.
Previously Jacobs et al reported that the use of CA125 and US led to the identification
of 16 ovarian cancers in the screening group, yet 11 of 16 cancers were late stage
(III/IV). CA125 has a positive predictive value of less than 10% as a single marker. The
addition of ultrasound screening to CA125 measurement has improved the positive
11
predictive value to approximately 20% range.43-46 Three-dimensional power Doppler
ultrasound improves the diagnostic accuracy for ovarian cancer prediction.15
Unfortunately neither CA125 nor ultrasound has as yet been proven to be sensitive nor
specific enough to accurately detect stage I ovarian cancer.42
The Tumor Microenvironment- Growth Factors
By individualizing the components of the metastatic cascade into tumor cell
adhesion (cadherins and integrins), migration, matrix degradation (MMP’s and uPA),
invasion and proliferation, a number of investigators have identified new serum and
plasma biomarkers, such as lysophospholipids, and other biologically relevant markers
that may be useful in the early detection of early stage epithelial ovarian cancer. The
interrelationship between specific growth factors (lysophospholipids, epidermal growth
factor(s)), 1 integrin ligation, MMPs, and uPA play critical roles in the metastatic
dissemination of ovarian cancer.9,13,16,28,34,36,37,47-63 The clinical relevance of these
proteins and lipids by regulating ovarian carcinogenesis may enable the detection of
early stage disease and the development of new ovarian cancer specific therapies.
Lysophosphatidic Acid
Recent attention has focused on phospholipids, such as lysophosphatidic acid
(LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC), as
potential serum biomarkers for the early detection of epithelial ovarian carcinoma.
These phospholipids function extracellularly to activate cells through specific cell
membrane receptors and have been found to induce proliferation of ovarian and breast
cancer cells. Mills et al reported that LPA induces a rapid and transient increase in
cytosolic free calcium, and stimulates tyrosine phosphorylation, including mitogen12
activated protein kinase activation.47 In ovarian cancer cells but not in normal ovarian
surface epithelial cells, LPA increases cell proliferation, cell survival, resistance to
cisplatin, and the production of vascular endothelial growth factor (VEGF), interleukin 8,
urokinase plasminogen activator (uPA), the urokinase plasminogen activator receptor
(uPAR) and of LPA itself. The Edg2, Edg4 and Edg7 members of the endothelial
differentiation gene (Edg) family of G protein-coupled receptor family have been
proposed to mediate LPA signaling in mammalian cells. Normal ovarian epithelial cells
and ovarian cancer cell lines have variable Edg2 mRNA and protein levels. Edg4
protein and mRNA levels are modestly elevated in ovarian cancer cells, whereas, Edg7
mRNA levels are markedly elevated suggesting that Edg4 and Edg7 may contribute to
the deleterious effects of LPA in ovarian cancer. Indeed, selective agonists of Edg7
induce cell activation, proliferation, increased survival, and uPA production in ovarian
cancer cells further implicating Edg7 in the pathophysiology of ovarian cancer.
Activation of the Edg2 LPA receptor on ovarian cancer cells may induce cells to
undergo apoptosis rather than to proliferate. Thus, the development of agonists and
antagonists for specific LPA receptors may alter proliferation, apoptosis or the response
to therapy. As almost half of all current drugs are receptor selective agonists or
antagonists for G protein coupled receptors, the LPA receptor family represents a highly
“drugable” target. Mills et al are using expression of specific LPA receptors in yeast,
insect and mammalian model systems to determine the structure activity relationships
for the different Edg receptors as well as to screen for potential agonists and
antagonists of specific LPA receptors. These agonists and antagonists can be used as
13
lead compounds for the development of molecular therapeutics aimed at ovarian
cancer.
Certain phospholipids appear to increase urinary type plasminogen activator
(uPA) and matrix metalloproteinase (MMP) expression and activation. 48 Therefore, it
appears that phospholipids may play a significant role in ovarian metastasis. We have
found that LPA treatment of ovarian cancer cells increases membrane fluidity, cellular
adhesion to type I collagen and 1 integrin expression. A significant upregulation of
MMP-dependent proMMP-2 activation was also observed in LPA-treated cells, leading
to enhanced pericellular MMP activity. As a result of increased MMP activity, haptotactic
and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement
membrane are enhanced. These data suggested that LPA contributes to metastatic
dissemination of ovarian cancer cells via upregulation of MMP activity and subsequent
downstream changes in MMP-dependent migratory and invasive behavior.47,48,49
Previous data has shown that invasion and metastasis of ovarian cancer cells
are facilitated by the presence of the lipid mitogen lysophosphatidic acid (LPA). LPA
levels are elevated in the majority of ovarian cancer patients, and this lipid, through an
interaction with a family of G-protein-coupled receptors, stimulates the activity of various
intracellular signaling molecules including focal adhesion kinases and mitogen-activated
protein kinases. The effects of LPA-induced signaling in tumor cells include cellular
proliferation, survival, invasion, and the upregulation of proteolytic enzymes. The
gelatinolytic enzyme matrix metalloproteinase-2 (MMP-2) is implicated in ovarian cancer
cell invasion, and previous data from our lab has demonstrated that LPA induces pericellular MMP-2 activity. Using the DOV13 ovarian cancer cell line suggest a major role
14
of PI3-kinase in LPA-induced MMP-2 activation, with a contribution from p38 MAPK.
Furthermore, the obstruction of these pathways results in partial inhibition of LPAinduced cellular invasion, supporting a role for MMP-2 activity in this process. PI3kinase also appears to be necessary for LPA-stimulated urokinase plasminogen
activator (uPA) activity in DOV13 cells. Inhibition of p44/p42 MAPK slightly decreases
LPA-stimulated MMP-2 activation, and inhibits LPA-stimulated uPA activity and
invasion. As MMP-2 activation is dependent upon both MT1-MMP and 1 integrin
clustering, we are also investigated the effects of LPA on the expression and
localization of these molecules, as well as determining the signaling pathways through
which LPA exerts these effects. We have found that LPA treatment results in increased
production and cell surface localization of 1 integrin. The results of these studies will
aid in understanding the role of LPA in ovarian cancer metastasis, and may provide
targets for therapeutic intervention to prevent the activation of MMP-2 and thus inhibit
cellular invasion.
Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with
ovarian carcinoma including 90% of patients with stage I disease, suggesting that LPA
may promote early events in ovarian carcinoma dissemination.49 Expression of matrix
metalloproteinases (MMPs) is also upregulated in ovarian cancer tissues and ascites,
and numerous studies have provided evidence for a direct role of MMPs in
intraperitoneal invasion and metastasis. Using three-dimensional type I collagen
cultures or immobilized β1 integrin subunit-specific antibodies, we previously
demonstrated that β1 integrin clustering promotes activation of proMMP-2 and
processing of membrane type 1 (MT1-)-MMP in ovarian cancer cells (Ellerbroek et al.,
15
Cancer Research 59:1635, 1999). We evaluated the effect of LPA on MMP expression
and invasive activity. Treatment of ovarian cancer cells with pathophysiological levels of
LPA increased cellular adhesion to type I collagen and β1 integrin expression. A
significant upregulation of MMP-dependent proMMP-2 activation was observed in LPAtreated cells, leading to enhanced pericellular MMP activity. As a result of increased
MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion
of a synthetic basement membrane were enhanced. These data indicate that LPA
contributes to metastatic dissemination of ovarian cancer cells via upregulation of MMP
activity and subsequent downstream changes in MMP-dependent migratory and
invasive behavior.
The clinical application of LPA in ovarian cancer detection was initially reported
by Xu et al. Women with ovarian cancer had elevated plasma levels of LPA as
compared to healthy controls and most importantly elevated levels were observed in 9
of 10 women with stage I disease.49 Our ongoing multi-institutional international study
has also found elevated levels of LPA in the plasma and serum of women with
advanced and early stage epithelial ovarian cancer despite normal CA125 values.
Atairgin
Corporation
has
investigated
a
series
of
enzymatic
and
liquid
chromatography/mass spectrometry (LC-MS/MS) approaches to the development of a
commercial OvCA assay. The most recent approach utilizes concentrations of LPA and
LPI molecular species determined by LC-MS/MS and a proprietary algorithm to predict
OvCA. Serum samples were extracted using a semi-automated, proprietary, organic
extraction and filtration method. Extracts were analyzed by LC-MS/MS; the run time of
each sample was within commercially feasible limits (i.e., less than 6 minutes). The
16
concentrations of 6 LPA molecular species and 5 LPI molecular species, as well as total
LPA and total LPI, were determined. ROC curves were constructed to describe the
sensitivity and specificity of total LPA or total LPI concentrations to detect OvCA. At
concentration cut-off levels that yielded 100% specificity, the sensitivity of the total LPA assay
was 40% and the sensitivity of the total LPI assay was 60%. These results suggest that
plasma/serum LPA levels may be of value in the early detection of stage disease.
Concentrations of LPA and LPI molecular species, as well as total concentrations of LPA and
LPI, were inputs into the model. The model output was a prediction of OvCA or not OvCA. The
model output was translated into sensitivity results by determining the number of correct OvCA
predictions in the group with OvCA. Specificity was determined by the number of “not OvCA”
predictions in the group of benign gynecological condition patients and age-matched controls.
The sensitivity and specificity of the LPA model was 92% and 76%, respectively. The LPI model
sensitivity was 83% and specificity was 94%. The LPA and LPI model achieved 100% sensitivity
and specificity. A commercially feasible LC-MS/MS assay has been developed that measures
LPA and LPI molecular species in serum to diagnose ovarian cancer. The assay performance
was enhanced by using proprietary multivariate algorithms to predict the presence or absence
of ovarian cancer.
EGF/ErbB Receptor Family
While physical examination, ultrasonography, and serum CA-125 levels aid in
distinguishing EOC from benign adnexal masses, they are not truly diagnostic. Moreover, no
definitive screening test for early stage EOC has been developed yet. Risk factors, and screening
and preoperative diagnostic tests for this disease, therefore, are needed urgently.
Overexpression of ErbB1, ErbB2 and ErbB3 is common in human ovarian carcinomaderived cell lines and tumors, and this growth factor receptor family is thought to play a
17
critical
role
in
tumor
etiology
and
progression.28,52-53,64
Furthermore,
ErbB1
overexpression is associated with disease recurrence and decreased survival in ovarian
cancer patient. Several studies demonstrate that normal and malignant cells synthesize
soluble forms of ErbB1 receptors in addition to the transmembrane form of this
molecule.28,52-53,64 Maihle and Baron et al recently discovered a 3.0 kb alternative transcript of
the EGFR gene that encodes a 110-kDa glycoprotein, and have shown that women with
advanced stage EOC have significantly lower serum p110 sErbB1 concentrations than healthy
women. They have demonstrated that serum p110 sErbB1 is a product of the 3.0 kb c-ERBB1
alternate transcript and that preoperative serum p110 sErbB1 concentrations of 225 women with
stage I-IV EOC are substantially lower than those of 144 healthy women (P < 0.0001).
Moreover, serum p110 sErbB1 concentrations differ with respect to stage and grade, being
moderately lower in stage III/IV versus stage I/II EOC patients (P = 0.0237), in grade II versus I
tumors (P = 0.0076), and in grade III versus I tumors (P = 0.0227). Dichotomization of serum
p110 sErbB1 concentrations into high ( median) and low ( median) levels reveals that women
with low levels have a significantly greater risk of having EOC than women with high levels
(OR = 16.4; 95% CI: 9.4, 28.8). Further stratification of p110 sErbB1 concentrations into
quartiles revealed that women with p110 sErbB1 concentrations between ND and 272 fmol/ml
(1st quartile), 273 and 1,418 fmol/ml (2nd quartile), and 1,419 and 5,731 fmol/ml (3rd quartile)
have a 282.6 (95% CI: 61.3, 1302.1), 26.0 (95% CI: 11.7, 57.7), and 7.1 (95% CI: 3.4, 14.9)
higher odds, respectively, of having EOC compare to women with p110 sErbB1 concentrations
between 5,732 and 82,436 fmol/ml (4th quartile). ROC analyses indicate an AUC of 0.88 for all
women, 0.91 for premenopausal women, and 0.81 for postmenopausal women. Sensitivity and
specificity for all EOC patients versus all healthy women are 55.6% and 94.4%, respectively. For
18
premenopausal EOC patients versus premenopausal healthy women, sensitivity and specificity
are 60.0% and 98.8%, respectively; whereas for postmenopausal EOC patients versus
postmenopausal healthy women, sensitivity and specificity are 53.6% and 88.1%, respectively.
Maihle et al. found that serum p110 sErbB1 levels are significantly lower in women with
stage III or IV disease prior to and shortly after cytoreductive staging laparotomy, in
comparison to healthy women of similar age.28,52-53,64 Serum sErbB1 levels also
increase after cytoreductive surgery, and decreasing serum sErbB1 levels may predict
disease recurrence. These studies demonstrate that altered and/or changing serum
p110 sErbB1 levels may provide important diagnostic and/or prognostic information
useful for the patient management. Preoperative serum sErbB1 levels in women with
Stage III/IV carcinoma are significantly lower than serum sErbB1 levels in healthy
women of similar ages, suggesting that epithelial ovarian tumors affect circulating
sErbB1 levels and implies that low sErbB1 levels may be useful as diagnostic
biomarkers for epithelial ovarian cancer.
The Tumor Microenvironment- Enzymes
Plasminogen Activators, Matrix Metalloproteinases and Integrins
Predominant among the proteinases produced by invading tumor cells are
enzymes in the plasminogen activator (PA) and matrix metalloproteinase (MMP)
families.13,29-31,65-69 Plasminogen activators are serine proteinases, which catalyze the
conversion of the plasma zymogen plasminogen to the active proteinase plasmin.
Plasmin is a broad spectrum serine proteinase capable of degrading numerous
extracellular matrix and matrix-associated proteins including fibrin, laminin, fibronectin
and vitronectin. Since a large reservoir of potential proteolytic activity is available in the
19
form of plasminogen, production of PAs provides a mechanism by which tumor cell
degradation of the extracellular matrix is amplified. Although the invasion and spread of
hematogenously metastasizing tumors is known to be mediated via the action of
extracellular matrix-degrading proteinases the role of proteolysis in intraperitoneal
metastasis remains unclear.13,29-31,65-69
Proteolytic activity may be required for disruption of the mesothelial cell layer, during
invasion of the implanted tumor through the submesothelial basement membrane into
the visceral organ stroma, and for subsequent tumor-mediated angiogenesis. MMPs are
a family of zinc-dependent metalloendopeptidases, which function in the degradation of
collagen, gelatin and other extracellular matrix macromolecules. Expression of
gelatinolytic MMPs such as MMP-2 (gelatinase A, 72 kDa type IV collagenase) and
MMP-9 (gelatinase B, 92 kDa type IV collagenase) has been linked to enhanced tumor
invasion in numerous model systems.13-18,32 Established cultures of epithelial ovarian
carcinoma cells secrete elevated quantities of urinary-type PA (u-PA, urokinase),
coexpress the cellular u-PA receptor, and overexpress gelatinolytic MMPs relative to
normal ovarian epithelium.13,29-31,65-69 However gelatinolytic MMPs including MMP-2 and
MMP-9 were secreted in large amounts by primary cells, regardless of original anatomic
source of the culture. Furthermore, whereas u-PA production was regulated in part by
cellular growth substratum, MMP secretion was not diminished by the cellular adhesive
microenvironment. Together these data suggest that initial production of MMP(s) may
mediate the invasive behavior of
epithelial ovarian carcinoma cells. Matrix
metalloproteinase (MMP) production is upregulated in ovarian cancer and has been
found to contribute to tumor invasion.
20
Ovarian carcinogenesis and metastasis require a complex cascade of interrelated
genetic, molecular, and biochemical events. Ovarian cancer causes morbidity and
mortality due to the malignant epithelial cells ability to adhere to distant sites foreign to
the ovary which allow for migration, proteolytic degradation of the extracellular matrix,
tumor
cell
invasion
into
host
tissues,
proliferation,
and
ultimately
tumor
neovascularization. The acquisition of a vascular supply from pre-existing host venules
stimulates exponential tumor growth and exfoliation with further hematogenous and
lymphatic dissemination. Vascular endothelial growth factor (VEGF) is a potent
angiogenic factor overexpressed by ovarian cancer cells that mediates angiogenesis
and ascites formation yet its specific function on ovarian metastasis is unknown.In our
study, we evaluated the specific efects of VEGF on the individual components of the
ovarian metastatic cascade. We utilized the established human ovarian cancer cell line,
DOV-13, to examine the effects of VEGF121, a biologically active form of VEGF which
has 121 amino acids, on the regulation of adhesion, proliferation,proteinase expression
and activation, cellular migration, and in vitro invasion.The DOV-13 cells were treated
with recombinant VEGF121 (rVEGF121) within the concentration range of 0-100ng/ml.
VEGF had no effect on tumor cell proliferation (Promega assay), or tumor cell adhesion
to extracellular matrix proteins (laminin, collagens I and IV, fibronectin, vitronectin, or
bovine serum albumin). Cellular migration (colloidal gold tract assay) was significantly
increased by exposure to VEGF as compared to control. VEGF treatment significantly
increased DOV13 cell invasion through an artificial basement membrane (Matrigel
invasion assay), with a plateau observed at 25ng/ml. Zymographic, Western, and RTRTPCR analyses demonstrated no increase in expression or activation of the matrix
21
metalloproteinase, MMP-2. In contrast, urinary-type plasminogen activator (uPA) activity
and expression increased approximately two-fold after VEGF treatment. Addition of anticatalytic uPA antibody inhibited VEGF-induced cellular invasion in a dose-dependent
fashion. The generic MMP inhibitor(GM6001) alone decreased DOV13 invasion yet in
the presence of VEGF the inhibition was not as significant as that observed with the
uPA-antibody. In conclusion, uPA activity seems to play a more important role than
MMPs in VEGF-mediated cellular invasion of human ovarian cancer DOV-13 cells. The
suspected signal transduction pathways involved in VEGF-mediated invasion are
currently under investigation.
Lysophosphatidic acid (LPA), or 1-acyl-glycerol 3-phosphate, is a phosphoplipid
which signals through G protein-coupled receptors to induce a wide repertoire of
responses in multiple cell types. LPA levels are elevated in the serum and ascitic fluid of
ovarian cancer patients, and may, in conjunction with other growth factors, mediate the
peritoneal spread of ovarian cancer. Furthermore, LPA activates the small GTPase,
Rho, which has been demonstrated to play a pivotal part in cancer cell migration and
invasion. To determine if LPA activation of Rho-regulated pathways is required for
ovarian cancer cell migration and invasion, we investigated the effects of inhibiting Rhokinase (ROCK) on LPA-mediated changes in actin cytoskeleton morphology, proteinase
activation, haptotactic migration, and cellular invasion. LPA treatment of DOV13 cells
results in a reduction in stress fibers but an increase in peripheral actin bundles,
suggesting that cells are contracting. This is consistent with our observation that cells
appear to round up and lose adhesion upon LPA treatment. Since Rho GTPases are
thought to regulate stress fiber formation, we reasoned that inhibiting ROCK would
22
further enhance LPA-induced stress fiber disassembly and consequently, adhesion.
These events should, consequently, have an impact on cell migration and invasion.
Phalloidin staining of actin filaments in cells treated with both LPA and Y-27632, a
specific inhibitor of ROCK, revealed that compromising ROCK function augments the
loss of stress fibers observed with LPA treatment. Interestingly, the inhibition of ROCK
with Y-27632 enhances LPA-mediated activation of the metalloproteinase, MMP-2, but
has no significant effect on uPA activity. Since previous studies have shown that LPA
increases haptotactic migration and invasion through a synthetic matrix, we wanted to
determine the effects of Y-27632 treatment on cell migration and invasion. Y-27632
treatment alone does not significantly affect haptotactic migration on colloidal goldcoated coverslips, but in the presence of both Y-27632 and LPA, migration is reduced
close to basal levels. Furthermore, inhibition of ROCK abrogates LPA-mediated
invasion through a complex matrix, indicating that ROCK activity is required for LPAstimulated invasion, perhaps via its role in mediating cell migration. Taken together,
these results suggest that inhibition of ROCK blocks LPA-mediated migration and
invasion, but enhances LPA-stimulated proteinase activation. This enhanced MMP-2
activation, however, does not promote cell invasion above basal levels.
Ovarian tumor growth and metastasis requires a coordinated series of events that
include cellular adhesion, migration, extracellular matrix degradation, invasion into host
parenchyma, proliferation, and neovascularization that are influenced by numerous
regulatory molecules, such as lysophosphatidic acid (LPA) epidermal growth factor
(EGF) and receptors (EGF-R/ErbB), urinary-type plasminogen activator (uPA) and
receptor (uPAR), and matrix metalloproteinases (MMP). Many stimulatory factors such
as lysophosphatidic acid, matrix metalloproteinases, and IL-8 have been detected at
elevated levels in malignant ascites from ovarian cancer patients. In melanoma cells IL8 up-regulates MMP 2 expression, activity and invasiveness, and IL-8 expression
directly correlates with metastatic potential. We presently are evaluating the effects of
IL-8 on the individual components of the ovarian metastatic cascade. In vitro, ovarian
23
cancer cells grown under hypoxic or acidic conditions or exposed to
lysophosphosphatidic acid (LPA) increased IL-8 gene expression. We found that IL-8
treatment did not have any effect on cellular proliferation (Promega assay) or uPA
activity (colorimetric assay) in ovarian carcinoma cells (DOV13). Acidic pH was found to
have no effect on MMP or uPA activity and expression. However, exposure to IL-8
significantly increased the amount of pro- MMP-2 (72kDa), which was confirmed by
Western blotting and ELISA. IL-8 treatment alone did not up-regulate MMP-2 activity.
We previously reported that LPA treated ovarian carcinoma cells (DOV13) resulted in a
statistically significant increase in the active form of MMP-2 (62kDa). A synergistic effect
was observed when cells were treated with both LPA and IL-8, with the combination
resulting in increased MMP-2 activation as determined by gelatin zymography. Cellular
migration was unaffected by IL-8 as quantified by a modification of the Albrecht- Buehler
colloidal gold assay. However cellular invasion through a Matrigel membrane was
significantly stimulated by IL-8. Our findings indicate that IL-8 increases pro-MMP-2
expression and stimulates Matrigel invasion. Since LPA and IL-8 coexist within the
ovarian carcinoma microenvironent with critical roles in metastatic dissemination further
studies will be aimed at understanding the mechanism of IL-8-induced MMP-2
expression and cellular invasion.
New Technologies
Proteomics
Low molecular weight serum protein profiling may reflect the pathologic state of
organs and aid in the early detection of cancer. Matrix-assisted laser desorption and
ionization time-of-flight (MALDI-TOF) and surface-enhanced laser desorption and
ionization time-of-flight (SELDI-TOF) mass spectroscopy can profile proteins in this
range.70-72 These profiles can contain thousands of data points, necessitating
sophisticated analytical tools. Bioinformatics has been employed to study physiological
outcomes and cluster gene microarray transcript profiles.73-76 Emanuel Petricoin MD
(FDA) and Lance Liotta MD PhD (NCI) et al have linked SELDI-TOF spectral analysis
with a high order analytical approach to define an optimal discriminatory proteomic
pattern. A bioinformatics tool was developed and used to identify proteomic patterns in
serum that distinguish neoplastic from non-neoplastic disease within the ovary. The
discriminatory pattern was developed from spectra from a training set of women with
24
ovarian cancer and applied to a blinded series of samples from unaffected women,
those with early and late stage ovarian cancer, and those with benign disorders. This
pattern identified the presence of ovarian cancer in affected women using less than a
drop of blood.
In their Lancet report Petricoin and Liotta found that ovarian cancers (50/50) were correctly
classified and distinguished from non-malignant disorders.77 All stage I cancers (cancer
confined to the ovary) were correctly classified. The PPV in their validation set was 94 percent
(95% CI: 84%-99%). By comparison, the PPV of CA125, the most widely used serum marker
for ovarian cancer was 34 percent in this same validation set. The algorithm identified a cluster
pattern that, in the training set, completely segregated cancer from unaffected. The
discriminatory pattern correctly identified all 50 ovarian cancer cases in the validation set,
including all 18 stage I cases. Of the 66 cases of nonmalignant disease, 63 were recognized
as not cancer. This yielded a sensitivity of 100 percent (95% Confidence Interval [CI], 93% 100%), specificity of 95 percent (95% CI, 87%-99%), and positive predictive value (PPV) of 94
percent (95% CI, 84% - 99%) compared with a positive predictive value of 34 percent for CA125 in this same cohort.(77) These findings have led to the recently initiated prospective
population-based evaluation of proteomic pattern technology, as a screening tool for ovarian
cancer in both the high-risk and general population.
The Ovarian Pap Test
In 1984, Gusberg and Deligdisch described the histologic evaluation of an ovary that
revealed significant epithelial abnormalities that did not qualify as malignant nor low
malignant potential and thus introduced “ovarian dysplasia.” 78 Ovarian dysplasia has
been further characterized by morphometric methods revealing specific changes in the
25
architecture
and
cytologic
characteristics
of
ovarian
surface
epithelium. 78-81
Retrospective analysis of ovarian tissues from women with stage I carcinoma assessed
the presence of cellular and nuclear atypia in noncancerous tissue adjacent to the
primary tumor. Atypia was more common in the cancer patients and was defined as the
presence of nuclear pleomorphism or irregular chromatin distribution and the presence
of stratification or loss of polarity. The presence of this nuclear or cellular atypia is
termed ovarian intraepithelial neoplasia (OIN), which is believed to precede the
development of ovarian cancer. Methodology for the direct sampling of ovarian tissue
(in vivo testing) from those women deemed at increased risk for the development of
ovarian cancer is now available as an outpatient office laparoscopic procedure. With the
advent of microlaparoscopic technology, visualization of the adnexal and peritoneal
cavity can be afforded via laparoscopes less than 0.9 mm in diameter. The procedure
requires anesthesia similar to that offered for egg retrieval. Ovarian cytology can
accurately discern malignant ovarian epithelium from normal and the specialty of
cytopathology can benefit the Ovarian Pap Test. The question as yet to be answered is
can the molecular taxonomy of ovarian carcinoma be applied to histologically normal
appearing epithelium to detect occult carcinoma- this is to be answered within the
NOCEDP.
Conclusion
The clinical application of the biochemical, genetic and molecular basis of
ovarian carcinogenesis, invasion, and metastasis is required to affect change in the
morbidity and mortality from epithelial ovarian cancer. The clinical evaluation and
validation of new technologies permit the opportunity to challenge established scientific
26
paradigms. Presently the scientific community continues to identify biologically relevant
lipids and proteins, gene mutations, aberrant DNA methylation, and specific low
molecular weight proteins (fragments) in serum and/or plasma that may not only
acheive early detection but also ovarian cancer specific therapies. It is anticipated that
women’s healthcare will be significantly improved by the combination of biologically
relevant biomarkers, use of novel technologies such as proteomics (SELDI-TOF, AI), in
combination with innovative diagnostic imaging, to arrive at the accurate detection of
early stage epithelial ovarian carcinoma.
Supported by NIH/ NCI Early Detection Research Network Grant NCI UO1CA85133,
NCI P50 CA83639, Friends of Prentice, Northwestern Memorial Foundation, Stenn
Fund for Ovarian
Cancer Research, Joanne
Silverman
Cancer Foundation,
Kaleidoscope of Life Foundation, Illinois Department of Public Health, and the Robert H.
Lurie Comprehensive Cancer Center of Northwestern University.
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