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Circulating levels of insulin-like growth factor-II and IGF-binding protein 3 in cervical cancer
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Gynecologic Oncology 91 (2003) 486 – 493
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Circulating levels of insulin-like growth factor-II and IGF-binding protein 3 in cervical cancer夞 Subbi P. Mathur, Ph.D.,* Rajesh S. Mathur, Ph.D, Paul B. Underwood, M.D., Matthew F. Kohler, M.D., and William T. Creasman, M.D. Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC 29425, USA Received 3 December 2002
Abstract Objective. We aimed to further document that elevated levels of circulating insulin-like growth factor II (IGF-II) are associated with cervical cancer and to test the hypothesis that there may be an inverse association between IGF-II and IGF-binding protein 3 (IGF-BP3). Method. Serum IGF-II and IGF-BP3 levels were measured, using ELISA kits (Diagnostic Systems Laboratories), in 23 controls; 16 ASC-US with normal biopsies; 14 ASC-US with advanced CIN; 2 pretherapy CIN-I; 8 successfully treated CIN-I; 24 persistent CIN I; 14 pretherapy CIN II/III; 10 posttherapy CIN II/III with normal biopsies; 18 persistent CIN-II/III; 7 with pretherapy cervical cancer; 19 with posttherapy cervical cancer under remission; 15 with posttherapy persistent/recurrent cervical cancer; 10 with persistent ovarian or endometrial cancer; and 3 with endometrial or vulvar with cervical cancer. Student’s t test and linear regression analysis were used. Results. Compared to controls (493 ⫾ 90 ng/ml) and women with other gynecological cancers, serum IGF-II levels were significantly increased in women with ASC-US, with advanced CIN on biopsy (P ⬍ 0.0001), persistent CIN-I (993 ⫾ 262 ng/ml; P ⬍ 0.0001), pretherapy advanced CIN (1086 ⫾ 240; P ⬍ 0.0001), pretherapy cervical cancer patients (1746 ⫾ 318 ng/ml; P ⬍ 0.0001) and posttherapy persistent/recurrent CIN (1094 ⫾ 300; P ⬍ 0.0001); and cervical cancer (1395 ⫾ 189; P ⬍ 0.0001). After therapy, the IGF-II levels returned to normal in both CIN and cervical cancer patients under remission. Elevated serum IGF-II levels had 100% sensitivity and 87% specificity for cervical cancer and 81% sensitivity and 82% specificity for CIN. The levels of IGF-BP3 were significantly reduced in women with CIN before and after therapy (P ⬍ 0.0001) and in cervical cancer patients before and after therapy (P ⬍ 0.001). There was an inverse relationship between serum IGF-II and BP-3 levels (P ⬍ 0.01). Decreased serum IGF-BP3 levels had a sensitivity of 72% and specificity of 75% for cervical cancer and 81% sensitivity and 83% specificity for CIN. When both markers were considered together the sensitivity was 72% and specificity 84% for cervical cancer, while for CIN, the sensitivity was 57% and specificity 81%. Conclusion. Serum IGF-II may be a reliable marker for early diagnosis and monitoring therapy efficacy (sensitivity and specificity of 100% versus normal controls), while IGF-BP3 levels can be reliably used to predict prognosis. © 2003 Elsevier Inc. All rights reserved.
Introduction It is estimated that nearly 10 to 15% of women have abnormal Pap smears. Although cytological screening and earlier intervention have reduced cervical cancer deaths,
夞 This paper was presented as a Focused Plenary Oral Presentation during the 34th Annual Meeting of the SGO held in New Orleans, LA, January 31–February 4, 2003. * Corresponding author. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 634, Charleston, SC 29425. E-mail address: [email protected] (S.P. Mathur). 0090-8258/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2003.08.023
cervical cancer is still the leading cause of death for women in third world countries. In the USA, 14,500 new cases and 4800 deaths are annually reported [1]. About 25% of women with high-grade intraepithelial neoplasia are not identified to be at risk during routine gynecological examination due to diagnosis of ASC-US (atypical squamous cells of undetermined significance) [2,3]. Repetitive diagnoses of ASC-US delays prompt surgical therapy that may otherwise curb malignant proliferation and metastasis. Not all women infected with human papilloma virus (HPV) develop cervical cancer. Hence, HPV testing cannot specifically identify women in whom an established HPV infection, combined with other risk factors and a compromised
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immunity, has progressed into malignant cellular proliferation. Metastasis into other locations is hard to diagnose until the cancer is advanced [4]. A basic understanding of cervical cancer cell biology may provide us with a translational research tool to assess patients with malignant proliferation and hence at risk for metastasis. This in turn will enable us to devise focused treatment protocols, with possible reduction of morbidity and mortality. A rapid blood test that may help identify those women with CIN who are at risk for developing squamous cell cervical cancer and help monitor the therapy efficacy in these patients will be valuable. Immortalization of HPV-infected cervical squamous epithelial cells may be initiated by the inactivation of cell cycle regulatory p53 and retinoblastoma genes by HPV-E6 and E7 early genes, respectively. This in turn may trigger an up-regulation of growth factors such as epidermal growth factor (EGF) or insulin-like growth factor (IGF) [5]. These peptide growth factors that are in the extracellular space may stimulate a cascade of molecular events leading to cellular proliferation by binding to cell membrane receptors. The two tyrosine kinase receptors that may be active in cervical cancer are EGF-R [6] and HER-2/neu. Messenger RNA to EGF-R is present in the human uterus [7]. Normal, dysplastic, and malignant cervical epithelium expresses EGF-R protein to varying degrees [8 –10]. Consensus of work done in this area points to the importance of assessing the levels of EGF-R in patients with cervical cancer and, lately, women with advanced stages of CIN [11–14]. Insulin-like growth factor II (IGF-II) is a 7.5-kDa, 67amino-acid peptide that mediates some of the actions of growth hormone [15]. It is secreted by the liver and other tissues and is postulated to have a paracrine role through its mitogenic and metabolic actions at or near the sites of synthesis. IGF-II in circulation is in a combined form as a high-molecular-weight ternary complex with IGF-binding protein 3, IGF-binding protein 5, and acid-labile protein. The proportion of unbound IGF-II in the circulation of healthy men and women is less than 5%. IGF-II mRNA expression is highest during fetal life, after which it declines with age [16 –18]. Interest in IGFs and their effects on carcinogenesis has increased recently because high concentrations of IGF-I are associated with increased risk for breast, endometrial, prostate, colorectal, and lung cancers [19 –27]. The availability of unbound IGF-I for interaction with IGF-I receptor is modulated by IGF-binding proteins 1 to 6, especially IGFBP3. Interestingly, the latter has independent proapoptotic effects on cell growth that are dependent and independent of p53 and IGFs. Indeed, mutants of IGF-BP3 that do not bind IGF-I or -II, but stimulate apoptosis in human prostate cancer cells have been reported [28]. The latter finding suggests that IGF-BP3 can mediate apoptosis in an IGFindependent manner. Interestingly, IGF-II rather than IGF-I seems to play a significant role in cervical cancer. For example, the presence of IGF-II in cervical cancer cell culture supernatants
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was indirectly assessed by use of a radioreceptor assay [29]. Relaxation of insulin-like growth factor-II gene imprinting in human gynecologic tumors has been reported [30]. In our earlier in vitro studies, we found that the presence of HPV was associated with elevated levels of EGF-R [31] and that the addition of EGF and nicotine resulted in further increase [32]. We also observed an elevation in the levels of IGF-II in culture supernatants after stimulation with EGF that positively correlated with the increase in EGF-R level [32]. In a logical extension of these findings, we documented elevated levels of EGF-R and IGF-II in the cervical epithelial cells and IGF-II in the serum samples of women with cervical cancer and women with advanced CIN [33,34]. In the present study, we aimed to verify if significantly elevated levels of serum IGF-II are specific to cervical cancer among gynecological cancers and if levels of IGFBP3 may fluctuate with the changes in IGF-II in cervical cancer. Our hypothesis is that levels of IGF-BP3 may be reduced in cervical cancer patients and along with IGF-II may be a good measure of the cervical cancer patients’ prognosis.
Materials and methods Study groups and samples All the study subjects were recruited in the past 2 years and are different from those used in our earlier studies [33,34]. We focused our study on women with CIN or squamous cell cervical cancer. The study controls were 23 women who had consistently normal Pap smears. The study groups were (1) 16 women with recurrent abnormal Pap smears and a diagnosis of ASC-US for a period of 1 to 2 years who had normal cervical biopsies; (2) 14 women with recurrent abnormal Pap smears and a diagnosis of ASC-US for 1 to 2 years who had advanced CIN on cervical biopsy; (3) women with abnormal Pap smears who underwent colposcopy in the Colpo-Clinic and were clinically diagnosed with CIN I (n ⫽ 34; 2 were newly diagnosed, 24 had persistent CIN-I, and 8 had successfully treated CIN-I with normal biopsy), CIN II, and/or III (n ⫽ 14 pretherapy; n ⫽ 10 posttherapy with normal biopsy and 18 with persistent CIN-II/III); (4) 7 women with recently diagnosed cervical cancer; (5) 19 women in posttherapy cervical cancer remission; (6) 15 women with persistent/recurrent cervical cancer posttherapy; (7) 10 women with active and persistent ovarian and/or endometrial cancers; and (8) 3 women with primary endometrial, overian, or vulvar cancer with cervical cancer. Surgical therapy was the standard of care in all CIN patients, whereas adjuvant chemo- and radiation therapies were administered to women with recurrent/persistent cervical cancer. All women with ovarian and/or endometrial cancers had undergone surgical, chemo- and/or radiation
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therapy and were in active disease when the blood samples were obtained. All women signed an informed consent (protocol approved by the Office for Research Risk Protection) before donating blood samples. The clinics at Charleston cater to a mixed racial population (50% white, 40% black, and 10% American Indian, Asian, or Hispanic) and hence our studies are not racially biased. The laboratory was blinded to the clinical diagnosis of the patient until the conclusion of testing. We collated the clinical (for the past 5 to 10 years) and research data at the end of the study and entered them in a database for statistical evaluation. Determination of serum IGF-II levels We used a nonextraction IGF-II ELISA kit (DSL-102600) from Diagnostics Systems Laboratories, Inc. (Webster, TX) to determine serum IGF-II levels in our study participants. This is an enzymatically amplified “two-step” sandwich-type immunoassay [33]. In the assay, eight standards, two low-level and high-level controls and the pretreated unknown samples, were incubated in triplicate in microtiter plate wells that had been coated with an antiIGF-II antibody. After the first incubation for 2 h with shaking in an Orbital shaker at room temperature and washing with PBS–Tween solution, the wells were incubated with a second anti-IGF-II antibody raised in a different species and labeled with horseradish peroxidase. After a second incubation for 1 h with shaking and washing with PBS–Tween, the wells were incubated with the substrate tetramethylbenzidine (TMB). We determined the degree of enzymatic turnover of the substrate by dual-wavelength absorbance measurement at 450 nm. The absorbance measured is directly proportional to the concentration of IGF-II present. A set of IGF-II standards was used to plot a standard curve of absorbance versus IGF-II concentration from which the IGF-II concentrations in the unknown samples were calculated. We have assessed the reproducibility of the assays by performing the assays on standards and controls provided by the supplier and by using the same kit on different days, comparing different batches of kits and performing the assays in triplicate. The serum levels of IGF-II in normal women have previously been assessed to be in the range of 375 to 650 ng/ml (n ⫽ 250 samples) and are known to have minor variations during the menstrual cycle [35,36]. Determination of serum IGF-BP3 levels The Active IGF-BP3 ELISA kit (DSL-10-6600) from Diagnostic System Laboratories was used for measuring the levels of IGF-BP3 in the serum samples from our study groups. The test is an enzymatically amplified two-step sandwich-type ELISA. The standards, controls, and unknown samples (no pretreatment, as for IGF-II measure-
ment) were incubated in microtitration wells coated with anti-IGF-BP3 polyclonal antibody. After incubation and washing, the wells were treated with another anti-IGF-BP3 polyclonal antibody labeled with horseradish peroxidase. After a second incubation and washing step, the wells were incubated with the substrate TMB. An acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual-wavelength absorbance measurement at 450 nm. The absorbance measured was directly proportional to the concentration of IGF-BP3 present. A set of IGF-BP3 standards was used to plot a standard curve of absorbance versus IGF-BP3 concentration from which the IGF-BP3 concentration in the unknown sample could be calculated. Since the current information available on the concentrations of IGF-BP3 in serum samples of normal women is based on a radioimmunoassay, with a different sample preparation protocol [35], the manufacturers of the kit emphasize that each laboratory should establish its own expected value ranges. The assay is highly specific to IGF-BP3 and is guaranteed not to have interference by IGF-I, IGF-II, hGH, and insulin. Cross-reactivity with IGF-BPI was nondetectable. Statistics A Student’s t test was used to determine the levels of significance of values between the controls and the two patient groups and among the patient groups. Excel software from Microsoft was employed for this purpose. The sensitivity and specificity of both the markers were determined using conventional statistical methods [37]. Sensitivity is defined to mean the ability to detect an abnormal state (true positives) by a particular test and specificity means the ability to obtain normal results in nondiseased populations (true negatives). A Bayesian formula is used for these calculations: Sensitivity ⫽ Number of subjects with the disease and who test negative/Number of subjects with the disease, any test result, and Specificity ⫽ Number of subjects without the disease and who test negative/Number of subjects without the disease, any test result.
Results Levels of serum IGF-II in the study samples Serum IGF-II levels were significantly increased over those of the controls in women with ASC-US, with advanced CIN on biopsy, pretherapy CIN-I, CIN-II/III, and cervical cancer, as well as persistent/recurrent posttherapy CIN I, II/III, or cervical cancer. The levels were similar to those of the controls in successful posttherapy CIN-II/III, posttherapy remission of cervical cancer, and in active and persistent ovarian and/or endometrial cancer patients (Table 1; Fig. 1).
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Table 1 Levels of serum insulin-like growth factor II (IGF-II) and IGF-binding protein 3 (IGFBP3) in women with cervical cancer, ASCUS, and CIN Study Group
IGF-II (mean ⫾ SD ng/ml)
P value vs control
IGFBP3 (mean ⫾ SD ng/ml)
P value vs control
Controls (n ⫽ 23) ASC-US, normal biopsy (n ⫽ 16) ASC-US, advanced CIN on biopsy (n ⫽ 14) New CIN-I (n ⫽ 2) Successfully treated CIN-I (n ⫽ 8) Persistent CIN-I (n ⫽ 24) CIN-II/III pretherapy (n ⫽ 14) Successfully treated CIN-II/III (n ⫽ 10) Persistent CIN-II/III (n ⫽ 18) Cervical cancer, pretherapy (n ⫽ 7) Cervical cancer remission posttherapy (n ⫽ 19) Cervical cancer persistent/recurrent after therapy (n ⫽ 15) Persistent endometrial/ovarian cancers (n ⫽ 10) Metastastic endometrial adenocarcinoma with cervical cancer (n ⫽ 1) Vulvar cancer with CIN-III (n ⫽ 1) Endometrial and cervical cancer (n ⫽ 1)
493 ⫾ 90 691 ⫾ 149 998 ⫾ 251 771 ⫾ 65 768 ⫾ 191 993 ⫾ 262 1086 ⫾ 240 667 ⫾ 207 1094 ⫾ 300 1746 ⫾ 318 727 ⫾ 273
— 0.004 ⬍0.0001 NS 0.02 ⬍0.0001 ⬍0.0001 (0.06 vs Pretherapy) NS (0.03 vs pretherapy) ⬍0.0001 (NS vs pretherapy) ⬍0.0001 0.02 (⬍0.0001 vs pretherapy)
8152 ⫾ 1216 7096 ⫾ 1771 7317 ⫾ 1325 5726 ⫾ 565 6227 ⫾ 913 5418 ⫾ 1028 5251 ⫾ 923 5930 ⫾ 704 5753 ⫾ 883 4769 ⫾ 685 6183 ⫾ 1307
— NS NS NS 0.002 (NS vs pretherapy ⬍0.0001; NS vs pretherapy ⬍0.0001 ⬍0.001 (NS vs pretherapy) 0.0001 (NS vs pretherapy) ⬍0.0001 0.0003 (0.006 vs pretherapy)
1395 ⫾ 189 557 ⫾ 83
⬍0.00001 (NS vs pretherapy) N.S.
5723 ⫾ 1503 6609 ⫾ 1107
0.0003 (NS vs pretherapy) NS
1158 1726 912
— — —
4899 5284 6200
— — —
Positive cutoff values for serum IGF-II (mean in controls ⫹ 2 SD), 673 ng/ml; serum IGF-BP3 (mean in controls ⫺2 SD), 5720 ng/ml.
Levels of serum IGF-BP3 in the study groups In contrast to IGF-II levels, serum IGF-BP3 levels were significantly reduced over the controls in women with CIN-I, II, and III (pre- or posttherapy) and pre- or posttherapy cervical cancer (Table 1; Fig. 2). Sensitivity and specificity of IGF-II (positive marker) and IGF-BP3 (negative) markers We used serum IGF-II levels of ⱖ 673 ng/ml (mean ⫹ 2 SD in normal controls) and IGF-BP3 levels of ⱕ5720 ng/ml (mean ⫺ 2 SD normal controls) as our values for disease indication. Significantly elevated serum IGF-II levels and cervical cancer All of the 23 controls, 13 of 16 ASC-US patients with normal biopsies, 13 of 19 cervical cancer patients under remission after therapy, and 10 persistent endometrial or ovarian cancer patients had normal levels of serum IGF-II. All pretherapy cervical cancer patients, 15 of 15 cervical cancer patients with persistent cancer in spite of therapy, and 3 of 3 patients with primary cervical and secondary endometrial or vulvar cancer had elevated levels of IGF-II (ⱖ 1000 ng/ml). If we use all cervical cancer-free patients as negatives, the sensitivity of serum IGF-II was 100%, with a specificity of 87%. If we use only the normal controls as negatives (which is probably more valid) we had a specificity and sensitivity of 100% for serum IGF-II levels to detect cervical cancer.
Significantly elevated serum IGF-II levels and CIN When we evaluated the sensitivity and specificity of elevated serum IGF-II levels to detect CIN, using controls, patients with ASC-US with normal biopsy, and successfully treated (normal biopsy results) CIN I, II, and III as our disease-free groups, the sensitivity of serum IGF-II for detecting CIN was 81%, with a specificity of 82%. If we used just the controls as the disease-free group, the sensitivity was 81% and the specificity was 100%. Significantly decreased IGF-BP3 levels and cervical cancer The sensitivity of IGF-BP3 measurement for cervical cancer using just the controls as the disease-free group was 90% and specificity was 100%, whereas if we use all cervical cancer (pretherapy, successfully treated, and persistent cervical cancer) groups for the calculation, the sensitivity fell to 72% and specificity was 75%. In CIN, the low IGF-BP3 levels had a sensitivity of 81% and specificity of 81% if we included the treated groups and 52% sensitivity and 100% specificity if we excluded treated groups. Significantly decreased serum IGF-BP3 levels and CIN None of the 23 controls, 3 of 16 ASC-US patients with normal biopsies, 4 of 10 treated CIN-1, and 3 of 10 treated CIN/II/III patients had significantly decreased levels of serum IGF-BP3. Eleven of 14 women with ASC-US/CIN, 1 of 2 pretherapy CIN-1, 17 of 24 pretherapy CIN-I, 13 of 14 pretherapy CIN-II/III, and 16 of 18 persistent CIN-II/III had significantly decreased levels of serum IGF-BP3. The sensitivity of decreased serum IGF-BP3 levels for CIN was 81% and the specificity 83%.
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Fig. 1. Serum IGF-II levels in the study groups (CONT, controls; ASC-N, atypical squamous cells of undetermined significance; normal biopsy; ASC-C, ASC-US, advanced CIN on biopsy; PRE-CIN-I, pretherapy cervical intraepithelial neoplasia I; TRE CIN-I, treated CIN-I; PER CIN-I, persistent CIN-I; pre CIN II/III, pretherapy CIN II/III; TRE CIN-II/III, treated CIN-II/CIN III; PER CIN-II/III, persistent CIN II/III; PRE CERVICAL CAN, pretherapy cervical cancer; REM CERVICAL CAN, treated cervical cancer patients under remission; PER CERVICAL CAN, persistent/recurrent cervical cancer after therapy; EN/OV CAN, endometrial/ovarian cancer. The bold horizontal line depicts the mean ⫹ 2 SD of controls.
Combination of both markers and cervical cancer We determined the numbers of subjects in each study group that had both elevated serum IGF-II and decreased serum IGF-BP3. None of the 23 controls, 5 of 16 ASC-US with normal biopsy, 4 of 19 treated cervical cancer patients under remission, and 2 of 10 women with endometrial or ovarian cancers had this combination of criteria. Nine of 10 pretherapy cervical cancer patients and 9 of 15 persistent cervical cancer patients were positive for this combination. The sensitivity was 72% and specificity was 84%. Combination of both markers and CIN None of the 23 controls, 5 of 16 ASC-US with normal biopsy, 3 of 10 treated CIN-I (with normal biopsy results), and 3 of 10 successfully treated CIN-II/III (with normal biopsy results) had this combination of criteria. Five of 14 ASC-US with advanced CIN on biopsy, p1 of 2 CIN-I (pretherapy), 14 of 24 persistent CIN-I, 10 of 14 pretherapy
CIN-II/III, and II of 18 persistent CIN-II/III had this combination of criteria. The sensitivity of both markers together was 57 and the specificity was 81% for CIN.
Discussion Insulin-like growth factors I and II and their binding proteins, especially IGF-BP1 and 3, are recognized to be vital for human reproduction and fetal well-being. For example, IGF-II secreted by the ovarian granulosa cells at the time of follicular maturation enhances the growth and development of the dominant follicle [38] and promotes implantation of the embryo, while IGF-I secreted by the placental trophoblast enables fetal well-being [38]. The IGFbinding proteins and their associated proteases orchestrate these events in a paracrine manner. Recently, there has been an increasing awareness of the role played by IGF-I in the
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Fig. 2. Serum IGF-BP3 levels (mean ⫾ SD ng/ml) in the study groups (CONT, controls; ASC-N, atypical squamous cells; normal biopsy; ASC-C, ASC-US CIN biopsy; PRE-CIN-I, pretherapy cervical intraepithelial neoplasia I; TRE CIN-I, treated CIN-I; PER CIN-I, persistent CIN-I; PRE CIN II/III, pretherapy CIN II/III; TRE CIN-II/III, treated CIN-II/CIN III; PER CIN-II/III, persistent CIN II/III; PRE CER CAN, pretherapy cervical cancer; REM CERVICAL CAN, treated cervical cancer patients under remission; PER CER CAN, persistent/recurrent cervical cancer after therapy; EN/OV CAN, endometrial/ovarian cancer. The bold horizontal line depicts the mean ⫺ 2 SD of controls.
tumorigenesis of prostate, ovarian, colon, endometrial, and breast cancers [19 –30] and IGF-II in the malignant proliferation of cervical cancer, suggested by our earlier studies [33,34]. The present finding of elevated levels of serum IGF-II (levels ⱖ 673 ng/ml) in women with ASC-US with advanced CIN on biopsy, advanced CIN and persistent/recurrent cervical cancer before and after therapy (Table 1; Fig. 1) supports our data obtained earlier [33]. Serum IGF-II levels have a 100% sensitivity and 87% specificity in identifying women with active cervical cancer. They have 81% sensitivity and 82% specificity for identifying active CIN. This makes serum IGF-II a potential positive marker for cervical cancer. It is interesting that women with active ovarian and endometrial cancers do not have serum levels in the range of cervical cancer patients. Knowing serum levels of IGF-II may help in early detection of malignant proliferation in women with ASC-US/ASC-H and CIN and may lead to an earlier than normal clinical intervention, as shown by the high degree of sensitivity and specificity of these levels for CIN. These levels can also be effectively used with a high
degree of sensitivity and specificity to monitor therapy efficacy in treated patients. Larger studies with large patient populations are needed and will be undertaken. Human IGF-BP3 is a 264-amino-acid peptide, with a 27-amino-acid signal peptide and 18 cysteine residues [39]. IGF-BP3 is the major IGFBP in postnatal serum, where its molar concentration in normal serum approximates the molar concentration of total IGF peptides I and II [40]. Serum levels arise gradually during childhood, increase more rapidly during puberty, reaching peak levels at mid or late puberty, and fall during adult life [41]. The levels, however, may not be age-dependent and can vary with nutrition and levels of IGFs and growth hormone. We had hypothesized that the increased levels of serum IGF-II may be associated with decreased levels of its binding proteins. We chose human IGF-BP3 since it forms the major portion of the IGF-BP1– 6 pool and has great affinity to IGF-II. The hypothesis is proven by the present finding of significantly decreased levels of IGF-BP3 in women with cervical cancer and advanced CIN before therapy (Table 1; Fig. 2). Interestingly, these levels returned to normal in many women with cervical cancer after therapy. The inverse
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correlation between serum IGF-II and IGF-BP3 levels in all study subjects was significant (P ⬍ 0.01). Significantly decreased serum IGF-BP3 levels have sensitivity and specificity of 72 and 75%, respectively, for cervical cancer and 81 and 83%, respectively, for CIN. Our data clearly show that decreased levels of IGF-BP3 are associated with precancerous CIN through cervical cancer. A combination of elevated IGF-II and decreased IGF-BP3 has 72% sensitivity and 84% specificity in cervical cancer and 57% sensitivity and 81% specificity in CIN. This is logical since a natural remission of CIN-I is common in healthy women. These women will never progress to cervical cancer. The tissues that normally secrete the IGFs also secrete IGF-BP proteases that keep the levels of IGF-BPs under check to allow IGFs to perform their cell proliferative actions. In cervical cancer patients, the IGF-BP3 levels may be controlled by the increased levels of IGF-BP3 proteases, leading to an unopposed increase in IGF-II levels. Besides binding to IGFs, IGF-BP3 is known to have proapoptotic properties both dependent on and independent of p53 and IGFs [20]. IGF-BP3 can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating IGF-I receptor to stimulate cell survival and proliferation. For example, IGF-BP3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in PC-3 human prostate cancer cells [18]. Hence, IGF-BP3 may have an IGF-independent antiproliferative and antitumorigenic action on cancer cells. Normal levels of IGF-BP3 in the serum of cervical cancer patients may be a good prognosis for their recovery. However, an isolated report describes elevated levels of serum IGF-I, IGF-II, and IGFBP3 to be associated with an increased risk of colon cancer in the oriental population [42]. We observed a significant decrease in IGF-BP3 levels in 4 of 10 women with ovarian and/or endometrial cancer. This may perhaps be due to the fact that these women had active metastatic disease as well as having previously undergone chemo- or radiation therapy that might have compromised the normal functions of the liver, which is the main source of the secretion of these proteins. Also, IGF-I production is higher in ovarian and endometrial cancers and may be responsible for binding with free IGF-BP3 and reducing its levels in the circulation. Enumeration of serum levels of IGF-II may be valuable for early diagnosis and monitoring of therapy in cervical cancer, while serum IGF-BP3 levels may serve for prognosis of cervical cancer patients. Further studies using large patient populations are needed.
Acknowledgments This research was supported by National Cancer Institute Grant R21 CA 92085-01 and a Department of Defense
Phase V Grant to Hollings Cancer Center. The assistance rendered by Philip F. Rust, Ph.D., Department of Biometry, and Ms. Pamela Smith, M.S.R.N., is deeply appreciated.
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S.P. Mathur et al. / Gynecologic Oncology 91 (2003) 486 – 493 [18] Rachlar M. Insulin-like growth factor-binding proteins. Vitam Horm 1993;47:1–114. [19] Zumkeller W. IGFs and IGFBPs: surrogate markers for diagnosis and surveillance of tumor growth? Mol Pathol 2001;54:285– 8. [20] Furstenberger G, Senn HJ. Insulin-like growth factors and cancer. Lancet Oncol 2002;3:298 –302. [21] Ayabe T, Tsutsumi O, Sakai H, Yoshikawa H, Yano T, Kurimoto F, Taketani Y. Increased circulating levels of insulin-like growth factor-I and decreased circulating levels of insulin-like growth factor binding protein-1 in postmenopausal women with endometrial cancer. Endocr J 1997;44(3):419 –24. [22] Chan JM, Stampfer MJ, Giovannucci E. Plasma insulin-like growth factor I and prostate cancer risk: a prospective study. Science 1998; 279:563– 6. [23] Hankinson SE, Willett WC, Colditz GA. Circulating concentrations of insulin-like growth factor I and risk of breast cancer. Lancet 1998;351:1393– 6. [24] Peyrat JP, Bonneterre J, Hecquet B, et al. Plasma insulin-like growth factor I (IGF-I) concentrations in human breast cancer. Eur J Cancer 1993;29:492–7. [25] Bruning PF, van Doorn J, Bonfrer JMG. Insulin-like growth factorbinding protein 3 is decreased in early stage operable premenopausal breast cancer. Int J Cancer 1995;2:266 –70. [26] Bohlike K, Cramer DW, Trichopoulos D, et al. Insulin-like growth factor-I in relation to premenopausal ductal carcinoma in situ of the breast. Epidemiology 1998;9:570 –3. [27] Wolk A, Mantzoros CS, Andersson SO, et al. Insulin-like growth factor I and prostate cancer risk: a population-based case-control study. J Natl Cancer Inst 1998;90:911–5. [28] Hong J, Zhang G, Dong F, Rechler MM. Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells. J Biol Chem 2002;277:10489 –97. [29] Naik P, Christofori G, Hanahan D. Insulin-like growth factor II is focally upregulated and functionally involved as a second signal of oncogene-induced tumorigenesis. Cold Spring Harbor Symposia on Quantitative Biology. Vol. LIX. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1994, p. 459 –70. [30] Yaginuma Y, Nishiwaki K, Kitamura S, Hayashi H, Sengoku K, Ishikawa M. Relaxation of insulin-like growth factor-II gene imprinting in human gynecologic tumors. Oncology 1997;54(6):502–7. [31] Mathur SP, Mathur RS, Young RC. Human papilloma virus (HPV) protein E6/E7 and epidermal growth factor receptor (EGF-R) levels
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Gynecologic Oncology 91 (2003) 486 – 493
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Circulating levels of insulin-like growth factor-II and IGF-binding protein 3 in cervical cancer夞 Subbi P. Mathur, Ph.D.,* Rajesh S. Mathur, Ph.D, Paul B. Underwood, M.D., Matthew F. Kohler, M.D., and William T. Creasman, M.D. Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC 29425, USA Received 3 December 2002
Abstract Objective. We aimed to further document that elevated levels of circulating insulin-like growth factor II (IGF-II) are associated with cervical cancer and to test the hypothesis that there may be an inverse association between IGF-II and IGF-binding protein 3 (IGF-BP3). Method. Serum IGF-II and IGF-BP3 levels were measured, using ELISA kits (Diagnostic Systems Laboratories), in 23 controls; 16 ASC-US with normal biopsies; 14 ASC-US with advanced CIN; 2 pretherapy CIN-I; 8 successfully treated CIN-I; 24 persistent CIN I; 14 pretherapy CIN II/III; 10 posttherapy CIN II/III with normal biopsies; 18 persistent CIN-II/III; 7 with pretherapy cervical cancer; 19 with posttherapy cervical cancer under remission; 15 with posttherapy persistent/recurrent cervical cancer; 10 with persistent ovarian or endometrial cancer; and 3 with endometrial or vulvar with cervical cancer. Student’s t test and linear regression analysis were used. Results. Compared to controls (493 ⫾ 90 ng/ml) and women with other gynecological cancers, serum IGF-II levels were significantly increased in women with ASC-US, with advanced CIN on biopsy (P ⬍ 0.0001), persistent CIN-I (993 ⫾ 262 ng/ml; P ⬍ 0.0001), pretherapy advanced CIN (1086 ⫾ 240; P ⬍ 0.0001), pretherapy cervical cancer patients (1746 ⫾ 318 ng/ml; P ⬍ 0.0001) and posttherapy persistent/recurrent CIN (1094 ⫾ 300; P ⬍ 0.0001); and cervical cancer (1395 ⫾ 189; P ⬍ 0.0001). After therapy, the IGF-II levels returned to normal in both CIN and cervical cancer patients under remission. Elevated serum IGF-II levels had 100% sensitivity and 87% specificity for cervical cancer and 81% sensitivity and 82% specificity for CIN. The levels of IGF-BP3 were significantly reduced in women with CIN before and after therapy (P ⬍ 0.0001) and in cervical cancer patients before and after therapy (P ⬍ 0.001). There was an inverse relationship between serum IGF-II and BP-3 levels (P ⬍ 0.01). Decreased serum IGF-BP3 levels had a sensitivity of 72% and specificity of 75% for cervical cancer and 81% sensitivity and 83% specificity for CIN. When both markers were considered together the sensitivity was 72% and specificity 84% for cervical cancer, while for CIN, the sensitivity was 57% and specificity 81%. Conclusion. Serum IGF-II may be a reliable marker for early diagnosis and monitoring therapy efficacy (sensitivity and specificity of 100% versus normal controls), while IGF-BP3 levels can be reliably used to predict prognosis. © 2003 Elsevier Inc. All rights reserved.
Introduction It is estimated that nearly 10 to 15% of women have abnormal Pap smears. Although cytological screening and earlier intervention have reduced cervical cancer deaths,
夞 This paper was presented as a Focused Plenary Oral Presentation during the 34th Annual Meeting of the SGO held in New Orleans, LA, January 31–February 4, 2003. * Corresponding author. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 634, Charleston, SC 29425. E-mail address: [email protected] (S.P. Mathur). 0090-8258/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2003.08.023
cervical cancer is still the leading cause of death for women in third world countries. In the USA, 14,500 new cases and 4800 deaths are annually reported [1]. About 25% of women with high-grade intraepithelial neoplasia are not identified to be at risk during routine gynecological examination due to diagnosis of ASC-US (atypical squamous cells of undetermined significance) [2,3]. Repetitive diagnoses of ASC-US delays prompt surgical therapy that may otherwise curb malignant proliferation and metastasis. Not all women infected with human papilloma virus (HPV) develop cervical cancer. Hence, HPV testing cannot specifically identify women in whom an established HPV infection, combined with other risk factors and a compromised
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immunity, has progressed into malignant cellular proliferation. Metastasis into other locations is hard to diagnose until the cancer is advanced [4]. A basic understanding of cervical cancer cell biology may provide us with a translational research tool to assess patients with malignant proliferation and hence at risk for metastasis. This in turn will enable us to devise focused treatment protocols, with possible reduction of morbidity and mortality. A rapid blood test that may help identify those women with CIN who are at risk for developing squamous cell cervical cancer and help monitor the therapy efficacy in these patients will be valuable. Immortalization of HPV-infected cervical squamous epithelial cells may be initiated by the inactivation of cell cycle regulatory p53 and retinoblastoma genes by HPV-E6 and E7 early genes, respectively. This in turn may trigger an up-regulation of growth factors such as epidermal growth factor (EGF) or insulin-like growth factor (IGF) [5]. These peptide growth factors that are in the extracellular space may stimulate a cascade of molecular events leading to cellular proliferation by binding to cell membrane receptors. The two tyrosine kinase receptors that may be active in cervical cancer are EGF-R [6] and HER-2/neu. Messenger RNA to EGF-R is present in the human uterus [7]. Normal, dysplastic, and malignant cervical epithelium expresses EGF-R protein to varying degrees [8 –10]. Consensus of work done in this area points to the importance of assessing the levels of EGF-R in patients with cervical cancer and, lately, women with advanced stages of CIN [11–14]. Insulin-like growth factor II (IGF-II) is a 7.5-kDa, 67amino-acid peptide that mediates some of the actions of growth hormone [15]. It is secreted by the liver and other tissues and is postulated to have a paracrine role through its mitogenic and metabolic actions at or near the sites of synthesis. IGF-II in circulation is in a combined form as a high-molecular-weight ternary complex with IGF-binding protein 3, IGF-binding protein 5, and acid-labile protein. The proportion of unbound IGF-II in the circulation of healthy men and women is less than 5%. IGF-II mRNA expression is highest during fetal life, after which it declines with age [16 –18]. Interest in IGFs and their effects on carcinogenesis has increased recently because high concentrations of IGF-I are associated with increased risk for breast, endometrial, prostate, colorectal, and lung cancers [19 –27]. The availability of unbound IGF-I for interaction with IGF-I receptor is modulated by IGF-binding proteins 1 to 6, especially IGFBP3. Interestingly, the latter has independent proapoptotic effects on cell growth that are dependent and independent of p53 and IGFs. Indeed, mutants of IGF-BP3 that do not bind IGF-I or -II, but stimulate apoptosis in human prostate cancer cells have been reported [28]. The latter finding suggests that IGF-BP3 can mediate apoptosis in an IGFindependent manner. Interestingly, IGF-II rather than IGF-I seems to play a significant role in cervical cancer. For example, the presence of IGF-II in cervical cancer cell culture supernatants
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was indirectly assessed by use of a radioreceptor assay [29]. Relaxation of insulin-like growth factor-II gene imprinting in human gynecologic tumors has been reported [30]. In our earlier in vitro studies, we found that the presence of HPV was associated with elevated levels of EGF-R [31] and that the addition of EGF and nicotine resulted in further increase [32]. We also observed an elevation in the levels of IGF-II in culture supernatants after stimulation with EGF that positively correlated with the increase in EGF-R level [32]. In a logical extension of these findings, we documented elevated levels of EGF-R and IGF-II in the cervical epithelial cells and IGF-II in the serum samples of women with cervical cancer and women with advanced CIN [33,34]. In the present study, we aimed to verify if significantly elevated levels of serum IGF-II are specific to cervical cancer among gynecological cancers and if levels of IGFBP3 may fluctuate with the changes in IGF-II in cervical cancer. Our hypothesis is that levels of IGF-BP3 may be reduced in cervical cancer patients and along with IGF-II may be a good measure of the cervical cancer patients’ prognosis.
Materials and methods Study groups and samples All the study subjects were recruited in the past 2 years and are different from those used in our earlier studies [33,34]. We focused our study on women with CIN or squamous cell cervical cancer. The study controls were 23 women who had consistently normal Pap smears. The study groups were (1) 16 women with recurrent abnormal Pap smears and a diagnosis of ASC-US for a period of 1 to 2 years who had normal cervical biopsies; (2) 14 women with recurrent abnormal Pap smears and a diagnosis of ASC-US for 1 to 2 years who had advanced CIN on cervical biopsy; (3) women with abnormal Pap smears who underwent colposcopy in the Colpo-Clinic and were clinically diagnosed with CIN I (n ⫽ 34; 2 were newly diagnosed, 24 had persistent CIN-I, and 8 had successfully treated CIN-I with normal biopsy), CIN II, and/or III (n ⫽ 14 pretherapy; n ⫽ 10 posttherapy with normal biopsy and 18 with persistent CIN-II/III); (4) 7 women with recently diagnosed cervical cancer; (5) 19 women in posttherapy cervical cancer remission; (6) 15 women with persistent/recurrent cervical cancer posttherapy; (7) 10 women with active and persistent ovarian and/or endometrial cancers; and (8) 3 women with primary endometrial, overian, or vulvar cancer with cervical cancer. Surgical therapy was the standard of care in all CIN patients, whereas adjuvant chemo- and radiation therapies were administered to women with recurrent/persistent cervical cancer. All women with ovarian and/or endometrial cancers had undergone surgical, chemo- and/or radiation
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therapy and were in active disease when the blood samples were obtained. All women signed an informed consent (protocol approved by the Office for Research Risk Protection) before donating blood samples. The clinics at Charleston cater to a mixed racial population (50% white, 40% black, and 10% American Indian, Asian, or Hispanic) and hence our studies are not racially biased. The laboratory was blinded to the clinical diagnosis of the patient until the conclusion of testing. We collated the clinical (for the past 5 to 10 years) and research data at the end of the study and entered them in a database for statistical evaluation. Determination of serum IGF-II levels We used a nonextraction IGF-II ELISA kit (DSL-102600) from Diagnostics Systems Laboratories, Inc. (Webster, TX) to determine serum IGF-II levels in our study participants. This is an enzymatically amplified “two-step” sandwich-type immunoassay [33]. In the assay, eight standards, two low-level and high-level controls and the pretreated unknown samples, were incubated in triplicate in microtiter plate wells that had been coated with an antiIGF-II antibody. After the first incubation for 2 h with shaking in an Orbital shaker at room temperature and washing with PBS–Tween solution, the wells were incubated with a second anti-IGF-II antibody raised in a different species and labeled with horseradish peroxidase. After a second incubation for 1 h with shaking and washing with PBS–Tween, the wells were incubated with the substrate tetramethylbenzidine (TMB). We determined the degree of enzymatic turnover of the substrate by dual-wavelength absorbance measurement at 450 nm. The absorbance measured is directly proportional to the concentration of IGF-II present. A set of IGF-II standards was used to plot a standard curve of absorbance versus IGF-II concentration from which the IGF-II concentrations in the unknown samples were calculated. We have assessed the reproducibility of the assays by performing the assays on standards and controls provided by the supplier and by using the same kit on different days, comparing different batches of kits and performing the assays in triplicate. The serum levels of IGF-II in normal women have previously been assessed to be in the range of 375 to 650 ng/ml (n ⫽ 250 samples) and are known to have minor variations during the menstrual cycle [35,36]. Determination of serum IGF-BP3 levels The Active IGF-BP3 ELISA kit (DSL-10-6600) from Diagnostic System Laboratories was used for measuring the levels of IGF-BP3 in the serum samples from our study groups. The test is an enzymatically amplified two-step sandwich-type ELISA. The standards, controls, and unknown samples (no pretreatment, as for IGF-II measure-
ment) were incubated in microtitration wells coated with anti-IGF-BP3 polyclonal antibody. After incubation and washing, the wells were treated with another anti-IGF-BP3 polyclonal antibody labeled with horseradish peroxidase. After a second incubation and washing step, the wells were incubated with the substrate TMB. An acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual-wavelength absorbance measurement at 450 nm. The absorbance measured was directly proportional to the concentration of IGF-BP3 present. A set of IGF-BP3 standards was used to plot a standard curve of absorbance versus IGF-BP3 concentration from which the IGF-BP3 concentration in the unknown sample could be calculated. Since the current information available on the concentrations of IGF-BP3 in serum samples of normal women is based on a radioimmunoassay, with a different sample preparation protocol [35], the manufacturers of the kit emphasize that each laboratory should establish its own expected value ranges. The assay is highly specific to IGF-BP3 and is guaranteed not to have interference by IGF-I, IGF-II, hGH, and insulin. Cross-reactivity with IGF-BPI was nondetectable. Statistics A Student’s t test was used to determine the levels of significance of values between the controls and the two patient groups and among the patient groups. Excel software from Microsoft was employed for this purpose. The sensitivity and specificity of both the markers were determined using conventional statistical methods [37]. Sensitivity is defined to mean the ability to detect an abnormal state (true positives) by a particular test and specificity means the ability to obtain normal results in nondiseased populations (true negatives). A Bayesian formula is used for these calculations: Sensitivity ⫽ Number of subjects with the disease and who test negative/Number of subjects with the disease, any test result, and Specificity ⫽ Number of subjects without the disease and who test negative/Number of subjects without the disease, any test result.
Results Levels of serum IGF-II in the study samples Serum IGF-II levels were significantly increased over those of the controls in women with ASC-US, with advanced CIN on biopsy, pretherapy CIN-I, CIN-II/III, and cervical cancer, as well as persistent/recurrent posttherapy CIN I, II/III, or cervical cancer. The levels were similar to those of the controls in successful posttherapy CIN-II/III, posttherapy remission of cervical cancer, and in active and persistent ovarian and/or endometrial cancer patients (Table 1; Fig. 1).
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Table 1 Levels of serum insulin-like growth factor II (IGF-II) and IGF-binding protein 3 (IGFBP3) in women with cervical cancer, ASCUS, and CIN Study Group
IGF-II (mean ⫾ SD ng/ml)
P value vs control
IGFBP3 (mean ⫾ SD ng/ml)
P value vs control
Controls (n ⫽ 23) ASC-US, normal biopsy (n ⫽ 16) ASC-US, advanced CIN on biopsy (n ⫽ 14) New CIN-I (n ⫽ 2) Successfully treated CIN-I (n ⫽ 8) Persistent CIN-I (n ⫽ 24) CIN-II/III pretherapy (n ⫽ 14) Successfully treated CIN-II/III (n ⫽ 10) Persistent CIN-II/III (n ⫽ 18) Cervical cancer, pretherapy (n ⫽ 7) Cervical cancer remission posttherapy (n ⫽ 19) Cervical cancer persistent/recurrent after therapy (n ⫽ 15) Persistent endometrial/ovarian cancers (n ⫽ 10) Metastastic endometrial adenocarcinoma with cervical cancer (n ⫽ 1) Vulvar cancer with CIN-III (n ⫽ 1) Endometrial and cervical cancer (n ⫽ 1)
493 ⫾ 90 691 ⫾ 149 998 ⫾ 251 771 ⫾ 65 768 ⫾ 191 993 ⫾ 262 1086 ⫾ 240 667 ⫾ 207 1094 ⫾ 300 1746 ⫾ 318 727 ⫾ 273
— 0.004 ⬍0.0001 NS 0.02 ⬍0.0001 ⬍0.0001 (0.06 vs Pretherapy) NS (0.03 vs pretherapy) ⬍0.0001 (NS vs pretherapy) ⬍0.0001 0.02 (⬍0.0001 vs pretherapy)
8152 ⫾ 1216 7096 ⫾ 1771 7317 ⫾ 1325 5726 ⫾ 565 6227 ⫾ 913 5418 ⫾ 1028 5251 ⫾ 923 5930 ⫾ 704 5753 ⫾ 883 4769 ⫾ 685 6183 ⫾ 1307
— NS NS NS 0.002 (NS vs pretherapy ⬍0.0001; NS vs pretherapy ⬍0.0001 ⬍0.001 (NS vs pretherapy) 0.0001 (NS vs pretherapy) ⬍0.0001 0.0003 (0.006 vs pretherapy)
1395 ⫾ 189 557 ⫾ 83
⬍0.00001 (NS vs pretherapy) N.S.
5723 ⫾ 1503 6609 ⫾ 1107
0.0003 (NS vs pretherapy) NS
1158 1726 912
— — —
4899 5284 6200
— — —
Positive cutoff values for serum IGF-II (mean in controls ⫹ 2 SD), 673 ng/ml; serum IGF-BP3 (mean in controls ⫺2 SD), 5720 ng/ml.
Levels of serum IGF-BP3 in the study groups In contrast to IGF-II levels, serum IGF-BP3 levels were significantly reduced over the controls in women with CIN-I, II, and III (pre- or posttherapy) and pre- or posttherapy cervical cancer (Table 1; Fig. 2). Sensitivity and specificity of IGF-II (positive marker) and IGF-BP3 (negative) markers We used serum IGF-II levels of ⱖ 673 ng/ml (mean ⫹ 2 SD in normal controls) and IGF-BP3 levels of ⱕ5720 ng/ml (mean ⫺ 2 SD normal controls) as our values for disease indication. Significantly elevated serum IGF-II levels and cervical cancer All of the 23 controls, 13 of 16 ASC-US patients with normal biopsies, 13 of 19 cervical cancer patients under remission after therapy, and 10 persistent endometrial or ovarian cancer patients had normal levels of serum IGF-II. All pretherapy cervical cancer patients, 15 of 15 cervical cancer patients with persistent cancer in spite of therapy, and 3 of 3 patients with primary cervical and secondary endometrial or vulvar cancer had elevated levels of IGF-II (ⱖ 1000 ng/ml). If we use all cervical cancer-free patients as negatives, the sensitivity of serum IGF-II was 100%, with a specificity of 87%. If we use only the normal controls as negatives (which is probably more valid) we had a specificity and sensitivity of 100% for serum IGF-II levels to detect cervical cancer.
Significantly elevated serum IGF-II levels and CIN When we evaluated the sensitivity and specificity of elevated serum IGF-II levels to detect CIN, using controls, patients with ASC-US with normal biopsy, and successfully treated (normal biopsy results) CIN I, II, and III as our disease-free groups, the sensitivity of serum IGF-II for detecting CIN was 81%, with a specificity of 82%. If we used just the controls as the disease-free group, the sensitivity was 81% and the specificity was 100%. Significantly decreased IGF-BP3 levels and cervical cancer The sensitivity of IGF-BP3 measurement for cervical cancer using just the controls as the disease-free group was 90% and specificity was 100%, whereas if we use all cervical cancer (pretherapy, successfully treated, and persistent cervical cancer) groups for the calculation, the sensitivity fell to 72% and specificity was 75%. In CIN, the low IGF-BP3 levels had a sensitivity of 81% and specificity of 81% if we included the treated groups and 52% sensitivity and 100% specificity if we excluded treated groups. Significantly decreased serum IGF-BP3 levels and CIN None of the 23 controls, 3 of 16 ASC-US patients with normal biopsies, 4 of 10 treated CIN-1, and 3 of 10 treated CIN/II/III patients had significantly decreased levels of serum IGF-BP3. Eleven of 14 women with ASC-US/CIN, 1 of 2 pretherapy CIN-1, 17 of 24 pretherapy CIN-I, 13 of 14 pretherapy CIN-II/III, and 16 of 18 persistent CIN-II/III had significantly decreased levels of serum IGF-BP3. The sensitivity of decreased serum IGF-BP3 levels for CIN was 81% and the specificity 83%.
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Fig. 1. Serum IGF-II levels in the study groups (CONT, controls; ASC-N, atypical squamous cells of undetermined significance; normal biopsy; ASC-C, ASC-US, advanced CIN on biopsy; PRE-CIN-I, pretherapy cervical intraepithelial neoplasia I; TRE CIN-I, treated CIN-I; PER CIN-I, persistent CIN-I; pre CIN II/III, pretherapy CIN II/III; TRE CIN-II/III, treated CIN-II/CIN III; PER CIN-II/III, persistent CIN II/III; PRE CERVICAL CAN, pretherapy cervical cancer; REM CERVICAL CAN, treated cervical cancer patients under remission; PER CERVICAL CAN, persistent/recurrent cervical cancer after therapy; EN/OV CAN, endometrial/ovarian cancer. The bold horizontal line depicts the mean ⫹ 2 SD of controls.
Combination of both markers and cervical cancer We determined the numbers of subjects in each study group that had both elevated serum IGF-II and decreased serum IGF-BP3. None of the 23 controls, 5 of 16 ASC-US with normal biopsy, 4 of 19 treated cervical cancer patients under remission, and 2 of 10 women with endometrial or ovarian cancers had this combination of criteria. Nine of 10 pretherapy cervical cancer patients and 9 of 15 persistent cervical cancer patients were positive for this combination. The sensitivity was 72% and specificity was 84%. Combination of both markers and CIN None of the 23 controls, 5 of 16 ASC-US with normal biopsy, 3 of 10 treated CIN-I (with normal biopsy results), and 3 of 10 successfully treated CIN-II/III (with normal biopsy results) had this combination of criteria. Five of 14 ASC-US with advanced CIN on biopsy, p1 of 2 CIN-I (pretherapy), 14 of 24 persistent CIN-I, 10 of 14 pretherapy
CIN-II/III, and II of 18 persistent CIN-II/III had this combination of criteria. The sensitivity of both markers together was 57 and the specificity was 81% for CIN.
Discussion Insulin-like growth factors I and II and their binding proteins, especially IGF-BP1 and 3, are recognized to be vital for human reproduction and fetal well-being. For example, IGF-II secreted by the ovarian granulosa cells at the time of follicular maturation enhances the growth and development of the dominant follicle [38] and promotes implantation of the embryo, while IGF-I secreted by the placental trophoblast enables fetal well-being [38]. The IGFbinding proteins and their associated proteases orchestrate these events in a paracrine manner. Recently, there has been an increasing awareness of the role played by IGF-I in the
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Fig. 2. Serum IGF-BP3 levels (mean ⫾ SD ng/ml) in the study groups (CONT, controls; ASC-N, atypical squamous cells; normal biopsy; ASC-C, ASC-US CIN biopsy; PRE-CIN-I, pretherapy cervical intraepithelial neoplasia I; TRE CIN-I, treated CIN-I; PER CIN-I, persistent CIN-I; PRE CIN II/III, pretherapy CIN II/III; TRE CIN-II/III, treated CIN-II/CIN III; PER CIN-II/III, persistent CIN II/III; PRE CER CAN, pretherapy cervical cancer; REM CERVICAL CAN, treated cervical cancer patients under remission; PER CER CAN, persistent/recurrent cervical cancer after therapy; EN/OV CAN, endometrial/ovarian cancer. The bold horizontal line depicts the mean ⫺ 2 SD of controls.
tumorigenesis of prostate, ovarian, colon, endometrial, and breast cancers [19 –30] and IGF-II in the malignant proliferation of cervical cancer, suggested by our earlier studies [33,34]. The present finding of elevated levels of serum IGF-II (levels ⱖ 673 ng/ml) in women with ASC-US with advanced CIN on biopsy, advanced CIN and persistent/recurrent cervical cancer before and after therapy (Table 1; Fig. 1) supports our data obtained earlier [33]. Serum IGF-II levels have a 100% sensitivity and 87% specificity in identifying women with active cervical cancer. They have 81% sensitivity and 82% specificity for identifying active CIN. This makes serum IGF-II a potential positive marker for cervical cancer. It is interesting that women with active ovarian and endometrial cancers do not have serum levels in the range of cervical cancer patients. Knowing serum levels of IGF-II may help in early detection of malignant proliferation in women with ASC-US/ASC-H and CIN and may lead to an earlier than normal clinical intervention, as shown by the high degree of sensitivity and specificity of these levels for CIN. These levels can also be effectively used with a high
degree of sensitivity and specificity to monitor therapy efficacy in treated patients. Larger studies with large patient populations are needed and will be undertaken. Human IGF-BP3 is a 264-amino-acid peptide, with a 27-amino-acid signal peptide and 18 cysteine residues [39]. IGF-BP3 is the major IGFBP in postnatal serum, where its molar concentration in normal serum approximates the molar concentration of total IGF peptides I and II [40]. Serum levels arise gradually during childhood, increase more rapidly during puberty, reaching peak levels at mid or late puberty, and fall during adult life [41]. The levels, however, may not be age-dependent and can vary with nutrition and levels of IGFs and growth hormone. We had hypothesized that the increased levels of serum IGF-II may be associated with decreased levels of its binding proteins. We chose human IGF-BP3 since it forms the major portion of the IGF-BP1– 6 pool and has great affinity to IGF-II. The hypothesis is proven by the present finding of significantly decreased levels of IGF-BP3 in women with cervical cancer and advanced CIN before therapy (Table 1; Fig. 2). Interestingly, these levels returned to normal in many women with cervical cancer after therapy. The inverse
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correlation between serum IGF-II and IGF-BP3 levels in all study subjects was significant (P ⬍ 0.01). Significantly decreased serum IGF-BP3 levels have sensitivity and specificity of 72 and 75%, respectively, for cervical cancer and 81 and 83%, respectively, for CIN. Our data clearly show that decreased levels of IGF-BP3 are associated with precancerous CIN through cervical cancer. A combination of elevated IGF-II and decreased IGF-BP3 has 72% sensitivity and 84% specificity in cervical cancer and 57% sensitivity and 81% specificity in CIN. This is logical since a natural remission of CIN-I is common in healthy women. These women will never progress to cervical cancer. The tissues that normally secrete the IGFs also secrete IGF-BP proteases that keep the levels of IGF-BPs under check to allow IGFs to perform their cell proliferative actions. In cervical cancer patients, the IGF-BP3 levels may be controlled by the increased levels of IGF-BP3 proteases, leading to an unopposed increase in IGF-II levels. Besides binding to IGFs, IGF-BP3 is known to have proapoptotic properties both dependent on and independent of p53 and IGFs [20]. IGF-BP3 can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating IGF-I receptor to stimulate cell survival and proliferation. For example, IGF-BP3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in PC-3 human prostate cancer cells [18]. Hence, IGF-BP3 may have an IGF-independent antiproliferative and antitumorigenic action on cancer cells. Normal levels of IGF-BP3 in the serum of cervical cancer patients may be a good prognosis for their recovery. However, an isolated report describes elevated levels of serum IGF-I, IGF-II, and IGFBP3 to be associated with an increased risk of colon cancer in the oriental population [42]. We observed a significant decrease in IGF-BP3 levels in 4 of 10 women with ovarian and/or endometrial cancer. This may perhaps be due to the fact that these women had active metastatic disease as well as having previously undergone chemo- or radiation therapy that might have compromised the normal functions of the liver, which is the main source of the secretion of these proteins. Also, IGF-I production is higher in ovarian and endometrial cancers and may be responsible for binding with free IGF-BP3 and reducing its levels in the circulation. Enumeration of serum levels of IGF-II may be valuable for early diagnosis and monitoring of therapy in cervical cancer, while serum IGF-BP3 levels may serve for prognosis of cervical cancer patients. Further studies using large patient populations are needed.
Acknowledgments This research was supported by National Cancer Institute Grant R21 CA 92085-01 and a Department of Defense
Phase V Grant to Hollings Cancer Center. The assistance rendered by Philip F. Rust, Ph.D., Department of Biometry, and Ms. Pamela Smith, M.S.R.N., is deeply appreciated.
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