Validation of a Biomarker Panel and Longitudinal Biomarker Performance for Early Detection of Ovarian Cancer.

OBJECTIVES: Longitudinal multimarker combinations have the potential to improve sensitivity while maintaining the high specificity required for the early detection of ovarian cancer. The use of multiple markers to improve sensitivity over cancer antigen 125 (CA125) in longitudinal algorithms for early ovarian cancer detection requires the selection of markers with optimal discriminatory power and low longitudinal variance relative to disease-initiated changes. Our objective was to identify a multimarker panel suitable for ovarian cancer, where each individual marker has its own baseline, permitting longitudinal algorithm development. MATERIALS AND METHODS: In this retrospective study, we measured CA125, human epididymis protein 4 (HE4), matrix metalloproteinase-7 (MMP-7), CA72-4, CA19-9, CA15-3, carcinoembryonic antigen, and soluble vascular cell adhesion molecule (sVCAM) concentrations using immunoassays in pretreatment sera from 142 stage I ovarian cancer cases and 5 annual samples each from 217 healthy controls. After random division into training and validation sets, all possible biomarker combinations were explored exhaustively using linear classifiers to identify the panel with the greatest sensitivity for stage I disease at a high specificity of 98%. To evaluate longitudinal performance of the individual markers, the within-person over time and the between-person coefficient of variation (CV) were estimated. Hierarchical modeling across women of log-concentrations enabled the borrowing of information across subjects to moderate variance estimates given the small number of observations per subject. RESULTS: The 4-marker panel comprising CA125, HE4, MMP-7, and CA72-4 performed with the highest sensitivity (83.2%) at 98% specificity. The within-person CVs were lower for CA125, HE4, MMP-7, and CA72-4 (15%, 25%, 25%, and 21%, respectively) compared with their corresponding between-person CV (49%, 20%, 35%, and 84%, respectively) indicating baselines in healthy volunteers. After simple log-transformations, the within-volunteer variation across volunteers was modeled with a normal distribution permitting parsimonious hierarchical modeling. CONCLUSIONS: The multiplex panel chosen is suitable for the early detection of ovarian cancer and the individual markers have their own baseline permitting longitudinal algorithm development.

Proteomic biomarkers apolipoprotein A1, truncated transthyretin and connective tissue activating protein III enhance the sensitivity of CA125 for detecting early stage epithelial ovarian cancer.

OBJECTIVE: The low prevalence of ovarian cancer demands both high sensitivity (>75%) and specificity (99.6%) to achieve a positive predictive value of 10% for successful early detection. Utilizing a two stage strategy where serum marker(s) prompt the performance of transvaginal sonography (TVS) in a limited number (2%) of women could reduce the requisite specificity for serum markers to 98%. We have attempted to improve sensitivity by combining CA125 with proteomic markers. METHODS: Sera from 41 patients with early stage (I/II) and 51 with late stage (III/IV) epithelial ovarian cancer, 40 with benign disease and 99 healthy individuals, were analyzed to measure 7 proteins [Apolipoprotein A1 (Apo-A1), truncated transthyretin (TT), transferrin, hepcidin, ß-2-microglobulin (ß2M), Connective Tissue Activating Protein III (CTAPIII), and Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4)]. Statistical models were fit by logistic regression, followed by optimization of factors retained in the models determined by optimizing the Akaike Information Criterion. A validation set included 136 stage I ovarian cancers, 140 benign pelvic masses and 174 healthy controls. RESULTS: In a training set analysis, the 3 most effective biomarkers (Apo-A1, TT and CTAPIII) exhibited 54% sensitivity at 98% specificity, CA125 alone produced 68% sensitivity and the combination increased sensitivity to 88%. In a validation set, the marker panel plus CA125 produced a sensitivity of 84% at 98% specificity (P=0.015, McNemar's test). CONCLUSION: Combining a panel of proteomic markers with CA125 could provide a first step in a sequential two-stage strategy with TVS for early detection of ovarian cancer.

Development of a multimarker assay for early detection of ovarian cancer.

PURPOSE: Early detection of ovarian cancer has great promise to improve clinical outcome. PATIENTS AND METHODS: Ninety-six serum biomarkers were analyzed in sera from healthy women and from patients with ovarian cancer, benign pelvic tumors, and breast, colorectal, and lung cancers, using multiplex xMAP bead-based immunoassays. A Metropolis algorithm with Monte Carlo simulation (MMC) was used for analysis of the data. RESULTS: A training set, including sera from 139 patients with early-stage ovarian cancer, 149 patients with late-stage ovarian cancer, and 1,102 healthy women, was analyzed with MMC algorithm and cross validation to identify an optimal biomarker panel discriminating early-stage cancer from healthy controls. The four-biomarker panel providing the highest diagnostic power of 86% sensitivity (SN) for early-stage and 93% SN for late-stage ovarian cancer at 98% specificity (SP) was comprised of CA-125, HE4, CEA, and VCAM-1. This model was applied to an independent blinded validation set consisting of sera from 44 patients with early-stage ovarian cancer, 124 patients with late-stage ovarian cancer, and 929 healthy women, providing unbiased estimates of 86% SN for stage I and II and 95% SN for stage III and IV disease at 98% SP. This panel was selective for ovarian cancer showing SN of 33% for benign pelvic disease, SN of 6% for breast cancer, SN of 0% for colorectal cancer, and SN of 36% for lung cancer. CONCLUSION: A panel of CA-125, HE4, CEA, and VCAM-1, after additional validation, could serve as an initial stage in a screening strategy for epithelial ovarian cancer.

Urinary levels of Bcl-2 are elevated in ovarian cancer patients.

OBJECTIVE(S): The poor prognosis associated with ovarian cancer is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors overexpress anti-apoptotic proteins, the purpose of this study was to determine whether elevated levels of the anti-apoptotic protein Bcl-2 were present in urine from patients with ovarian cancer. METHODS: Bcl-2 was assayed by ELISA in urine samples from two cohorts consisting of a total of 77 healthy women, 161 women with benign gynecologic disease and 150 women with ovarian cancer, 13 with early and 137 with late stage disease, respectively. Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. RESULTS: Urinary levels of Bcl-2 from healthy individuals or women with benign disease averaged 0.59 ng/ml+/-0.61 and 1.12 ng/ml+/-0.79, respectively. In contrast, urinary levels of Bcl-2 averaged 2.60 ng/ml+/-2.23 and 3.58 ng/ml+/-1.55 from women with early (N=13) and late (N=137) stage ovarian cancer. Further, urinary levels of Bcl-2 were elevated in ovarian cancer patients regardless of tumor grade, stage, size, histologic subtype, creatinine levels or patient age, but appeared to complement CA125 measurements. CONCLUSION(S): Levels of Bcl-2 are elevated in the urine of patients with ovarian cancer and may be of diagnostic and/or prognostic clinical importance. Further studies of urinary Bcl-2 as a biomarker for ovarian cancer alone or in combination with other markers are warranted.

Utility of a novel serum tumor biomarker HE4 in patients with endometrioid adenocarcinoma of the uterus.

OBJECTIVE: Tumor markers with increased sensitivity and specificity for endometrial cancer are needed to help monitor response to therapy and to detect recurrent disease. Currently, the tumor maker CA125 is utilized in this role with limited value. The objectives of this study were to examine the levels of several novel tumor markers HE4, SMRP, CA72.4 and CA125 as potential markers in patients diagnosed with endometrioid adenocarcinoma of the uterus. METHODS: Pre-operative serum samples from surgically staged patients with endometrioid adenocarcinoma of the uterus were analyzed for levels of HE4, SMRP, CA72-4 and CA125. Control samples were obtained from healthy postmenopausal women. Logistic regression models and receiver operating characteristic (ROC) curves were constructed for each tumor marker and for all combinations, with cross-validation analyses to obtain average sensitivities at set specificities of 90%, 95%, and 98%. RESULTS: Serum samples from 156 healthy subjects and 171 patients with endometrial cancer (122 stage I, 17 stage II, 26 stage III, and 6 stage IV) were analyzed. At a 95% specificity, the sensitivities for differentiating between healthy subjects and all stages of cancer were 45.5% for HE4 and 24.6% for CA125. For stage I disease, HE4 yielded a 17.1% improvement in sensitivity compared with CA125. CONCLUSION: HE4 is elevated in all stages of endometrial can100cer and is more sensitive in early-stage endometrial cancer compared to CA125. Further investigation of HE4 as a marker for early detection of recurrent endometrial cancer and monitoring response to therapy is warranted.

Early detection of ovarian cancer.

Despite advances in therapy, ovarian cancer remains the most deadly of the gynecological cancers. Less than 30% of women with advanced stage disease survive long-term. When diagnosed in stage I, up to 90% of patients can be cured with conventional surgery and chemotherapy. At present, only 25% of ovarian cancers are detected in stage I due, in part, to the absence of specific symptoms and to lack of an effective screening strategy. Early detection of ovarian cancer might significantly improve the overall survival rate of women with ovarian cancer if 1) most cancers are clonal and unifocal, arising in the ovary rather than in the peritoneum, 2) metastatic disease results from progression of clinically detectable stage I lesions, and 3) cancers remain localized for a sufficient interval to permit cost-effective screening. Given the prevalence of ovarian cancer, strategies for early detection must have high sensitivity for early stage disease (> 75%), but must have extremely high specificity (99.6%) to attain a positive predictive value of at least 10%. Transvaginal sonography (TVS), serum markers and a combination of the two modalities have been evaluated for early detection of ovarian cancer. Among the serum markers, CA125 has received the most attention, but lacks the sensitivity or specificity to function alone as a screening test. Greater specificity can be achieved by combining CA125 and TVS and/or by monitoring CA125 over time. Two stage screening strategies promise to be cost effective, where abnormal serum assays prompt TVS to detect lesions that require laparotomy. Accrual has been completed for a 200,000 woman trial in the United Kingdom that will test the ability of a rising CA125 to trigger TVS and subsequent exploratory surgery. Given the heterogeneity of ovarian cancer, it is unlikely that any single marker will be sufficiently sensitive to provide an effective initial screen. Sensitivity of serum assays might be enhanced by utilizing a panel of biomarkers. Candidate biomarkers have been discovered through empirical development of monoclonal antibodies, studies of gene expression, cloning of gene families and proteomic techniques. The development of technologies that measure multiple serum markers simultaneously, linked to the creation of statistical methods that enhance sensitivity without sacrificing specificity hold great promise.

Urinary mesothelin provides greater sensitivity for early stage ovarian cancer than serum mesothelin, urinary hCG free beta subunit and urinary hCG beta core fragment.

OBJECTIVES: Early detection of ovarian cancer should improve overall survival. Multiple serum markers have been evaluated as possible tests to detect early stage disease, but few urine markers have been studied. Mesothelin has been detected in serum from patients with ovarian cancer, but has not been previously reported in urine. METHODS: Mesothelin was assayed in the serum and in the urine from 28 patients with early stage (I/II) invasive epithelial ovarian cancers, 111 with advanced stage (III/IV) invasive disease and 19 with tumors of low malignant potential. Marker values have been compared to those in healthy controls and 115 patients with benign pelvic masses. Thresholds were set to include 95% of mesothelin values for 127 sera and 89 urines from healthy women. Urine values were considered: (1) as assayed; (2) normalized using the ratio of serum to urine creatinine; and (3) normalized using the Cockroft-Gault formula for glomerular filtration rate (GFR). Urines were also assayed for human chorionic gonadotropin (hCG) free beta subunit and beta subunit core fragment and similarly normalized. RESULTS: Optimal sensitivity for early stage disease was obtained when urine mesothelin was normalized using GFR. A greater fraction of patients with early stage disease was detected with the mesothelin urine assay (42%) than with the serum assay (12%). Similarly, 75% of patients with advanced ovarian cancer had elevated mesothelin in urine compared to 48% in serum. Serum and urine levels of mesothelin correlated for early (p=0.02) and late (p<0.001) disease. Urine mesothelin exhibited greater sensitivity for early stage ovarian cancer than did hCG free beta subunit or beta subunit core fragment and complementarity was not observed. CONCLUSION: Urine mesothelin deserves further evaluation as a biomarker for detection of early stage ovarian cancer in combination with other urinary markers.

Prevention and early detection of ovarian cancer: mission impossible?

Epithelial ovarian cancer is neither a common nor a rare disease. In the United States, the prevalence of ovarian cancer in postmenopausal women (1 in 2,500) significantly affects strategies for prevention and detection. If chemoprevention for ovarian cancer were provided to all women over the age of 50, side effects would have to be minimal in order to achieve an acceptable ratio of benefit to risk. This ratio might be improved by identifying subsets of individuals at increased risk or by bundling prevention of ovarian cancer with treatment for other more prevalent conditions. Approximately 10% of ovarian cancers are familial and relate to mutations of BRCA1, BRCA2, and mismatch repair genes. More subtle genetic factors are being sought in women with apparently sporadic disease. Use of oral contraceptive agents for as long as 5 years decreases the risk of ovarian cancer in later life by 50%. In one study, fenretinide (4-HPR) delayed development of ovarian cancer in women at increased risk of developing breast and ovarian cancer. Accrual to confirmatory studies has been prohibitively slow and prophylactic oophorectomy is recommended for women at increased genetic risk. Vaccines may have a role for prevention of several different cancers. Breast and ovarian cancers express mucins that could serve as targets for vaccines to prevent both cancers. Early detection of ovarian cancer requires a strategy with high sensitivity (> 75% for stage I disease) and very high specificity (> 99.6%) to achieve a positive predictive value of 10%. Transvaginal sonography (TVS) has achieved these values in some studies, but is limited by the cost of annual screening in a general population. Two-stage strategies that incorporate both serum markers and TVS promise to be more cost-effective. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risks for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the United Kingdom that will critically test the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125. Recent candidates include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s) and soluble EGF receptor. Proteomic approaches have been used to define a distinctive pattern of peaks on mass spectroscopy or to identify a limited number of critical markers that can be assayed by more conventional methods. Several groups are placing known markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

Biochemistry and biology of ARHI (DIRAS3), an imprinted tumor suppressor gene whose expression is lost in ovarian and breast cancers.

ARHI is a maternally imprinted tumor suppressor gene that is downregulated in 60% of ovarian and breast cancers. Loss of ARHI expression is associated with tumor progression in breast cancer and decreased disease-free survival in ovarian cancer. ARHI encodes a 26-kDa protein with 55-62% homology to Ras and Rap. In contrast to Ras, ARHI inhibits growth, motility, and invasion. ARHI contains a unique 34 amino-acid extension at its N-terminus and differs from Ras in residues critical for GTPase activity and for its putative effector function. Deletion of ARHI's unique N-terminal extension markedly reduces its inhibitory effect on cell growth. The gene maps to chromosome 1p31 at a site of LOH in 40% of ovarian and breast cancers. Mutations have not been detected, but the remaining allele is silenced by methylation in approximately 10-15 % of cases. In the remaining cancers, ARHI is downregulated by transcriptional mechanisms that involve E2F1 and E2F4, as well as by the loss of RNA binding proteins that decrease the half-life of ARHI mRNA. Transgenic expression of human ARHI in mice produces small stature, induces ovarian atrophy, and prevents postpartum milk production. Reexpression of ARHI in cancer cells inhibits signaling through Ras/Map and PI3 kinase, upregulates P21(WAF1/CIP1), downregulates cyclin D1, induces JNK, and inhibits signaling through STAT3. Marked overexpression of ARHI with a dual adenoviral vector induces caspase-independent, calpain-dependent apoptosis. When ARHI is expressed from a doxycycline-inducible promoter at more physiological levels, autophagy is induced, rather than apoptosis. Growth of ovarian and breast cancer xenografts is reversibly suppressed by ARHI, but expression of the NTD mutant produced only a limited inhibitory effect on growth of xenografts.

IL-6 signaling via the STAT3/SOCS3 pathway: functional analysis of the conserved STAT3 N-domain.

The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences.

Ethanol and aloe emodin alter the p53 mutational spectrum in ultraviolet radiation-induced murine skin tumors.

Mutations in the p53 tumor-suppressor gene contribute to the development of skin cancer, and the spectrum of mutations in this gene correlates with specific physical and chemical carcinogens in the environment. Cosmetics may contain alcohols and/or aloe emodin (AE). Although these compounds are not carcinogenic when applied to the skin, they may increase the carcinogenicity of ultraviolet (UV) radiation. We investigated whether ethanol (EtOH) and AE alone or combined with UV radiation cause mutations in the p53 gene. In the absence of UV radiation, C3H/HeN mice chronically treated for up to 33 wk with AE in 25% EtOH-in-water vehicle or vehicle alone failed to develop tumors and had no mutations in exons 4-8 of the p53 gene. UV radiation alone induced skin tumors, which had mutations predominantly in p53 exons 5 and 8. In contrast, mutations arising in UV + EtOH-or UV + AE-treated groups were more broadly distributed throughout the p53 gene. Mutations were found in exons 4, 6, and 7, as well as in exons 5 and 8. This altered distribution of mutations across the p53 DNA sequence more closely resembles the pattern observed in TP53 from human skin tumors at sun-exposed sites than that in the p53 gene of mice treated with UV alone. Thus, treatment with UV radiation in combination with two chemicals not thought to be carcinogenic, alcohol, and AE results in a broader distribution of mutations in a critical tumor-suppressor gene.