Influence of ovarian cancer risk status on the diagnostic performance of the serum biomarkers mesothelin, HE4, and CA125.

OBJECTIVE: To evaluate the effect of ovarian cancer risk on the performance of the serum biomarkers mesothelin, human epididymis protein 4 (HE4), and CA125. METHODS: We measured mesothelin, HE4, and CA125 levels from women with invasive ovarian cancer (n = 143), benign gynecologic conditions (n = 124), and controls (n = 344). Demographic, epidemiologic, reproductive, medical, and family history data were collected using a standardized questionnaire. Pedigree and BRCA 1/2 test results were used to stratify women into average and high-risk groups. The diagnostic accuracy of each biomarker was characterized using receiver operating characteristic curve methods. RESULTS: Baseline characteristics did not vary by risk or case status. The distribution of stage and histology was similar in average and high-risk women. All three markers discriminated ovarian cancer cases from risk-matched healthy and benign controls. Marker performance did not vary by risk status. The sensitivity at 95% specificity for discriminating cases from risk-matched healthy control women in the average and high-risk groups, respectively, was 53.9% and 39.0% for mesothelin, 80.4% and 87.8% for HE4, and 79.4% and 82.9% for CA125. The performance of the markers was not as robust when cases were compared with benign controls. Area under the curve values for cases versus healthy and benign controls did not vary by risk status. CONCLUSIONS: The ability of serum mesothelin, HE4, and CA 125 levels to discriminate ovarian cancer cases from healthy and benign controls is not influenced by risk status. Our findings support the pursuit of additional studies evaluating the early detection potential of these markers in high-risk populations.

Use of yeast-secreted in vivo biotinylated recombinant antibodies (Biobodies) in bead-based ELISA.

PURPOSE: To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative. EXPERIMENTAL DESIGN: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls. RESULTS: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38). CONCLUSION: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead-based assays for use in large validation studies.

Validation of LRG1 as a potential biomarker for detection of epithelial ovarian cancer by a blinded study.

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets. METHODS AND RESULTS: Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (p=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (p=0.018) in detecting early stage EOC cases from non-cases using the larger sample set. CONCLUSIONS: LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.

JAK2 expression is associated with tumor-infiltrating lymphocytes and improved breast cancer outcomes: implications for evaluating JAK2 inhibitors.

Janus kinase-2 (JAK2) supports breast cancer growth, and clinical trials testing JAK2 inhibitors are under way. In addition to the tumor epithelium, JAK2 is also expressed in other tissues including immune cells; whether the JAK2 mRNA levels in breast tumors correlate with outcomes has not been evaluated. Using a case-control design, JAK2 mRNA was measured in 223 archived breast tumors and associations with distant recurrence were evaluated by logistic regression. The frequency of correct pairwise comparisons of patient rankings based on JAK2 levels versus survival outcomes, the concordance index (CI), was evaluated using data from 2,460 patients in three cohorts. In the case-control study, increased JAK2 was associated with a decreasing risk of recurrence (multivariate P = 0.003, n = 223). Similarly, JAK2 was associated with a protective CI (<0.5) in the public cohorts: NETHERLANDS CI = 0.376, n = 295; METABRIC CI = 0.462, n = 1,981; OSLOVAL CI = 0.452, n = 184. Furthermore, JAK2 was strongly correlated with the favorable prognosis LYM metagene signature for infiltrating T cells (r = 0.5; P < 2 × 10(-16); n = 1,981) and with severe lymphocyte infiltration (P = 0.00003, n = 156). Moreover, the JAK1/2 inhibitor ruxolitinib potently inhibited the anti-CD3-dependent production of IFN-γ, a marker of the differentiation of Th cells along the tumor-inhibitory Th1 pathway. The potential for JAK2 inhibitors to interfere with the antitumor capacities of T cells should be evaluated.

Impact of screening test performance and cost on mortality reduction and cost-effectiveness of multimodal ovarian cancer screening.

Ongoing ovarian cancer screening trials are investigating the efficacy of a two-step screening strategy using currently available blood and imaging tests [CA125 and transvaginal sonography (TVS)]. Concurrently, efforts to develop new biomarkers and imaging tests seek to improve screening performance beyond its current limits. This study estimates the mortality reduction, years of life saved, and cost-effectiveness achievable by annual multimodal screening using increasing CA125 to select women for TVS, and predicts improvements achievable by replacing currently available screening tests with hypothetical counterparts with better performance characteristics. An existing stochastic microsimulation model is refined and used to screen a virtual cohort of 1 million women from ages 45 to 85 years. Each woman is assigned a detailed disease course and screening results timeline. The preclinical behavior of CA125 and TVS is simulated using empirical data derived from clinical trials. Simulations in which the disease incidence and performance characteristics of the screening tests are independently varied are conducted to evaluate the impact of these factors on overall screening performance and costs. Our results show that when applied to women at average risk, annual screening using increasing CA125 to select women for TVS achieves modest mortality reduction (~13%) and meets currently accepted cost-effectiveness guidelines. Screening outcomes are relatively insensitive to second-line test performance and costs. Identification of a first-line test that does substantially better than CA125 and has similar costs is required for screening to reduce ovarian mortality by at least 25% and be reasonably cost-effective.

Irradiation effect after mastectomy on breast cancer recurrence in patients presenting with locally advanced disease.

BACKGROUND: Current guidelines recommend postmastectomy irradiation (PMI) for patients with tumors >5 cm and/or ≥4 positive lymph nodes. This study evaluates the effect of PMI on recurrence and survival within tumor size and node status groups. METHODS: Locoregional and distant recurrences and survival for different tumor and treatment characteristics were analyzed in 2,797 patients with invasive breast cancer treated with mastectomy. RESULTS: Tumor size, positive nodes, extranodal extension, lymphatic/vascular invasion, estrogen receptor/progesterone negative, HER-2 positive, and high grade were associated with significantly increased recurrence. In patients with ≥4 positive nodes and patients with tumors >5 cm and positive nodes, PMI decreased local and distant recurrence (overall 53% vs 24%, P < .001) and increased disease-free survival (P < .001) but not overall survival. In patients with less disease, a benefit from PMI irradiation could not be identified. CONCLUSIONS: PMI is indicated for patients with ≥4 positive nodes or patients with tumors >5 cm and positive nodes.

Potential role of HE4 in multimodal screening for epithelial ovarian cancer.

In screening for epithelial ovarian cancer, unnecessary surgery can be reduced by limiting use of transvaginal ultrasound (TVU) to women with increasing CA125 serum levels. Replacing or augmenting TVU with measurement of a serum marker specific for malignancy might further improve screening performance. Serum samples from 112 invasive ovarian cancer patients and 706 matched control subjects from the Prostate, Lung, Colorectal, and Ovarian trial were used to evaluate human epididymis protein 4 (HE4), mesothelin, matrix metalloproteinase 7 (MMP7), SLPI, Spondin2, and insulin-like growth factor binding protein 2 (IGFBP2) for their potential use in screening. TVU results were available for a subset of 84 patients and 516 control subjects used to compare the best marker with TVU. HE4 was found to perform better than TVU as a second-line screen, confirming 27 of 39 cancers with increasing CA125 serum levels compared with 17 cancers confirmed by TVU (P = .03). Serum HE4 levels were found to increase with age and smoking status, suggesting that a longitudinal algorithm might improve its performance.

Assessing lead time of selected ovarian cancer biomarkers: a nested case-control study.

BACKGROUND: CA125, human epididymis protein 4 (HE4), mesothelin, B7-H4, decoy receptor 3 (DcR3), and spondin-2 have been identified as potential ovarian cancer biomarkers. Except for CA125, their behavior in the prediagnostic period has not been evaluated. METHODS: Immunoassays were used to determine concentrations of CA125, HE4, mesothelin, B7-H4, DcR3, and spondin-2 proteins in prediagnostic serum specimens (1-11 samples per participant) that were contributed 0-18 years before ovarian cancer diagnosis from 34 patients with ovarian cancer (15 with advanced-stage serous carcinoma) and during a comparable time interval before the reference date from 70 matched control subjects who were participating in the Carotene and Retinol Efficacy Trial. Lowess curves were fit to biomarker levels in cancer patients and control subjects separately to summarize mean levels over time. Receiver operating characteristic curves were plotted, and area-under-the curve (AUC) statistics were computed to summarize the discrimination ability of these biomarkers by time before diagnosis. RESULTS: Smoothed mean concentrations of CA125, HE4, and mesothelin (but not of B7-H4, DcR3, and spondin-2) began to increase (visually) in cancer patients relative to control subjects approximately 3 years before diagnosis but reached detectable elevations only within the final year before diagnosis. In descriptive receiver operating characteristic analyses, the discriminatory power of these biomarkers was limited (AUC statistics range = 0.56-0.75) but showed increasing accuracy with time approaching diagnosis (eg, AUC statistics for CA125 were 0.57, 0.68, and 0.74 for > or = 4, 2-4, and <2 years before diagnosis, respectively). CONCLUSION: Serum concentrations of CA125, HE4, and mesothelin may provide evidence of ovarian cancer 3 years before clinical diagnosis, but the likely lead time associated with these markers appears to be less than 1 year.

Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.

BACKGROUND: The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery. METHODOLOGY/PRINCIPAL FINDINGS: We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease. CONCLUSIONS/SIGNIFICANCE: Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

Systematic evaluation of candidate blood markers for detecting ovarian cancer.

BACKGROUND: Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer. METHODS AND FINDINGS: We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers--MUC16, WFDC2, MSLN and MMP7--warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer. CONCLUSIONS: By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

Effects of blood collection conditions on ovarian cancer serum markers.

BACKGROUND: Evaluating diagnostic and early detection biomarkers requires comparing serum protein concentrations among biosamples ascertained from subjects with and without cancer. Efforts are generally made to standardize blood processing and storage conditions for cases and controls, but blood sample collection conditions cannot be completely controlled. For example, blood samples from cases are often obtained from persons aware of their diagnoses, and collected after fasting or in surgery, whereas blood samples from some controls may be obtained in different conditions, such as a clinic visit. By measuring the effects of differences in collection conditions on three different markers, we investigated the potential of these effects to bias validation studies. METHODOLOGY AND PRINCIPLE FINDINGS: We analyzed serum concentrations of three previously studied putative ovarian cancer serum biomarkers-CA 125, Prolactin and MIF-in healthy women, women with ovarian cancer undergoing gynecologic surgery, women undergoing surgery for benign ovary pathology, and women undergoing surgery with pathologically normal ovaries. For women undergoing surgery, a blood sample was collected either in the clinic 1 to 39 days prior to surgery, or on the day of surgery after anesthesia was administered but prior to the surgical procedure, or both. We found that one marker, prolactin, was dramatically affected by collection conditions, while CA 125 and MIF were unaffected. Prolactin levels were not different between case and control groups after accounting for the conditions of sample collection, suggesting that sample ascertainment could explain some or all of the previously reported results about its potential as a biomarker for ovarian cancer. CONCLUSIONS: Biomarker validation studies should use standardized collection conditions, use multiple control groups, and/or collect samples from cases prior to influence of diagnosis whenever feasible to detect and correct for potential biases associated with sample collection.