Human epididymis protein 4 (HE4) has been shown to be a promising serum biomarker for epithelial ovarian cancer (EOC). Recently, Hellstrom et al. reported the detection of HE4 in urine as a biomarker for ovarian neoplasms (Cancer Lett.2010;296:43–8). We investigated the feasibility of urinary HE4 in detection of EOC in a pilot study. METHODS: This retrospective study measured the HE4 levels in urine samples obtained from an institutional review board-approved sample bank. HE4 was measured with HE4 EIA (Fujirebio Diagnostics, Inc.). Creatinine was measured via colorimetric determination by the Jaffe Reaction and used to normalize the HE4 levels. Clinical diagnosis of the subjects was based on the pathological diagnosis. Single-point urine samples were analyzed from 232 subjects; 72 with EOC or low malignant potential tumors and 160 with benign disease. RESULTS: The medians of HE4 levels in subjects with EOC and with benign disease were 29833 pmol/L (95% Confidence Interval (CI): 21185–54728) and 8515 pmol/L (95% CI: 7077–9314) respectively. The medians of HE4/Creatinine ratios in subjects with EOC and with benign disease were 420 (95% CI: 269–491) and 75 (95% CI: 68–83), respectively. Both the medians of HE4 levels and HE4/Creatinine ratios in EOC subjects were significantly higher than that in subjects with benign disease (P<0.0001 in Median test). Receiver operator characteristic (ROC) analysis showed that areas under the curve (AUC) were 0.87 for HE4/Creatinine ratio (95% CI = 0.81–0.92) and 0.83 for HE4 level (95% CI = 0.76–0.89). There was no significant difference between the AUC for HE4 level and HE4/Creatinine ratio (P>0.05). Using the shoulder point of each ROC curve as the cut-off, the cut-off values were 152.5 for HE4/Creatinine ratio and 19161 pmol/L for HE4 level respectively. The sensitivity, specificity, positive predictive value and negative predictive value at the set cut-off values were 70.8% (95% CI = 58.9% – 81.0%), 93.1% (95% CI = 88.0% – 96.5%), 82.3% and 87.6% for HE4/Creatinine ratio, and 65.3% (95% CI = 53.1% – 76.1%), 89.4% (95% CI = 83.5% – 93.7%), 73.4% and 85.1% for HE4 level, respectively. Concordance rates versus clinical diagnosis were 86.2% for HE4/creatinine ratio and 81.9% for HE4 level. CONCLUSIONS: This pilot study demonstrated the utility of urinary HE4 level alone or HE4/Creatinine ratio as potential biomarkers for the detection of EOC. The HE4/Creatinine ratio appeared to be better than the HE4 level in correlating with the clinical pathological diagnosis of EOC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-451. doi:10.1158/1538-7445.AM2011-LB-451

Human epididymis protein 4 (HE4) is a whey acidic four-disulfide core protein. Serum HE4 has been shown to be a promising biomarker for epithelial ovarian cancer (EOC) with manual HE4 EIA assays. The ARCHITECT system is one of the major automated formats for cancer immunodiagnostics. This abstract describes the clinical performance of an investigational ARCHITECT HE4 assay in the measurement of serum HE4 from longitudinal samples of subjects with EOC and single-point samples from healthy subjects, and subjects with malignancy, benign diseases, EOC or other malignant diseases. PROCEDURES: The ARCHITECT HE4 Assay is a paramagnetic microparticle chemiluminescent assay adopting the sensitive CHEMIFLEX® technology. Single-point and longitudinal serum samples were tested with an investigational ARCHITECT HE4 Assay reagent lot on the ARCHITECT i2000SR. Single-point sera were collected from healthy subjects (N = 400), subjects with pregnancy (N = 50) and benign disease (N = 612), and subjects which were diagnosed with EOC (N = 314) and other cancers (N = 250) including endometrial, breast, gastrointestinal, lung, and bladder cancer. Longitudinal sera (N = 506), including initial time point and follow-up visits (average = 5.7 per subject), were collected from subjects (N = 76) which were diagnosed with EOC. All of the single-point and longitudinal samples were handled in accordance with IRB-approved and GCP-compliant protocols. RESULTS: Using an ARCHITECT HE4 cut-off value of >140 pmol/L, the percent distributions of ARCHITECT HE4 by cohort are: 3% of healthy subjects, 0% with pregnancy, 7.2% of subjects with benign diseases, 53.5% with EOC and 28.4% with other cancers. Using the upper 95% point of the distribution of the HE4 ratio at 14% to categorize the successive HE4 readings into those that were and were not significantly elevated for the longitudinal samples, the sensitivity, specificity and positive predictive value of HE4 elevation versus disease progression were 53.4% (95% CI: 44.1%-62.5%), 78.8% (95% CI: 74.7% - 82.4%) and 43.2% (95% CI: 32.9% - 54.1%) respectively. The total concordance between HE4 elevation and disease progression was 73%. Receiver Operator Characteristic analysis showed an area under the curve of 0.685 (SE = 0.033) for the diagnosis of progression from the ratio of successive HE4 readings. When comparing the change of the HE4 ratio with the diagnostic response to therapy, a decrease in HE4 reflected response to therapy with a sensitivity of 82% and a specificity of 53%. CONCLUSIONS: The study with single-point samples has shown the utility of ARCHITECT HE4 values as a serum biomarker for EOC. The longitudinal increase of ARCHITECT HE4 values in sera seemed to be effective in recognizing the progression of EOC. The longitudinal decrease of ARCHITECT HE4 values in sera seemed to correlate with the response of EOC subjects to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4653.

e15565 Background: CYFRA 21-1 is a known lung cancer biomarker. In subjects with ovarian cancer (OC) versus those with benign gynecological diseases, serum CYFRA 21-1 was reported sensitivities (SN) of 44% and 41% at specificity (SP) of 95% for OC detection using an ELISA assay by Hasholzner U (1994) and Tempfer C (1998), respectively. Nolen B (2010) reported a SN of 84.1% and 85.8% and a SP of 85% and 79.3% with a combination of CYFRA 21-1, CA 125 and HE4 using a multiplex bead-based immunoassay in a training and validation set, respectively. We carried out a cohort study to evaluate the serum CYFRA 21-1 as an ovarian cancer biomarker and compared it with ROMA (Risk of Ovarian Malignancy Algorithm), an FDA-cleared ovarian cancer algorithm. Methods: ARCHITECT CYFRA 21-1 assay was used to measure the levels of serum CYFRA 21-1. Single point serum samples from 170 female subjects with a pelvic mass were randomly selected from an archive cohort of female subjects. Among the 170 subjects, 47 had OC (42 with epithelial OC and 5 with low malignant potential tumor) and 123 with benign gynecological diseases. ROMA values were obtained with HE4 EIA and ARCHITECT CA 125 II. Results: For ARCHITECT CYFRA 21-1, the ROC-AUC, cut-off, SN, SP, PPV and NPV were 86%, 1.8 ng/mL, 70.2% (95% CI: 55.1% to 82.7%), 95.1% (95% CI: 89.7% to 98.2%), 84.6% and 89.3%, repectively. For ROMA algorithm, the ROC-AUC, cut-off, SN, SP, PPV and NPV were 92%, 2.44, 89.4% (95% CI: 76.9% to 96.5%), 84.6% (95% CI: 76.9% to 90.4%), 68.9% and 95.4%, repectively. Chi-square test showed an SN for ROMA was significantly higher than that of ARCHITECT CYFRA 21-1 (p < 0.05), and SP for ARCHITECT CYFRA 21-1 was significantly higher than that of ROMA (p < 0.01). Conclusions: In distinguishing OC from benign gynecological diseases, ROMA algorithm appears to be more sensitive than serum ARCHITECT CYFRA 21-1 (p < 0.05), and serum ARCHITECT CYFRA 21-1 appears to be more specific than (p < 0.01) in this cohort study.

5563 Background: Urinary HE4 has been reported as a promising biomarker for ovarian cancer (OC). In the current study, a large validation was performed to evaluate urinary HE4 as a biomarker for the stratification of OC from benign pelvic masses. Methods: Normalized HE4 was obtained from the ratio of HE4 (pmol/L measured with HE4 EIA) and creatinine (mg/dL measured with a Jaffe Reaction) in single-point urine samples from female subjects with a pelvic mass (N = 809). The samples were from one case-control prospective study (Moore RG, 2008) and two prospective clinical trials (NCT00315692 and NCT00987649). R Package was used to randomly distribute the subjects into the Training and Testing Sets. Analyze-it was used to analyze the clinical performance. Results: Medians of Normalized HE4, sensitivities (SN) and likelihood ratios (LR) (+) of Normalized HE4 at two levels of specificity (SP) are presented in the Table. Conclusions: Urinary HE4 normalized with urinary creatinine appears to be a valid biomarker for the stratification of OC from a benign pelvic mass. [Table: see text]

CYFRA 21-1 is a known lung cancer biomarker. In subjects with ovarian cancer (N = 159) and those with benign gynecological diseases (N = 53), serum CYFRA 21-1 reported a sensitivity (SN) of 44% and a specificity (SP) of 95% for ovarian cancer detection at a cut-off value of 2.4 ng/mL using an ELISA assay (Hasholzner U, et al, 1994). Another report showed a SN of 41% and a SP of 95% for ovarian cancer (N = 37) versus benign ovarian cysts, endometriosis, pelvic inflammatory disease, bowel disease and liver cirrhosis (N = 168) using an ELISA assay (Tempfer C, et al, 1998). We carried out a pilot study to evaluate the serum CYFRA 21-1 as a biomarker for stratification of ovarian cancer risk in women with a pelvic mass. Methods: ARCHITECT CYFRA 21-1 was used to measure the levels of serum CYFRA 21-1. Single point frozen serum samples from 171 female subjects with a pelvic mass were randomly selected from the archive clinical trial described by Moore R et al (2009). Of the 171 subjects, 42 had epithelial ovarian cancer (EOC), 5 with low malignant potential tumor (LMP) and 124 with benign pelvic masses. Results: To stratify EOC from benign pelvic masses and LMP, the following performance was obtained for serum CYFRA 21-1: Notes: The cutpoints were set at 1.2 and 1.8 ng/mL to reach a 75% and 95% SP, respectively. Conclusion: Serum CYFRA 21-1 appears to be a useful biomarker for stratification of EOC from benign pelvic mass and LMP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3574. doi:1538-7445.AM2012-3574