Based on the American Cancer Society, 21,880 new cases of ovarian cancer were expected to be diagnosed in the United States during 2010, and ovarian cancer will result in 13,850 deaths.1 Although the 1-year survival rate for patients with ovarian cancer is 75%, at 5 years, the survival rate declines to 46%. Yet, if this cancer is diagnosed and treated early-before the cancer has spread outside the ovary-the 5-year survival rate increases to 94%. Disconcertingly, only 15% of all ovarian cancers are detected at this early stage. Therefore, early diagnosis and aggressive treatment are of the highest priority in improving long-term survival in these women.
Cancer antigen 125 has been used as the criterion standard serum biomarker in the diagnosis and monitoring of ovarian cancer.2,3 However, CA 125 elevations are not specific to ovarian cancer; in fact, elevated levels of this antigen have been noted in numerous other conditions, both benign and malignant, such as the first trimester of pregnancy, breast cancer, endometriosis, and lesions that promote peritoneal irritation.4 Moreover, the specificity of CA 125 testing alone has been shown to be inadequate for effective early ovarian cancer diagnosis.5,6 In fact, approximately 20% of women with ovarian cancer display low concentrations of CA 125.7 Researchers have shown that these patients manifest relatively high levels of circulating immune complexes against CA 125, a finding that may contribute to lower-than-expected levels of the antigen in patients with ovarian cancer.7
In recent years, a large number of biomarkers for ovarian cancer have been examined as complements to CA 125.8,9 Human epididymis protein 4 (HE4, or WFDC2) represents an intriguing biomarker with a potential for improving early ovarian cancer diagnosis. Human epididymis protein 4, originally found in the epithelial cells of the human epididymis, is overexpressed in patients with ovarian carcinoma.10-12 In ovarian cancer, HE4 is up-regulated, with high levels detected in serous, endometrioid, and clear cell ovarian tumors.13 Moreover, unlike CA 125, HE4 has been found to be elevated in patients with mucinous ovarian tumors.14
In a study that examined which biomarkers of those typically used for ovarian cancer detection could boost the sensitivity of CA 125, HE4 was found to display the highest sensitivity of any single biomarker (73%), including CA 125 (43%). However, the highest sensitivity (76%) was achieved when HE4 was used in combination with CA 125; other biomarkers, such as CA 72-4, activin, inhibin, osteopontin, and epidermal growth factor, only minimally enhanced CA 125 sensitivity.15 These findings are consistent with those reported in recent studies by other investigators14,16 Intriguingly, increases in biomarkers for ovarian cancer, such as HE4, CA 125, and mesothelin, have been shown to occur up to 3 years before a clinical cancer diagnosis, offering the possibility at least for an extended lead time in the detection of early cancer with future biomarker refinements.17 However, the current likely lead time associated with these markers for the diagnosis of ovarian cancer, based on a substantial increase, is less than 1 year.
Certain cancer biomarkers have been shown to vary based on ethnicity.18,19 This study examined the role of HE4 in the diagnosis of ovarian cancer in Chinese subjects. We measured the levels of CA 125 and HE4 in Chinese women with ovarian cancer or with nonmalignant disorders (benign gynecologic diseases and surgical patients) and in healthy women to assess the value of HE4, both alone and in combination with CA 125 as a diagnostic marker for ovarian cancer.
MATERIALS AND METHODS
The trial, which was conducted in China at Peking University People's Hospital (PUPH), was exempt from institutional review board review owing to the fact that the biomarker analysis (HE4) was performed on leftover blood samples from routine blood testing; the testing did not pose any harm to patient, thus written informed consent was not obtained from patients; however, the blood samples were analyzed anonymously, protecting patient identity. The Medical Ethics Committee of PUPH made the following determinations related to this study:
- These abandoned samples did not injure patients. Thus, investigators did not need to obtain informed consent.
- Investigators were required to guarantee that the samples would be used only for medical scientific study purposes and not for any commercial purpose.
- Investigators were required to maintain the confidentiality of all personal patient information.
The serum sample population is shown in Table 1. Serum samples from 491 subjects were obtained from PUPH, Peking University First Hospital, and Beijing Jiuhua International Medical Center from January 2005 to January 2009. All subjects underwent surgical removal of an ovarian mass or a cyst. Tissue specimens from each subject were saved, examined, and centrally reviewed by 3 gynecologists and surgical pathologists to verify the diagnoses made by the onsite pathologists.
Interference samples were collected from the women who were pregnant or who were planning pregnancy with higher lipemic specimen at overall routine physical examination in PUPH.
Samples from controls were collected from Peking Jiuhua International Medical Center, which has a long history of cooperating with PUPH in scientific studies. These samples were collected and maintained in the same manner as those from patients in the tumor groups. Serum for the control group was obtained from women with normal physical examination findings.
All of the histologic evaluations and laboratory testing for serum CA 125 and HE4 were performed in a double-blind manner.
Blood was collected from each subject before surgery. Collected blood was allowed to clot and was centrifuged within 3 days (3000 rpm for 10-15 minutes). Sera were harvested and frozen at −70°C. The numbering of all of the samples was done double blinded. Any serum sample demonstrating hemolysis was considered invalid and excluded from the study.
Quantitative determination of HE4 and CA 125 was performed using the EIA kits from CanAg Technical Consulting Service Ltd (Beijing, China) according to manufacturer's instructions. Frozen sera were thawed slowly at 4°C, and the testing was performed at room temperature. The procedure was as follows: The well plate was washed, and 25 μL each of the HE4 calibration solution, controls, and specimens was pipetted into each well, respectively (duplicate replicate of the former two were used to assure its reliability); 100 μL of biotin anti-HE4 was added to each well, and the plate was incubated for 1 hour at room temperature (20°C-25°C) using a microplate shaker. Each strip was aspirated and washed 3 times, and 100 μL of tracer working solution was added to each well. The plate was incubated for 1 hour at room temperature (20°C-25°C) using a microplate shaker. Each strip was aspirated and washed 6 times, and 100 μL of TMB HRP-Substrate was added to each well as soon as possible. The plate was incubated for 30 minutes at room temperature (20°C-25°C) using a microplate shaker, avoiding exposure to direct sunlight; 100 μL of Stop Solution was added and mixed, and the absorbance at 405 nm was read immediately. The HE4 assay results were considered valid if the mean values of control duplicates were within the specified ranges. We repeated the experiment to confirm reliability. The result was presented as a median value. The reference ranges of HE4 and CA 125 were 0 to 150 pmol/L and 0 to 35 U/mL, respectively. A sample was treated as ovarian cancer-positive if either the value of HE4 or that of CA 125 was equal to or more than the cutoff value.
Quantitative assays were initially transformed into log scale owing to the wide range of the data points. Baseline characteristics of HE4 and CA 125 were compared via the Kruskal-Wallis test for nonparametric variables, followed by pairwise comparisons between the ovarian cancer cohort and each of the control groups using the Mann-Whitney U test. This comparison was also conducted between samples from subjects with epithelial cancer and those with nonepithelial cancer. Correlation between logarithmically transformed lnHE4 and lnCA125 was detected via the Spearman test. Sensitivities and specificities for both of the markers and the combination of the 2 markers were determined by 2 methods: (1) using given values as cutoffs (HE4 >150 pmol/L and CA 125 >35 U/mL) and (2) receiver operator characteristic (ROC) curves were constructed, and the area under the curve (AUC) was generated for the individual marker, and the markers were combined. Receiver operator characteristic curve analysis cutoff values with the specificities at 95% and 98% were determined. Sensitivities were then calculated using the ROC-derived cutoffs. Data involved in the ROC analysis included 2 individual marker assays and continuous combined marker assays generated via logistic regression analysis.
Two-sided P values were reported in all analyses. Differences and correlations were considered significant if P ≤ 0.05, unless otherwise indicated. Results are presented as median, with a range from the 25th to the 75th percentile for nonnormally distributive data.
Study data were analyzed using the Power Analysis and Sample Size software. This study statistically analyzed the patients with epithelial ovarian cancer (includes 4 serous fallopian tube carcinoma and 2 primary peritoneal carcinomas) at early stage (I-II, 12 cases) and advanced stage (III-IV, 30 cases) and calculated sample size and power. Analysis of variance results revealed that differences in marker levels based on histological grading of ovarian cancer at early stages (CA 125, 700.90 ± 1385.00; HE4, 460.56 ± 647.43) versus advanced (CA 125, 1921.57 ± 1707.91; HE4, 762.22 ± 583.53) were not statistically significant (P = 0.150). The study sample size was not sufficient to analyze differences among patients based on ovarian cancer stage. In this study, the power value was 0.65 (>0.8 was considered significant).
Serum levels of HE4 and CA 125 by group are shown in Table 2. At baseline, serum HE4 and CA 125 levels were significantly higher in the ovarian cancer group than in the other 5 reference groups (all P < 0.001).
The ovarian cancer group was divided into 2 subgroups: nonepithelial and epithelial. Serum HE4 and CA 125 levels were significantly higher in the epithelial ovarian cancer group; however, the increase was more robust with HE4 (P HE4 < 0.001, P CA125 = 0.005).
In subjects with ovarian cancer, both epithelial and nonepithelial, there was a significant and strong correlation between lnHE4 and lnCA125 (Spearman test; r = 0.708; P < 0.001; Fig. 1).
At the prespecified cutoffs (HE4 >150 pmol/L and CA 125 >35 U/mL), diagnostic sensitivity and specificity were calculated for the ovarian cancer group versus the 5 reference groups (Table 3). With healthy donors used as the controls, the sensitivity and specificity of the combined marker tests were 69% and 100%, respectively, and 92% and 98% for the parallel marker tests, respectively. Versus all reference groups individually, sensitivity for ovarian cancer was 73% for serum HE4 and 88% for serum CA 125. Across reference groups, HE4 specificity for ovarian cancer ranged from 90% to 100%, whereas CA 125 specificity for ovarian cancer ranged from 36% (in subjects with benign gynecologic disease) to 99%. The specificity of HE4 for distinguishing ovarian cancer from benign gynecologic disease was 100%. Combining both markers yielded specificity for distinguishing ovarian cancer from benign gynecological disease of 100%, with a sensitivity of 69%.
In the ROC curve analysis, healthy subjects were used as controls. Figure 2 displays the ROC curve for 3 diagnostic markers: lnHE4, lnCA125, and both markers combined. Areas under the curve of lnHE4, lnCA125, and the combination of the 2 markers were 0.917 (95% confidence interval [CI], 0.861-0.972), 0.919 (95% CI, 0.867-0.971), and 0.944 (95% CI, 0.8880.994), respectively (all P < 0.001).
Cutoffs for HE4 and CA 125 for the diagnosis of ovarian cancer at specificities of approximately 95% and 98% were also determined by ROC curve analysis (Table 4). Logistic regression analysis, using ROC-derived cutoff values, was used to determine the sensitivity of each tumor marker at predefined specificities of approximately 95% and 98% for all reference groups (Table 5). At predefined specificities of 95% and 98% for distinguishing ovarian cancer from absence of disease (healthy subjects), HE4 sensitivity was 84% and 82%, respectively; CA 125 sensitivity was 94% and 95%, respectively; and combined-marker sensitivity was 96% and 94%, respectively. Furthermore, when distinguishing the ovarian cancer group from the benign ovarian disease group at specificities of 95% and 98%, HE4 sensitivity was 74% and 72%, respectively; CA 125 sensitivity was 82% and 68%, respectively; and combined-marker sensitivity was 86% and 76%, respectively.
However, when differentiating ovarian cancer from benign gynecologic disease, HE4 demonstrated a 28% and 50% improvement over CA 125 in sensitivity at specificities of 95% and 98%, respectively. When distinguishing ovarian cancer from those who had undergone oncologic surgery (for breast, colorectal, or lung cancer), CA 125 showed a 22% and 44% improvement in sensitivity over HE4 at specificities of 95% and 98%, respectively. Using ROC-derived cutoffs, sensitivities for ovarian cancer were highest in the combined-marker group, except at 95% specificity in the benign gynecologic disease group using HE4 and at 95% and 98% specificities in the surgery group using CA 125 as a marker.
In this study, serum levels of HE4 and CA 125 were analyzed in Chinese women with ovarian cancer and in 5 control groups of subjects without ovarian cancer. Median levels of HE4 and CA 125 were found to be significantly higher in patients with ovarian cancer, compared with healthy controls and subjects with benign gynecological disorders, including ovarian disease. These findings are consistent with previously published reports in non-Chinese populations.15,20 Furthermore, in our study, lnHE4 and lnCA 125 levels displayed a significant and robust correlation (r = 0.78) in the detection of epithelial and nonepithelial ovarian cancers, and median levels of HE4 and CA 125 were significantly higher in the epithelial ovarian cancer group than in the nonepithelial ovarian cancer group, suggesting that both biomarkers are more sensitive to epithelial ovarian cancer. Other investigators also have shown a positive correlation between CA 125 and HE4 serum levels in patients with epithelial ovarian cancer in both Chinese21 and non-Chinese15 populations. In our study, HE4 displayed a sharper increase than CA 125 in patients with epithelial cancer, with levels increasing almost 6-fold in subjects with epithelial versus nonepithelial cancer, versus a 3-fold increase for CA 125 (P HE4 < 0.001, P CA125 = 0.005); thus, HE4 may be more sensitive to epithelial ovarian cancer than CA 125. This finding is not unexpected because HE4 expression seems to be limited to the epithelium, with robust and consistent overexpression in epithelial ovarian carcinomas.13,15 In contrast, CA 125 is expressed in the coelomic epithelium, amniotic fluid, pleura, peritoneum, pericardium, and in bronchial/cervical secretions, with relatively high levels present in noncancerous gynecologic conditions.22 Moreover, in the detection of epithelial ovarian cancer at a specificity of 95%, HE4 has been shown to display a sensitivity of 73% versus 43% for CA 125.15
Using predetermined cutoffs (HE4 >150 pmol/L and CA 125 >35 U/mL) to distinguish healthy subjects from subjects with ovarian cancer, HE4 displayed a sensitivity and specificity of 73% and 100%, respectively, and CA 125 displayed 88% and 99%, respectively. Combining HE4 and CA 125 typically diminished sensitivity compared with the use of either marker alone; the lower sensitivity for HE4 compared with CA 125 can be attributed to the cutoff value of 150 pmol/L used in this analysis. Other researchers have shown that lower HE4 cutoff values, such as 50 pmol/L, yield significantly higher sensitivity but lower levels of specificity for ovarian cancer than does the cutoff value of 150 pmol/L.23 In the current study, HE4 specificity at the 150-pmol/L cutoff was consistently higher than that seen with CA 125 for the detection of ovarian cancer versus all of the 5 reference groups. In fact, the specificity for differentiating ovarian cancer from benign gynecologic disease was 36% for CA 125 and 100% for HE4; however, the combined sensitivity was 100%, highlighting improved specificity for ovarian cancer with the use of both markers. This finding is consistent with findings reported by other investigators in non-Chinese14,15,20 and Chinese populations.23
When healthy donors were used as the controls, the ROC AUC yielded HE4 cutoffs of 102 and 150 pmol/L at 95% and 98% specificities, and CA 125 cutoffs of 127.2 and 325.5 U/mL, demonstrating that these values might provide clinically meaningful cutoffs in the diagnosis of ovarian cancer by providing an optimal sensitivity and specificity balance in Chinese patients.
Logistic regression analysis was used in our study to determine the sensitivity of each tumor marker to differentiate subjects with ovarian cancer from the control groups by incorporating the ROC-derived cutoffs to approximate specificities of 95% and 98%. These analyses were done for each marker independently as well as for a combination of the 2 markers. When distinguishing ovarian cancer from benign gynecologic disease, the sensitivity of CA 125 at the 95% and 98% specificity levels declined to 54% and 28%, respectively, from the 88% sensitivity seen with the preset cutoff of 35 U/mL. Sensitivity for ovarian cancer versus benign ovarian disease also declined using the higher ROC-derived cutoffs, highlighting the importance of cutoff levels as a consideration in achieving optimal sensitivity and specificity. When comparing patients with ovarian malignancy with those without ovarian malignancy, other researchers have shown that the specificity and accuracy of HE4 were 100% and 96%, respectively, at the 150- and 86-pmol/L cut points using ROC AUC data from Chinese subjects.23 These findings suggest that a lower cut point, closer to 86 pmol/L, may still be valuable in the screening for malignant ovarian cancer.
Consistent with previous findings in non-Chinese,15 notably higher sensitivities were seen for HE4 versus CA 125 at 95% and 98% specificities in distinguishing ovarian cancer from benign gynecologic disorders. However, higher sensitivities were seen with the use of CA 125 in distinguishing the ovarian cancer group from the oncologic surgery group. In almost all groups, the use of the combined marker yielded an increase in sensitivity, with 2 exceptions: HE4 performed better in distinguishing ovarian cancer group from benign gynecologic disease, and CA 125 performed better in distinguishing the ovarian cancer group from the oncologic surgery group.
This study had several limitations. Although the study included a total of 491 subjects, it was conducted at a single center in China, which may limit generalizability of the findings to the wider Chinese population. Furthermore, the study was not sufficiently powered to assess differences in marker levels between early and advanced ovarian cancer.
Overall, our findings in this Chinese population are generally consistent with those in non-Chinese populations in that HE4 was found to be a useful adjunct to CA 125 as a biomarker for differentiating patients with ovarian cancer from those with nonmalignant disorders, especially gynecologic disorders. The chief benefit seems to be an improvement in specificity with the use of both biomarkers. Our findings further indicate that the choice of a cutoff value for defining elevated HE4 serum levels has a clinically relevant impact on the sensitivity and specificity of HE4 alone and of the HE4/CA 125 combination. To maintain a high level of specificity (≥95%) in our study, the cutoff values for HE4 and CA 125 were approximately 103 pmol/L and 127 U/mL, respectively.
We thank Professor Xiaoping Kang of Peking University School of Public Health (Beijing, China), Dr Yuan-Yuan Fu of Peking University People's Hospital (Beijing, China), and Dr Zhong-Qian Li of Fujirebio Diagnostics, Inc (Malvern, PA) for statistical analyses and manuscript editing. Editorial assistance was also provided by James M. Kesslick, MS, Publication CONNEXION (Newtown, PA), which was contracted by Fujirebio Diagnostics, Inc for these services.
2. Yin BW, Dnistrian A, Lloyd KO. Ovarian cancer
antigen CA125 is encoded by the MUC16 mucin gene. Int J Cancer
3. Markman M, Federico M, Liu PY, et al. Significance of early changes in the serum CA-125 antigen level on overall survival in advanced ovarian cancer
. Gynecol Oncol
4. Sjovall K, Nilsson B, Einhorn N. The significance of serum CA 125
elevation in malignant and nonmalignant diseases. Gynecol Oncol
5. Menon U. Ovarian cancer
: challenges of early detection. Nat Clin Pract Oncol
6. Olivier RI, Lubsen-Brandsma MA, Verhoef S, et al. CA125 and transvaginal ultrasound monitoring in high-risk women cannot prevent the diagnosis of advanced ovarian cancer
. Gynecol Oncol
7. Cramer DW, O'Rourke DJ, Vitonis AF, et al. CA125 immune complexes in ovarian cancer
patients with low CA125 concentrations. Clin Chem
8. Kobayashi H, Yamada Y, Sado T, et al. A randomized study of screening for ovarian cancer
: a multicenter study in Japan. Int J Gynecol Cancer
9. Bast RC Jr, Urban N, Shridhar V, et al. Early detection of ovarian cancer
: promise and reality. Cancer Treat Res
10. Li J, Dowdy S, Tipton T, et al. HE4
as a biomarker for ovarian and endometrial cancer management. Expert Rev Mol Diagn
11. Schummer M, Ng WV, Bumgarner RE, et al. Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas. Gene
12. Hellstrom I, Raycraft J, Hayden-Ledbetter M, et al. The HE4
(WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res
13. Galgano MT, Hampton GM, Frierson HF Jr. Comprehensive analysis of HE4
expression in normal and malignant human tissues. Mod Pathol
14. Abdel-Azeez HA, Labib HA, Sharaf SM, et al. HE4
and mesothelin: novel biomarkers of ovarian carcinoma in patients with pelvic masses. Asian Pac J Cancer Prev
15. Moore RG, Brown AK, Miller MC, et al. The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol
16. Nolen B, Velikokhatnaya L, Marrangoni A, et al. Serum biomarker panels for the discrimination of benign from malignant cases in patients with an adnexal mass. Gynecol Oncol
17. Anderson GL, McIntosh M, Wu L, et al. Assessing lead time of selected ovarian cancer
biomarkers: a nested case-control study. J Natl Cancer Inst
18. Hein DW, Doll MA, Fretland AJ, et al. Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev
19. Yang G, Addai J, Ittmann M, et al. Elevated caveolin-1 levels in African-American versus white-American prostate cancer. Clin Cancer Res
20. Huhtinen K, Suvitie P, Hiissa J, et al. Serum HE4
concentration differentiates malignant ovarian tumours from ovarian endometriotic cysts. Br J Cancer
21. Liu YN, Ye X, Cheng HY, et al. [Measurement of serum human epididymis secretory protein 4 combined with CA125 assay in differential diagnosis of endometriosis cyst and ovarian benign and malignant tumors]. Zhonghua Fu Chan Ke Za Zhi
22. Gundogdu F, Soylu F, Erkan L, et al. The role of serum CA-125 levels and CA-125 tissue expression positivity in the prediction of the recurrence of stage III and IV epithelial ovarian tumors (CA-125 levels and tissue CA-125 in ovarian tumors). Arch Gynecol Obstet
. 2010;[Epub ahead of print].
23. Dong L, Chang XH, Ye X, et al. [The values of serum human epididymis secretory protein 4 and CA(125) assay in the diagnosis of ovarian malignancy]. Zhonghua Fu Chan Ke Za Zhi
Keywords:Copyright © 2011 by IGCS and ESGO
Ovarian cancer; Cancer biomarker; HE4; CA 125; Improved diagnosis