Abstract
Objectives
To evaluate the impact of different biologic, histopathologic and lifestyle factors on serum levels of human epididymis protein 4 (HE4) and Cancer antigen 125 (CA125) in the diagnostic work up of women with an ovarian cyst or pelvic tumor.
Methods
The statistical evaluation was performed on a population of 445 women diagnosed with a benign ovarian disease, included in a large Swedish multicenter trial (ClinicalTrials.gov NCT03193671). Multivariable logistic regression analyses were performed to distinguish between the true negatives and false positives through adjusting for biologic, histopathologic and lifestyle factors on serum samples of CA125 and HE4 separately. The likelihood ratio test was used to determine statistical significance and Benjamini-Hochberg correction to adjust for multiple testing.
Results
A total of 31% of the women had false positive CA125 but only 9% had false positive results of HE4. Smoking (OR 6.62 95% CI 2.93–15.12) and impaired renal function, measured by eGFR (OR 0.18 95% CI 0.08–0.39), were independently predictive of falsely elevated serum levels of HE4. Endometriosis was the only variable predictive of falsely elevated serum levels of CA125 (OR 7.96 95% CI 4.53–14.39). Age correlated with increased serum levels of HE4.
Conclusions
Smoking, renal failure, age and endometriosis are factors that independently should be considered when assessing serum levels of HE4 and CA125 in women with an ovarian cyst or pelvic mass to avoid false indications of malignant disease.
Introduction
When women are diagnosed with an ovarian cyst or tumor in the small pelvis, there is often an uncertainty about its malignancy potential. Current consensus suggests analysis of serum biomarkers such as cancer antigen 125 (CA125) and perhaps serum human epididymis 4 (HE4), a radiological examination and transvaginal ultrasound (TVU) [1]. Despite sufficient sensitivity for detection of ovarian cancer (OC) in postmenopausal (post-M) women, we lack acceptable specificity with current approaches [2, 3]. The above mentioned strategy results in false positive cases, unnecessary surgical procedures, and increased morbidity and, due to oophorectomy, hormonal loss and cardiovascular mortality [4].
Since the 1990s, CA125 has been used in the diagnostic work up despite a sensitivity of less than 50% in stage 1 OC in premenopausal (pre-M) women [5]. In addition, CA125 may be falsely elevated in several benign or non-cancer conditions [6]. The more recently detected HE4 [7], [8], [9] is also expressed in other tissues besides the female genital tract [10, 11]. HE4 has been suggested to have a higher sensitivity than CA125 in stage I OC but lacks, as CA125 does, expression in mucinous histology [11], [12], [13].
Despite the widely accepted preoperative usability of CA125 in post-MP women, it is only recommended by the U.S. Food and Drug administration (FDA) for monitoring OC treatment. To increase both sensitivity and specificity, several algorithms in combinations with or without TVU have been developed. These include risk of malignancy index (RMI), risk of ovarian malignancy algorithm (ROMA) and the International Ovarian Tumor Analysis (IOTA) group, second generation multivariate index assay (MIA2G), Copenhagen index (CPH-1), ROMA-P and GOT 1–3 [14], [15], [16], [17], [18], [19], [20], of which only ROMA and MIA2G have been FDA approved for differential diagnostics [21, 22].
The aim of the current study was to increase our knowledge and interpretation of falsely elevated tumor biomarkers CA125 and HE4. We have selected a subgroup of women with a benign ovarian histology (n=445) from a previously published large cohort of unselected women with an ovarian cyst or pelvic mass [2, 20]. False positives and true negatives according to recommended cut-offs have been analyzed by multivariable regression analyses comparing the various interfering biological and lifestyle factors, such as age, BMI, parity, heredity, hormonal replacement therapy (HRT), endometriosis, pelvic inflammatory disease (PID), other cancer, smoking, renal and heart function, and histology that may influence the performance of CA125 and HE4 to correctly diagnose an unknown ovarian cyst or pelvic tumor.
Materials and methods
Study population
We have evaluated 445 women with a tumor of benign ovarian histology, collected from September 2013 to February 2016. The patients were included in a Swedish multicenter study evaluating 638 women in the western healthcare region [2] registered in the National Institute of Health clinical trial registry Clinical Trial.gov (ClinicalTrials.gov NCT03193671).
Study design
Inclusion and exclusion criteria were previously described in detail [2]. The women included were clinically assessed with pelvic examination and TVU by both experienced doctors and doctors under training. Menopausal status was noted for all patients, and clinical data on biological and lifestyle factors for every patient was obtained as self-reported information or noted by the examining physician prior to surgery. The collected parameters included age, BMI, smoking (yes or no), heredity for ovarian or breast cancer, history of non-ovarian cancer, heart disease (hypertension, arrythmia, congestive heart failure, chronic and acute ischemic heart disease, valvopathy), kidney disease, parity and ongoing HRT (Table 1). Surgery was performed at all the hospitals in the western region (n=6) according to standardized procedures and pathologists specializing in gynecological diseases examined the surgically excised specimens.
Biological and lifestyle factors | Benign | ||
---|---|---|---|
Pre-M | Post-M | Total | |
n, % | 230 | 215 | 445 |
Age, years, std | 38.8a (9.77) | 63.6a (8.64) | 50.8a (15.48) |
BMI, kg/m2, std | 25.9a (4.70) | 25.8a (4.71) | 25.9a (4.70) |
Smoking | 36 (15.7%) | 32 (14.9%) | 68 (15, 3%) |
Heredity | 17 (7.4%) | 11 (5.1%) | 28 (6.3%) |
Other cancer disease | 18 (9.7%) | 25 (11.6%) | 43 (9.7%) |
Heart disease | 13 (5.7%) | 63 (29.3%) | 76 (17.1%) |
Kidney disease | 7 (3.0%) | 8 (3.7%) | 15 (3.4%) |
HRT | 1 (0.4%) | 16 (7.4%) | 17 (3.8%) |
Parity (0) | 87 (37.8%) | 35 (16.3%) | 122 (27.4%) |
Parity (1–2) | 110 (47.8%) | 119 (55.3%) | 229 (51.5%) |
Parity (>2) | 33 (14.3%) | 61 (28.4%) | 94 (21.1%) |
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aMean. Biological and lifestyle factors for the 445 women. Children are denoted as 0 = no children, 1–2 = one or two children, and >2 = more than two children. n, number of women; std, standard deviation; BMI, body mass index; HRT, hormone replacement therapy; Pre-M, premenopausal; Post-M, postmenopausal.
Serum analyses/sample collection
Serum for the analysis of CA125 (U/mL), HE4 (/L), creatinine (μmol/L) and pro-BNP (ng/L) were collected at the time of enrollment prior to surgery, centrifuged and stored in 4 °C before being shipped, according to standardized procedures, to Sahlgrenska University hospital as previously described [2]. The coded samples were analyzed at an accredited central laboratory (ISO 15189) Sahlgrenska University hospital in three separate batches. CA125, HE4, NT-pro-BNP and creatinine were analyzed using the Elecsys HE4 and Elecsys CA125 II with the electrochemiluminescence (ECLIA) technique (Cobas 6,000 and 8,000, RocheDiagnostics Scandinavia, Stockholm, Sweden) [2]. Recommended cut-offs according to the manufacturer were used in the study; CA125 > 35 U/mL, HE4 pre-M > 70 pmol/L, HE4 post-M > 140 pmol/L [23]. Glomerular filtration rate (eGFR) was estimated using creatinine as a measure of renal function using the revised Lund-Malmö equation [24]. Continuous levels of eGFR were used as a measure for impaired kidney function and continuous levels of NT-pro-BNP were used as a measure of heart disease in the multivariable analyses.
Histopathologic subgroups
The 445 women were divided into seven benign histological subgroups. For each histology, the median, range, mean and standard deviation for CA125 and HE4 were determined for both pre-M and post-M women (Table 2). The subgroup simple cysts included mesonephric, rete ovarii and inclusion cysts; PID included tubo-ovarian abscess, hemato-, sacto-, pyo- and hydro-salpinx; stromal included fibroma, fibro-thecoma and thecoma; serous and mucinous adenomas, adenofibromas and cystadenomas were denoted as serous or mucinous. The diagnosis endometrioma or endometriosis was set after surgical resection and pathologic evaluation. In 17 cases, endometriosis was noted in the pathology report concomitant with another cyst diagnosis, denoted concomitant endometriosis (CE). In two cases, endometriosis was diagnosed at a previous surgery.
Histology | n | Age, years, mean | HE4 | CA125 | ||||||
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Mean | Std | Median | Range | Mean | Std | Median | Range | |||
Pre-M | ||||||||||
Endometrioma + CEa | 53 + 12a | 39.9 | 50.9 | 11.4 | 48, 0 | 33.4–83.6 | 100.7 | 181.8 | 67.0 | 12.0–1, 427.0 |
Teratoma | 47 | 36.1 | 49.1 | 13.1 | 45.3 | 28.8–96.7 | 35.5 | 48.2 | 22.0 | 6.0–326.0 |
PID | 6 | 49.3 | 51.9 | 24.1 | 45.8 | 32.3–98.6 | 16.8 | 10, 0 | 13.0 | 11.0–37.0 |
Mucinous | 26 | 38.0 | 54.8 | 17.9 | 49.4 | 34.0–118.0 | 29.3 | 21.1 | 22.5 | 7.7–87.0 |
Myoma | 10 | 43.8 | 57.4 | 13.0 | 55.8 | 44.4–90.0 | 121.5 | 260.4 | 26.0 | 13.0–852.0 |
Serous | 21 | 40.9 | 51.2 | 13.2 | 47.8 | 35.1–86.3 | 24.8 | 23.2 | 17.0 | 6.2–95.0 |
Simple | 64 | 38.2 | 48.3 | 11.9 | 47.0 | 29.3–95.1 | 35.3 | 39.1 | 19.0 | 4.9–180.0 |
Stromal | 3 | 39.3 | 50.5 | 12.7 | 48.9 | 38.7–63.9 | 40, 0 | 33.2 | 30.0 | 13.0–77.0 |
Total | 177 | 38.8 | 50.5 | 13.5 | 47.9 | 28.8–118.0 | 55.5 | 118.4 | 25.0 | 4.9–1, 427.0 |
Post-M | ||||||||||
Endometrioma + CEa | 7 + 7a | 64.0 | 113.4 | 89.1 | 74.6 | 38.8–277 | 124.4 | 269.5 | 39.5 | 13.0–1, 044.0 |
Teratoma | 11 | 57.2 | 62.0 | 38.0 | 48.7 | 37.3–172 | 22.9 | 18.0 | 18.0 | 1.2–68.0 |
PID | 8 | 58.5 | 62.7 | 14.3 | 62.7 | 36.1–81.8 | 15.1 | 5.5 | 16.0 | 7.7–24.0 |
Mucinous | 33 | 62.4 | 95.4 | 85.5 | 70.0 | 41.2–486 | 44.8 | 143.3 | 16.0 | 1.9–840.0 |
Myoma | 13 | 64.6 | 61.0 | 21.2 | 55.0 | 40.4–116 | 19.7 | 10.9 | 17.0 | 4.7–40.0 |
Serous | 76 | 64.9 | 80.9 | 57.1 | 61.0 | 37.1–336 | 73.7 | 291.9 | 19.0 | 5.9–2, 443.0 |
Simple | 52 | 64.3 | 72.2 | 39.2 | 59.7 | 35.7–238 | 24.1 | 28.4 | 16.0 | 4.5–163.0 |
Stromal | 15 | 63.9 | 90.9 | 60.8 | 71.1 | 38.8–277 | 90.6 | 179.9 | 29.0 | 12.0–721.0 |
Total | 208 | 63.6 | 79.7 | 57.0 | 62.0 | 35.7–486.0 | 53.9 | 201.3 | 18.0 | 1.2–2, 443.0 |
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aConcomitant endometriosis (CE). Serum values presented as mean, standard deviation, median and range. All endometriosis cases were biopsy-verified as either endometriomas, or as concomitant endometriosis (CE) to other benign ovarian diagnosis at current surgery, or discovered at a previous surgery. n, number; Std, standard deviation; CE, concomitant endometriosis; PID, pelvic inflammatory disease; Pre-M, premenopausal; Post-M, postmenopausal.
Statistical analysis
Statistical analysis was performed in R version 3.6.1 (R Core Team 2019). Benign samples (n=445) were divided into two groups based on recommended cut-offs for CA125 and HE4. Samples below the cut-off were denoted as true negatives (TN) and samples above the cut-off, as false positives (FP). Logistic regression models were fitted to distinguish between the TN and FP for each marker, i.e. between CA125TN and CA125FP, and between HE4TN and HE4FP respectively. The following predictors were included in the models: PID, endometriosis, age, eGFR, NT-pro-BNP, HRT, BMI, smoking, parity, history of/or ongoing non-ovarian cancer disease and heredity for ovarian- and breast cancer. Of these, PID, endometriosis, HRT, smoking, other cancer disease and heredity were binary predictors and encoded with 0 (0=no) and 1 (1=yes). Missing values (n=67) were replaced by 0, indicating absence of a characteristic e.g. non-smoker. Statistical significance of predictors was calculated with the likelihood ratio test and p-values adjusted for multiple testing with Benjamini-Hochberg correction. An adjusted p-value less than 0.05 were considered significant (Table 3).
Predictors | HE4 | CA125 | ||||||
---|---|---|---|---|---|---|---|---|
OR | 95% CI | p-Value | adj p-Value | OR | 95% CI | p-Value | adj p-Value | |
PID | 0.79 | 0.04–5.49 | 0.837 | 0.921 | 0.28 | 0.01–1.49 | 0.153 | 0.336 |
Endometriosis | 1.70 | 0.64–4.14 | 0.273 | 0.600 | 7.96 | 4.53–14.39 | <0.001 | <0.001 |
Age | 0.97 | 0.94–1.00 | 0.058 | 0.159 | 0.98 | 0.96–1.00 | 0.100 | 0.336 |
eGFR | 0.18 | 0.08–0.39 | <0.001 | <0.001 | 0.82 | 0.50–1.38 | 0.456 | 0.502 |
NT-pro-BNP | 1.00 | 1.00–1.00 | <0.05 | <0.05 | 1.00 | 1.00–1.00 | <0.05 | 0.169 |
HRT | 1.02 | 0.05–6.12 | 0.986 | 0.986 | 0.58 | 0.09–2.19 | 0.457 | 0.502 |
BMI | 1.03 | 0.95–1.12 | 0.413 | 0.757 | 1.03 | 0.97–1.08 | 0.314 | 0.502 |
Smoking | 6.62 | 2.93–15.12 | <0.001 | <0.001 | 0.75 | 0.38–1.42 | 0.387 | 0.502 |
Parity | 0.96 | 0.69–1.32 | 0.814 | 0.921 | 0.87 | 0.71–1.05 | 0.148 | 0.336 |
Other cancer | 0.70 | 0.12–2.56 | 0.621 | 0.870 | 0.84 | 0.36–1.82 | 0.672 | 0.672 |
Heredity | 1.42 | 0.29–5.06 | 0.633 | 0.870 | 1.49 | 0.58–3.64 | 0.398 | 0.502 |
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Multivariate logistic regression analyses of different predictors for each biomarker separately. False positive (FP) and true negative (TN) cases were identified for each biomarker in the study population. 137 (31%) women had FP values of CA125 (47 Pre-M, 94 Post-M). 38 (9%) women had FP HE4 values (17% Pre-M, 21% Post-M). Multivariate logistic regression analyses were performed on the different predictors including: PID, endometriosis, eGFR (kidney disease n=15), NT-pro-BNP (heart disease n=76), age, smoking, parity, HRT, BMI, other cancer and heredity, for CA125 and HE4 separately. p-Values were calculated with likelihood ratio test and adjusted (adj p-value) for multiple testing with Benjamin-Hochberg correction. PID, pelvic inflammatory disease; eGFR, estimated glomerular renal filtration; NT-pro-BNP, N-terminal prohormone of brain natriuretic peptide; HRT, hormone replacement therapy; BMI, body mass index; OR, odds ratio; 95% CI = 95% confidence interval.
Results
Variations in HE4 and CA125 depending on age-intervals
In the analysis, 445 women with a benign ovarian cyst or pelvic tumor were included. The mean age was 50.8 in the study population (pre-M=38.8; post-M=63.6) (Table 1). HE4 serum levels increased continuously with age while the CA125 levels did not differ between the age groups (Figure 1; Table 4). The cohort was divided into age groups of 10 years. Using the group <30 years as a baseline, HE4 levels increased by 4.1% for the group 30–39 years, 6.1% for 40–49 years, 77% for 50–59 years, 52.9% for 60–69 years, 66.4% for 70–79 years and 85.8% for >80 years of age (Figure 1, Table 4).
Age group | Pre-M | Post-M | ||||||
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Biomarker | <30 | 30–39 | 40–49 | 50–59 | 60–69 | 70–79 | >80 | |
n (445) | 51 | 55 | 96 | 101 | 90 | 42 | 10 | |
HE4 | Mean | 47.0 | 48.4 | 50.9 | 66.7 | 75.4 | 105.5 | 86.1 |
Std | 12.2 | 11.1 | 12.7 | 49.2 | 48.6 | 72.1 | 19.3 | |
Median | 45.8 | 47.7 | 48.6 | 81.1 | 70.1 | 76.2 | 85.1 | |
Range | 28.8–79.9 | 31.1–82.7 | 31.5–118.0 | 34.0–486.0 | 36.1–293.0 | 38.7–336.0 | 59–120.0 | |
CA125 | Mean | 51.2 | 49.3 | 59.0 | 43.4 | 62.6 | 51.6 | 116.7 |
Std | 78.0 | 49.5 | 148.7 | 110.8 | 269.2 | 159.6 | 248.2 | |
Median | 27.0 | 29.0 | 27.0 | 55.5 | 24.5 | 20.5 | 28.0 | |
Range | 6.3–446 | 6.0–245.0 | 4.9–1, 427.0 | 1.2–852.0 | 1.9–2, 443.0 | 6.8–1, 044.0 | 9.1–818.0 |
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Distribution of serum CA125 and HE4 values, stratified by age divided into age groups of 10 years, presented as mean, standard deviation, median and range for every group. N, number; Std, standard deviation; Pre-M, premenopausal; Post-M, postmenopausal.
For all age groups, both mean and median HE4 values were beneath the determined cut-off values. This indicates that the recommended cut-offs (pre-M > 70 pmol/L, post-M > 140 pmol/L) are adequate and adjusted for increased age, for this cohort of women, representing an unselected cohort diagnosed with a pelvic tumor of unknown origin (Table 1).
HE4 and CA125 serum levels in different benign histology
In the pre-M, the most common histologies were endometriomas (n=53), teratomas (n=47), and simple cysts (n=64). Mean values of CA125 for endometriosis + CE, teratomas, myomas, simple cysts and stromal histology were above cut-off (>35). Only the endometriosis + CE values were above cut-off when the median was used (CA125=67.0). Neither mean nor median HE4 values were above cut-off (>70) in the pre-M group (Table 2).
In the post-M group, the most common histologies were serous adenoma (n=76), simple cysts (n=52), and mucinous adenoma (n=33). Mean values of CA125 for endometriosis + CE, mucinous, serous, and stromal histology were above cut-off (>35). Only the endometriosis + CE value remained elevated when the median was used instead of the mean (CA125=39.5). Neither mean nor median HE4 values were above cut-off (>140) in the postmenopausal group (Table 2).
Multivariable regression analysis
False positive (FP) and true negative (TN) cases were identified for each biomarker in the study population. For CA125, 137/445 (31%) false positive women (93 pre-M, 44 post-M) were identified, and for HE4 we identified 38/445 (9%) false positive women (21 pre-M, 17 post-M). Multivariable logistic regression analyses were performed with several biological and lifestyle predictors for each biomarker separately. Biology- and histology-verified variables included PID, endometriosis, eGFR and NT-pro-BNP. Lifestyle factors included age, smoking, parity, HRT, BMI, other cancer and heredity (Table 3).
Biology and histology verified variables and elevated serum levels of CA125 and HE4
In total, there were 79 women (pre-M=65; post-M=14) with endometriosis + CE (Table 2). CA125 was falsely elevated (>35 U/mL) in 57 (72%) women with endometriosis, distributed as 49 pre-M and eight post-M women. False positive HE4 (pre-M > 70 pmol/L, HE4 post-M > 140 pmol/L) was found in five pre-M and three post-M women. In fact, HE4 correctly classified 92% of the pre-M women with false positive CA125 values while 8% women had a simultaneous false positive HE4 and CA125. The endometriosis diagnosis was found to be a strong predictive factor of falsely elevated CA125 levels, with adjusted p-value<0.0001; odds ratio (OR) 7.96 (CI 95% 4.53–14.39). Endometriosis was not significantly associated to elevated HE4 levels, with adjusted p-value 0.6; OR 1.7 (CI 0.64–4.14) (Figure 2, Table 3). The distribution of serum values of HE4 and CA125 for the women diagnosed with endometriosis according to established cut-offs and menopausal status is presented in Figure 3.
Fifteen women reported that they had ongoing kidney disease (Table 1). eGFR was found to be significantly associated with falsely elevated HE4, with adjusted p-value<0.0001, OR 0.18 (CI 95% 0.08–0.39), but not to CA125 with adjusted p-value 0.502 (OR 0.82 CI 0.50–1.38). Seventy-six women responded that they had ongoing heart disease at the time of inclusion. Several different conditions related to heart disease were noted and increased levels of NT-pro-BNP (>100) were found in 39 of the 76 women with heart disease. Increased levels of NT-pro-BNP were found to be associated with elevated levels of HE4. After adjustment, a significant p-value (p<0.01) was noted, OR 1.0 (CI 1.0–1.0) (Figure 2, Table 3).
PID noted in the pathology report was found in 14 women, six pre-M and eight post-M (Table 2). PID was not found to be significantly associated to falsely elevated CA125 or HE4 (Figure 2, Table 3).
Lifestyle factors and elevated serum levels of CA125 and HE4
In total, 15% (68/445) reported that they were smokers. Smokers were divided equally within pre-M (n=36) and post-M (n=32) (Table 1). In total, 22% (15/68) of the women who stated they were smokers had falsely elevated HE4 levels. Smoking was shown to be significantly associated with elevated HE4 levels, with adjusted p-value<0.0001, OR 6.62 (CI 2.93–15.12), but not with CA125, adjusted p-value 0.502, OR 0.75 (CI 0.38–1.42). None of the other reported lifestyle factors were significantly associated with CA125 or HE4 levels (Figure 2, Table 3).
Discussion
The aim of the present study was to improve our knowledge about different biological and lifestyle factors possibly associated with falsely elevated CA125 and HE4 values investigating a population of women scheduled for surgical intervention of a diagnosed ovarian cyst/pelvic tumor. To our knowledge, this is one of the largest cohorts with presurgical data on lifestyle factors, biomarkers and histopathological verified diagnoses. The main findings revealed by multivariable regression analyses showed that smoking, endometriosis and impaired renal function are variables to consider, in the evaluation of women presenting with an ovarian cyst or a pelvic tumor and elevated CA125 or HE4. Falsely indicating ovarian cancer disease.
Smoking was associated with falsely elevated HE4 with an OR 6.62 (CI 02.93–15.12). In total, 22% of the women who stated that they were smokers had false positive serum levels of HE4, indicating the relevance of considering smoking when assessing serum values of HE4. This confirms prior data of 20–30% higher serum levels of HE4 among smokers compared to non-smokers [10, 25, 26]. The cause for this is not known, but recent data shows that in HE4 knockout mouse models, apoptosis increases in alveolar epithelium type I cells which lead to severe dyspnoea [27]. The elevation was higher among pre-M than post-M women, which could be explained by the relationship between increased age and elevated HE4 [10]. In a subgroup (n=112) from the prostate, lung, colorectal and ovarian (PLCO) trial, analyses showed increased HE4 levels among smokers and the elderly [25].
Endometriosis was the only significant variable associated with falsely elevated CA125 with an OR 7.96 (CI 4.53–14.39) in the current study. The overexpression of CA125 in women with endometriosis has been described repeatedly and further emphasizes the marker’s limitation in fertile women [28, 29]. While women with a history of endometriosis did not have elevated CA125 in the PLCO trial, CA125 has been suggested to act as a biomarker for diagnosis and treatment monitoring of endometriosis [30, 31]. In a study comparable to ours containing 17% endometriosis (176/1, 042), only 3% had elevated HE4 levels compared to the 67% with elevated CA125 (p<0.0001) [32]. To increase specificity, endometriosis should always be taken into consideration when assessing CA125 and HE4 in fertile women with an unknown pelvic mass.
HE4 is upregulated by the epithelium in the kidney, which is why elevated levels of HE4 could depend on an injury in the kidney tissue, which leads to renal fibrosis, followed by an increase in HE4 levels [13, 33]. Both age and body mass are factors that could affect the glomerular filtration with decreased eGFR and simultaneous increased HE4 levels, rather than renal failure as a single factor [10, 26, 34]. In our multivariable regression analysis, eGFR was found to be a significant predictive factor of false positive HE4 with OR 0.18 (CI 0.08–0.39) indicating that renal failure reflected by a decrease in glomerular filtration should be considered when interpreting HE4. Renal failure was the most common cause of falsely elevated levels of HE4 in patients with non-malignant disease [35]. HE4 was significantly elevated in a cohort of 113 women already at slight renal failure eGFR < 90 mL/min/1.73 m2 in both pre-M and post-M [26]. It may be argued that it is difficult to compare the results of renal failure and the effect of the biomarkers due to study group selection, technical analytic differences and choice of eGFR calculation [10, 25, 26]. However, the results of this study and other groups’ studies align, which strengthens the conclusion that renal failure and eGFR need to be considered when interpreting HE4 levels in women [10, 26, 34], [35], [36].
It was previously shown that concentrations of HE4 are higher in women >55 in age [37]. When using the age of 20 as a baseline, HE4 increased non-linearly for each decade until the age of 80 years [10, 25, 26, 38]. These results were confirmed in our cohort where HE4 levels increased, especially in the age groups above 50 years. HE4 rose by 77% in age group 50–59, 52.9% in age group 60–69 and 66.5% in age group 70–79. Age was accounted for prior to the multiregression analysis with different cut-offs for pre-M (>70 pmol/L) and post-M (>140 pmol/L) (OR 0.97). Recommended HE4 cut-offs may vary for different analytic platforms, pre-M (41.4–140) compared to post-M (140) [15, 32, 37, 38]. If the same cut-off (>70 pmol/L) would have been used for the entire cohort in the present study, false positive HE4 cases would increase to 102. Age was not found to be a significant factor for CA125 levels with OR 0.98, while others have shown a decline in CA125 after menopause [39, 40].
Elevation of serum NT-pro-BNP is an important biomarker for congestive heart failure [41]. It is also elevated by several comorbidities, such as renal failure. CA125 has been suggested as a biomarker for congestive heart failure and our results may have supported this if the reporting of heart-related illnesses had been more specific, or if we had included a larger cohort [42]. Since congestive heart failure was one of the types of all heart-related illnesses recorded (17%), the results are difficult to interpret. In the multivariable regression analysis, NT-pro-BNP was significantly associated with HE4, OR=1.0 (CI 1.0–1.0), indicating no clinical significance. BMI was not a predictive factor for false positive CA125 or HE4. Bolstad et al. reported decreased HE4 levels in women with high BMI, and low BMI was associated with increased HE4 levels (n=1, 591) [10], while several other groups report no correlation between HE4 and BMI [25, 34, 43].
This is one of the largest cohorts with presurgical data on both lifestyle factors, biomarkers and a histopathological verified diagnosis. The results are strengthened by the multivariable regression model used, compared to univariate statistical analysis, since regression allows analysis of the association between each factor and the marker concentration, while adjusting for the remaining factors [10, 25, 37, 43]. Only recommended cut-offs previously tested in other populations were used, which we see as strength of the current study and an important step towards understanding and implementing the use of HE4 in a clinical setting. All biomarker analyses have been performed with automated assays in an accredited university laboratory. It was reported that a manual assay (n=802) generated 14.0% higher values of HE4 compared to the automated assay (n=789) [10].
The original study also included malignant OC cases (n=162) but a similar analysis on false negatives could not be performed due to the small sample size. The study is a retrospective subgroup analysis and was initially not correctly powered to perform multivariable regression analyses and yet several factors were found to be significantly associated with false positive levels of CA125 or HE4. Other limitations include that lifestyle factors were mainly self-reported; heart disease could have been specified to having congestive heart failure or not; smoking was reported only as yes or no, and amount of cigarettes/day was not noted in our study. Reluctancy to admit to smoking should also be considered as a bias.
Our results confirm that smoking, endometriosis and impaired renal function are significant variables found in women presenting with an ovarian cyst or a pelvic tumor and elevated CA125 or HE4, falsely indicating ovarian cancer disease. These variables, age and possibly congestive heart failure should always be considered when assessing the biomarkers CA125 and HE4 in the differential diagnostics of an ovarian cyst or unknown pelvic mass in both pre- and postmenopausal women.
Funding source: Swedish Cancer Foundation
Funding source: The Assar Gabrielsson Foundation
Funding source: The Hjalmar Svensson Foundation
Funding source: The Gothenburg Medical Society
Funding source: Roche Diagnostics
Funding source: Cancerfonden
Acknowledgments
This study was conducted in collaboration with the Regional Cancer Center West and the Process Group for Ovarian Cancer.
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Research funding: The study was funded by the Swedish Cancer Foundation (KS), LUA-ALF (KS), the Assar Gabrielsson Foundation (ML, BK, CM), the Hjalmar Svensson Foundation (ML), and the Gothenburg Medical Society (ML). Roche Diagnostics Scandinavia covered the cost of running biomarker assays at the accredited laboratory at Sahlgrenska University Hospital, Gothenburg, Sweden. The funding organizations played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Author contribution: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors (ML, BU, JL, BK and KS) contributed substantially to the conception, planning, carrying out, analysing and writing. Data was collected by ML and BK in collaboration with the Regional Cancer Centre West Ovarian Cancer Group.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013). The Ethical committee of Gothenburg University approved the study (Ref 139–13). Date of approval 14 May 2013.
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