Serum HE4 as a prognostic marker in cervical cancer: a retrospective study

Background Human epididymis protein 4 (HE4) is a tumor marker that has been well-investigated in ovarian and endometrial cancers. The aim of this study was to evaluate the prognostic value of serum HE4 as a tumor marker in patients with cervical cancer. Serum HE4 levels from 67 cervical cancer patients were measured by immunoassay before starting primary treatment between September 2014 and May 2018. A mean serum HE4 level of 72.6 pmol/L was used to divide the patients into low and high HE4 groups. The patient characteristics, clinicopathological variables, and survival outcomes were compared between the 2 groups. Higher were with older at (age 0.0% vs. 100.0%; P 0.002), menopause (premenopause: 8.3% vs. postmenopause: 91.7%; P = 0.009), higher FIGO stage (stage I-II: 25.0% vs. III-IV: 75.5%; P = 0.008), large tumor size (< 4.0cm: 41.7% vs. ≥ 4.0 cm: 58.3%; P = 0.029), positive lymph node metastasis (negative: 41.7% vs. positive: 58.3%; P = 0.049), and involvement of the parametrium (negative: 25.0% vs. positive: 75.0%; P = 0.006). Higher levels of HE4 was a predictive factor for worse overall survival but not for progression-free survival, although elevated HE4 levels were not found to be independent factors for the prediction of either overall survival or progression-free survival. When subgroup analysis was performed by histological type, similar results were obtained for patients with cell carcinoma.


Abstract Background
Human epididymis protein 4 (HE4) is a tumor marker that has been well-investigated in ovarian and endometrial cancers. The aim of this study was to evaluate the prognostic value of serum HE4 as a tumor marker in patients with cervical cancer.

Methods
Serum HE4 levels from 67 cervical cancer patients were measured by immunoassay before starting primary treatment between September 2014 and May 2018. A mean serum HE4 level of 72.6 pmol/L was used to divide the patients into low and high HE4 groups. The patient characteristics, clinicopathological variables, and survival outcomes were compared between the 2 groups.

Conclusions
Our data revealed that high levels of HE4 expression were correlated with poor OS, indicating that elevated HE4 levels are associated with a poor prognosis for patients with cervical cancer.

Background
Over the past few decades, efforts have been made to reduce the incidence and mortality of cervical cancer through early detection and prevention. Nevertheless, cervical cancer remains the fourth most commonly diagnosed cancer and the fourth leading cause of cancerrelated death in women worldwide (1). Histologically, approximately 80% of cervical cancer cases are squamous cell carcinoma (SCC) and approximately 20% are adenocarcinoma (ADC) (2,3). Currently, squamous cell carcinoma antigen (SCC-Ag) and cancer antigen 125 (CA 125) are the most commonly used tumor markers for cervical cancer. Increased levels of SCC-Ag are present in only 64% of patients with SCC and 25% of patients with ADC, while increased levels of CA125 are present in only 42.6% of patients with SCC and in 18.9% of patients with ADC (3). Therefore, the identi cation of novel markers that improve the detection rate of cervical cancer is required.
Cervical cancer is a preventable disease and its morbidity and mortality has been dramatically reduced by the introduction of cervical cytology (4). However, cervical cytology has low sensitivity and a high false positive rate, partially due to inadequate specimen collection (3,5). Adjunctive tests such as colposcopy and screening for human papillomavirus (HPV) have been suggested to overcome this low accuracy (6). In addition, tumor markers for the diagnosis and follow up of cervical cancer are necessary. SCC-Ag is the most widely used tumor marker in the diagnosis of cervical cancer. Elevated levels of SCC-Ag are related to tumor size and stage of disease before treatment, as well as to the response to treatment (7). However, the diagnostic sensitivity is only 30% for early cervical cancer (8). As well, the utility of SCC-Ag in other histologic types is still unclear. With these limitations, identifying other tumor markers for cervical cancer is highly important.
Human epididymis protein 4 (HE4) is a promising biomarker that has been shown to have great potential for clinical use (9)(10)(11). HE4 was rst isolated from the epithelial cells of the human epididymis and can also be detected in serum (12). HE4 expression has also been identi ed in a number of normal human tissues outside of the male reproductive system, as well as in various types of malignancies (13,14). Previous studies have demonstrated that HE4 levels are higher in certain type of cancer (15)(16)(17). Increased HE4 is commonly found in tumors of gynecologic origin, and HE4 is well established in ovary and endometrial cancers (18,19). It has been reported that serum HE4, both alone and in combination with CA125, has great potential to predict prognosis (20)(21)(22).
Although it is known that serum levels of HE4 are signi cantly increased in ovarian and endometrial cancers, research on cervical cancer has been minimal. Some studies have shown that patients affected by cervical cancer may also have higher levels of HE4 (17,23). However, as these studies were limited by their small sample size, it remains unclear whether HE4 is a valuable marker for cervical cancer. A tumor marker that enables better strati cation of patients with cervical cancer may improve individualized primary treatment and would help to prevent over-or under-treatment of these patients. Therefore, we sought to evaluate the prognostic value of serum HE4 as a tumor marker for cervical cancer patients.

Methods
This retrospective study received approval from the Institutional Review Board of the Seoul National University Bundang Hospital (SNUBH; No. B-1602/336-103) and was performed in accordance with the principles of the Declaration of Helsinki. The requirement for informed consent was waived due to the retrospective nature of the study.

Study population
We retrospectively reviewed the medical records of 67 cervical cancer patients who were treated at Seoul National University Bundang Hospital, a tertiary hospital in Korea, from September 2014 to May 2018. All patients were restaged based on the revised 2018 International Federation of Gynecology and Obstetrics (FIGO) staging system for cervical cancer (24). We included patients who met the following inclusion criteria: (1) patients who had histologically con rmed cervical cancer of any stage; (2) those who underwent initial treatment, such as radical hysterectomy with lymphadectomy, concurrent chemoradiation therapy, radiation therapy or chemotherapy only; and (3) those who had HE4 levels assessed at diagnosis, before initiation of rst treatment. Patients with the following conditions were excluded: (1) patients who had an incomplete treatment for any reason; (2) those who had insu cient clinical and pathological data; and (3) those with other malignancies that had the potential to in uence survival outcomes.
For the patients who met the criteria mentioned above, we reviewed the medical records, surgical records, pathological ndings, and clinical characteristics. Histology was pathologically evaluated in all patients. Tumor size, lymphovascular space invasion (LVSI), lymph node (LN) metastasis, and involvement of the parametrium were evaluated pathologically only in patients who initially received surgery, while magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography computed tomography were evaluated in patients who received initial treatment other than surgery.

HE4 Immunoassay
The serum HE4 concentration was measured before initiation of patient treatment using the Architect Analyzer (Abbott Laboratories, USA). This is a two-step immunoassay that quantitatively measures HE4 levels in human serum using chemiluminescent microparticle technology.

Statistical analysis
The 67 cervical patients had a mean serum HE4 level of 72.6 pmol/L (standard deviation = 95.0) prior to the initiation of treatment. As no de nitive diagnostic thresholds for HE4 have been reported to date, we evaluated the differences in clinicopathological characteristics between patients who had lower HE4 levels (<72.6 pmol/L) and those who had higher HE4 levels (≥72.6 pmol/L) at initial diagnosis.

Results
In this study, we investigated the HE4 levels in 67 cervical cancer patients. The overall patient characteristics are presented in  (Fig. 1A). However, patients with higher HE4 levels had signi cantly worse OS than those with lower HE4 levels (HR: 3.726; 95% CI: 1.182-11.747; P=0.016) (Fig. 1B). We also performed subgroup analysis of patients with the SCC histological type (n=50), and the results were consistent with those of the overall study population. There was no signi cant difference observed in PFS (HR: 0.974; 95% CI: 0.198-4.805; P=0.975) (Fig. 1C), but patients with higher HE4 levels were associated with worse OS than those with lower HE4 levels (HR: 5.449; 95% CI: 1.460-20.332; P=0.005) (Fig. 1D).
In order to assess the prognostic signi cance of clinicopathologic variables of PFS and OS, we performed univariate Cox analysis. FIGO stage, tumor size, LN metastasis and involvement of parametrium were identi ed as unfavorable factor for PFS and OS (Table 3). In addition, higher HE4 levels were signi cantly associated with poor clinical outcomes for OS (HR: 3.726; 95% CI: 1.182-11.747; P=0.025; Table  3). On multivariate analysis, higher HE4 levels did not remained as an independent indicator of OS  Table 4).

Discussion
In this study, we found that high expression of HE4 is correlated with poor OS, indicating that HE4 is a potential candidate biomarker for predicting the prognosis of cervical cancer. Several recent studies investigating HE4 have reported that it is an effective tumor marker for predicting prognosis and is associated with poor OS and PFS, especially in ovarian cancer (25). Our ndings revealed that, in cervical cancer patients, elevated serum HE4 levels were signi cantly correlated with FIGO stage as well tumor size, LVSI, LN metastasis, and parametrium involvement, but not with histological type. Moreover, we found that in univariate analysis serum HE4 levels were associated with worse OS.
However, HE4 levels were not found to be an independent prognostic factor for PFS. Similar results were observed in the subgroup analysis of patients with histologically con rmed SCC.
Careful attention is required in interpreting serum HE4 levels, as they can be affected by numerous factors (26). Previous studies have shown that age, menopausal status, smoking, renal function, chronic liver disease, ethnicity, and detection method may in uence serum HE4 levels (17,(27)(28)(29). Thus, our results should be interpreted with caution. At current, there is no consensus established for optimal baseline serum HE4 cutoff values (30). Based on previous published investigations, a serum HE4 cut-off value of 70 pmol/L yields the best sensitivity and speci city (26). The positive serum HE4 cut-off values previously identi ed in ovarian and endometrial cancer patients were found to be similar to the mean value of HE4 in our study (23,31).
This study has several limitations. First, the existence of underlying selection bias is possible due to the retrospective design. Second, the sample size of the study population as well as the number of recurrence and death events might be insu cient for a comparison of survival outcomes between lower and higher serum levels of HE4. Third, no subgroup analysis was performed for the ADC histology type because of the small sample size of this population. Lastly, we did not attempt any strati cation of the study population.

Conclusions
In conclusion, our study showed that elevated serum HE4 was associated with poor prognostic factors for cervical cancer and was correlated with poor OS, suggesting the potential of HE4 as a novel biomarker for predicting the survival of patients with cervical cancer.