The Prognostic Value of High-risk Human Papillomavirus Infection Status in Surgically Treated Stage IB1-IIA2 Cervical Squamous Cell Carcinoma


 Background: High-risk HPV(hr-HPV) infection is important for the development of invasive cervical cancer. As a developing country with the largest population in the world, China has a great burden of cervical cancer. The cervical cancer screening strategies consisted of cytology and virology, which has been studied by HPV-typing test and HPV-DNA quantitative test since decades ago. The cervical cancer incidence rate has been declined due to the early treatment of precancerous lesions, but postoperative HPV infection is still an issue for cervical cancer patients. This study aims to investigate the association between HPV infection status and recurrence of early-stage cervical cancer through a novel PCR-based HPV test, which could do both HPV typing-test and DNA quantitative-test. Methods: Patients diagnosed with cervical cancer staged IB1- IIA2, who were treated by radical hysterectomy and lymphadenectomy at Cancer Hospital Chinese Academy of Medical Sciences(CAMS) between January 2014 and December 2016 were accrued. The clinicopathological factors, pre- and post-operative HPV infection status, and the prognosis were investigated. Cox regression was used to identify factors associated with LRFS, MFS and OS. Results: A total of 312 patients were enrolled in this study, who were treated by radical resection and accepted pre- and post-operative HPV tests, with a median follow-up time of 60 months(range 14~79 months). The 5-year LRFS rate, MFS rate, and OS rate were 97.8%, 98.4%, and 98.7%. The pre-operative HPV infection rate was 85.3%(266/312), 74 patients had a high level of HPV-DNA(>5x106 copy number/104 cells). Twenty-nine patients had a postoperative persistent high level of HPV-DNA(9.3%). Postoperative persistent high level of HPV-DNA within 12 months(p=0.013), postoperative persistence of HPV-16/18 within 24 months(p=0.004), and deep stromal invasion(>2/3)(p=0.007) were associated with a poor LRFS.Conclusion: Pre-operative HPV-16/18 infection and high level of HPV-DNA were not associated with local recurrence of cervical cancer. Most initial HPV-positive patients had HPV cleared within 24 months postoperatively. Postoperative HPV-16/18 persistence within 24 months, postoperative persistence of high HPV-DNA level within 24 months, and deep stromal invasion(>2/3) were independent risk factors for local recurrence of cervical cancer.


Background
Cervical cancer is the fourth most common cancer for women. In 2018, 570,000 new cases and 311,000 deaths were estimated worldwide, including 106,000 cases and 48,000 deaths in China. China contributed 18.6% of the new cases and 15.4% of the deaths of the global cervical burden [1]. The cervical cancer screening strategies were based on the HPV test and the Papanicolaou (Pap) test [2]. Most patients with stage IA1-IIA2 cervical cancer were treated by surgery. Patients with pathological risk factors will receive adjuvant radio(chemo)therapy. Persistent high-risk human papillomavirus(HR-HPV) infection of the low genital tract is an independent risk factor for the development of cervical intraepithelial neoplasms(CIN) and invasive cervical cancer [3,4]. Currently, most of the HPV tests were based on hybrid capture 2 (HC2) or real-time polymerase chain reaction(RT-PCR) [5]. Among the marketed HPV tests, the Cobas® HPV Test was based on the RT-PCR technique [6]. However, HPV viral load also had an impact on the prognosis of cervical cancer patients [7][8][9][10][11]. Though many previous studies try to clarify the in uence of HVP viral load, conclusions about the prognostic value of HPV viral load remained controversial for years [7,12,13].
The HPV qRT-PCR Kit(Liferiver, Shanghai, China), which covered 15 types of hr-HPV (16,18,31,33,35,39,45,51,52,56,58,59,66, 68 and 82), could do both typing and quantifying tests to con rm the HPV-DNA level of each type. In order to discover the relationship between HPV infection status and the prognosis of cervical cancer treated by radical surgery through this HPV test, we conduct a retrospective study by reviewing the data of stage IB1-IIA2 cervical cancer patients treated by radical hysterectomy.

Patients characteristics
Patients diagnosed with stage IB1-IIA2(FIGO stage, 2014) cervical cancer and treated at the Cancer Hospital, Chinese Academy of Medical Sciences (CAMS) from January 2014 to December 2016, were eligible for this study. The eligibility criteria included the following: (1)patients treated by radical hysterectomy with pelvic ± para-aortic lymphadenectomy; (2)accepted pre-and post-operative HPV tests by HPV qRT-PCR Kit(Liferiver, Shanghai, China); (3)squamous cell carcinoma. The exclusion criteria: (1) Initial treated by neo-adjuvant radio(chemo)therapy; (2) only accepted other HPV tests.

Treatment
All Patients were treated by radical hysterectomy(RH) and pelvic ± para-aortic lymphadenectomy, including laparotomy and laparoscopy. Ipsilateral ovary or bilateral ovaries was preserved for patients younger than 45 years old. Patients with LVSI, positive surgical margin, stromal invasion (> 2/3), or lymph node metastasis received adjuvant radiotherapy or concurrent radio(chemo)therapy(CCRT). The total dosage of radiotherapy was 45 ~ 50Gy. Cisplatin(40 mg/m2) was administered per week during CCRT.
The volume of external beam radiotherapy(EBRT) covered the region of previous gross disease, the parametrial space, uterosacral ligaments, the proximal 3 cm of the vagina, and all pelvic nodal volumes at risk.

Follow-up
The follow-up time was the duration from the date of surgical operation to the end of June 30th, 2020, or the date of death. Local recurrence-free survival(LRFS) was de ned as the time duration between the date of surgical operation and the detection of recurrent disease within the pelvis. Metastasis free survival(MFS) was de ned as the time duration between the date of surgical operation and the detection of distant metastasis. Overall survival(OS) was de ned as the time duration between the surgical operation and death.

HPV DNA testing
All patients accepted the HPV-DNA test within one month pre-operatively and at multiple time points during the follow-up period postoperatively. ThinPrep cytology specimens were tested by HPV qRT-PCR Kit (Liferiver, Shanghai, China), which detects viral DNA by nucleic acid hybridization with a pooled probe set for 15 high-risk types (16,18,31,33,35,39,45, 51, 52, 56, 58, 59, 66, 68 and 82). The nucleic acid was extracted by an automatic nucleic acid extracting machine(Autrax Bio-system®, Liferiver, Shanghai, China). All PCR reactions were performed by qRT-PCR machine(SLAN-96P Real-time PCR system, Shanghai Hongshi Corp, China) with a detection range of 5 × 10 2 copies to 5 × 10 7 copies/10 4 cells. All data collected was based on the o cial report by the laboratory test center of Cancer Hospital, Chinese Academy of Medical Sciences (CAMS).

HPV infection status
Patients infected by two or more genotypes of HPV were de ned as coinfection. HPV-DNA level > 5 × 10 6 copies/104 cells were de ned as high-level (For patients infected by multiple genotypes, any type of HPV-DNA level > 5 × 10 6 copies/10 4 cells). Pre-operatively, all patients were classi ed into negative, low-level, and high-level groups. According to the post-operative clearance time, patients were classi ed into ve groups: pre-and post-operative negative group, cleared within 12-month, during 12-24m, after 24 m, and uncleared groups. Based on the time of high HPV-DNA persistence, patients were classi ed into postoperative persistence within 12 months, 12-24 months, and longer than 24 months groups.

Statistics
Local recurrence-free survival, metastasis-free survival, overall survival, and follow-up time were continuous variables. Age, tumor size, FIGO stage, histological grade, lymphovascular space invasion(LVSI), lymph nodes metastasis, HPV infection status, HPV-DNA level were categorical variables.

Characteristics of patients
A total of 1030 cases data were reviewed, while 312 cases were met the inclusion criteria during the period from Jan 2014 to Dec 2016. All 312 patients were treated by radical hysterectomy with lymphadenectomy, while 123 patients(39.4%) with pathological risk factors received post-operative adjuvant radio(chemo)therapy. The median age of the 312 patients was 47(range, 25 ~ 73) years. No patients had parametrial invasion or positive resection margins, which were con rmed by pathologists.
The median follow-up time was 60(range 14 ~ 79) months. The characteristics of the patients were shown in Table 1.  According to the post-operative level of HPV-DNA, 59 patients had a low level of HPV-DNA, eleven patients had a persistence high-level within 12 months, eight patients up to 12-24 months, and ten patients had longer than 24 months. Compared with the pre-operative level of HPV-DNA, 227 patients had a reduction of the HPV-DNA level postoperatively (Table 2).

prognosis
The median follow-up time of the 312 patients was 60 months(range 14 ~ 79 months). Seven patients had local recurrence, and ve patients had distant metastasis. The median local recurrence time was 12 months(range 6 ~ 18 months). Three patients died of uncontrolled local recurrence, while one patient died of liver metastasis. The 5-year LRFS rate was 97.8%(305/312), the 5-year MFS rate was 98.4%(307/312) and the 5-year OS rate was 98.7%(308/312). The Kaplan-Meier curves showed that postoperative persistent high level of HPV-DNA within 12 months (p < 0.001) and postoperative persistence of HPV-16/18 within 24 months(P < 0.001) were associated with poor LRFS (Fig. 1.) The univariate and multivariate analysis were demonstrated in Table 3.

Discussion
Currently, about 40 types of HPV had been detected in the lower genital tract of women. The infection of hr-HPV was crucial for carcinogenesis in cervical cancer [4,10,14,15]. Though the association between HPV infection status and the prognosis of early-stage cervical had been studied through HPV tests, the conclusions remained controversial for decades [7,9,13,16,17]. HC2-based or PCR-based HPV tests were adopted by most studies, but HC2 could only do a typing test, while PCR could do both typing and quantifying tests [18]. In the present study, the HPV test was based on qRT-PCR, which con rmed that 85.3% of patients were HPV-positive(266/312). Among the pre-operative HPV-positive patients, 85.3% of them were HPV-16/18 positive, which was concordance with other studies(70%-90%) [13,15,[19][20][21].
The prognosis of early-stage cervical cancer was majorly dominated by the pathological risk factors, including lymph nodes metastasis, positive resection margin, tumor size, parametrial invasion, deep stromal invasion, and LVSI [22]. Early-stage patients had a better prognosis than advanced-stage patients(IIB-IV [23][24][25]. However, some patients without pathological risk factors could have recurrent disease occasionally. Thus, some studies focused on the HPV infection status and viral load, but the conclusions of the association between pre-treatment HPV infection status and the prognosis of cervical cancer were controversial. In the present study, the pre-operative HPV-DNA level and HPV-16/18 infection did not show impact on the LRFS, MFS, and OS (Table 3). Similar ndings have been reported by Chen et al. [26]. Yong et al. (2008) also found that pre-treatment HPV viral load did not have impact on clinicopathological factors and prognosis [16]. However, Wanram et al. (2009) found that patients with pre-treatment HPV-16 infection and a high viral load would increase the risk of progression of invasive cervical cancer after treatment [17].  [9]. These controversial conclusions might be caused by the difference in test methods, treatments, or pathological risk factors.
The conclusions of previous studies about the impact of post-treatment clearance of HPV and persistence on the prognosis of cervical cancer were controversial [12,27]. In the present study, though 86.3% of initial HPV-positive patients had HPV cleared within 24 months, the LRFS, MFS and OS of different post-operative clearance time groups did not shiw statiscal signi cance (Table 3) [12]. It would be necessary to monitor the post-treatment HPV status, including HPV viral load, to evaluate the association between HPV viral load and the prognosis of early-stage cervical cancer.
Compared to other studies, we con rmed 15 types of hr-HPV and the viral load of each type by using the HPV qRT-PCR Kit(Liferiver, Shanghai, China). This PCR-based HPV test has high sensitivity and speci city[ [28,29]. Although our study is a retrospective study with selection bias, it had a large sample size, initial HPV infection status, and consistent post-operative HPV infection status during the follow-up time. Besides, the data of the other 13 types of hr-HPV viral load could be used for future studies.

Conclusion
In summary, we have shown that the qRT-PCR is able to do both typing and quantifying tests of 15 types of hr-HPV. The result showed that pre-operative HPV infection status was not associated with the prognosis of early-stage cervical cancer. However, post-operative persistence of high HPV DNA level and HPV-16/18 could be a predictive factor of clinical outcome for surgically treated early-stage cervical cancer. Patients with post-operative persistence high HPV DNA level and hr-HPV might be screened strictly to detect the local recurrence earlier, which could be appropriately treated.

Declarations
Ethics approval and consent to participate This secondary analysis of de-identi ed data was determined not to be human subjects research by National Cancer Center/Cancer Hospital's research determination committee and thus was exempt from ethics committee review.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.