Gynecological Examination

The study was approved by the National Health Research Ethics Committee of Nigeria.
Women were recruited from 2 cervical cancer screening clinics within the National Hospital, Abuja and University of Abuja Teaching Hospital, Abuja, Nigeria during the period April 2012 to August 2012. To be eligible to enroll in the study, the participants had to be above the age of 18 and provide written informed consent for the study. We excluded women who had had a total hysterectomy, were pregnant or could not provide an informed consent. A total number of 278 women were screened, of which 267 (96%) had complete phenotype data and were successfully genotyped (as described below). These 267 women comprised 65 women with prevalent hrHPV (cases) and 202 controls.
Using interviewer administered questionnaires, participants provided information on demographics, socio-economic status, physical activity, smoking, alcohol use, sexual and reproductive history. A gynecologic examination was carried out on each participant. During the gynecologic examination, exfoliated cervical cells were collected from the cervical os. A cervical brush was inserted into the cervical os and rotated 3 full turns to collect exfoliated cells from the cervical os. The head of the brush was subsequently snapped off and placed at the bottom of a specimen transport tube containing 95% ethanol. In addition to the gynecologic specimens collected, blood samples were also collected from the antecubital veins. All samples were transported to the IHVN laboratory. The exfoliated cervical cells were stored at −80°C until the time of analysis. Buffy coat was separated from the whole blood samples and stored till the time of analysis. Data were collected and managed using REDCap electronic data capture tools hosted at the Institute of Human Virology, Nigeria [15] (link), [16] (link).

DFS was defined as the time from the initial diagnosis (histology) to disease recurrence or death from any cause. OS was defined as the time from initial diagnosis (histology) to death from any cause. PC is defined as the absence of local and nodal disease within the pelvis. LC was defined as the absence of disease in postoperative hysterectomy region, upper vagina, and parametria on gynecologic examination at follow-up. Data regarding patients with no evidence of recurrence or death were censored at the date of the last follow-up. Follow-up was defined as the time from the end of treatment to the relevant event (death from any cause, cancer-specific death, any recurrence, local recurrence, and pelvic recurrence).
All toxicity data were scored using the Common Terminology Criteria of Adverse Events (CTCAE) version 4.0.
Gastrointestinal (GI) and genitourinary (GU) radiotherapy-related toxicities were categorized into acute (symptoms experienced during or ≤ 3 months of completion of CRT-S) and chronic (> 3 months after CRT-S).
Surgical morbidity and mortality were evaluated and registered during hospitalization and postoperative (acute, ≤ 6 weeks postoperative) and at every visit thereafter (late). Based on CTCAE v4, the following data were extracted: urinary infection, wound infection, urinary fistula, digestive fistula, ileus, bowel subobstruction, and thromboembolic events.
Pathology results were analyzed with regard to resection margins and pathological response (residual tumor was defined as ≥ 10 mm grossly and < 10 mm microscopically); they were also compared with the imaging performed after CRT.

The study population comprised women who attended an annual gynaecological examination (including a cervical cancer‐screening program) between 20 September 2018 and 20 March 2020, at 10 local medical centres of mainland China, namely, (1) Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, (2) Pudong Hospital, Fudan University, Shanghai, (3) Songjiang Hospital (Songjiang Central Hospital), Songjiang, Shanghai, (4) Qilu Hospital, Shandong University, Jinan, Shandong, (5) Zouping People's Hospital, Zouping, Shandong, (6) The Central Hospital of Zibo Mining Group Co. Ltd., Zibo, Shandong, (7) Chifeng College Affiliated Hospital, Chifeng, Inner Mongolia, (8) The Second People's Hospital, Three Gorges University, Yichang, Hubei, (9) Yongcheng People's Hospital, Yongcheng, Henan and (10) Taiyuan Maternal and Child Health Care Hospital, Taiyuan, Shanxi. Women were excluded if they were pregnant, a virgin (or <16 years old), had undergone hysterectomy or had been treated for cervical intraepithelial neoplasia (CIN) or any malignant diseases during the past 5 years. Women with severe acute or chronic mental/infectious/internal/surgical diseases as well as those with autoimmune/posttransplant diseases who relied on daily immunosuppressive drugs or those with endocrinological syndromes/abnormal uterine bleeding who had to use hormone medicines long term were also excluded. The enrolment process contained three phases (Figure S1). For phase 1, 20 September 2018 to 20 March 2019, according to their HPV DNA and Pap testing results, HPV‐negative Pap‐normal women were consecutively enrolled from participating medical centres; signed informed consent was collected; clinical information was recorded; and cervical exfoliate samples (the remnant collection of Pap) were stored for later application of p16^INK4A FCM. At each medical centre, eligible HPV‐positive and/or Pap‐abnormal women who attended during the same period were systematically enrolled as a candidate pool, and the information and cervical samples of these women were properly kept until phase 2 or 3. During phase 2, after the age compositions of the enrolled HPV‐negative cervical healthy women were determined, age‐matched HPV‐positive women who had normal Pap results were selected from the candidate pool and called back to participate in our observational study; their informed consent was obtained before the first round of follow‐up. In addition, the candidate pool was continuously expanded by collecting eligible HPV‐positive and/or Pap‐abnormal women during the same phase. Phase 2 enrolment ceased on 20 September 2019, and the study quickly entered phase 3 as the HPV genotyping process had been completed for each of the phase 2 women (i.e., the age‐matched HPV‐positive Pap‐normal women). In phase 3, HPV‐positive/‐negative Pap‐abnormal women were selected from the pool based on the following rules: (1) the enrolled HPV‐negative Pap‐abnormal women should comprise a group with the same/similar age composition as that of the group of HPV‐negative Pap‐normal women; (2) the enrolled HPV‐positive Pap‐abnormal women should comprise a group with the same/similar age composition and HPV genotype composition as those of the group of HPV‐positive Pap‐normal women; (3) the women were randomly selected and called to determine their willingness to participate if they met the age and HPV genotype requirements; informed consent was obtained from the enrolled women before the first round of follow‐up; and (4) if there were still vacancies regarding a specific age and/or HPV genotype condition that could not be fulfilled by the pool, the candidates were selected and enrolled directly from outpatient departments of the participating centres before 20 March 2020. Upon the enrolment deadline, the remnant and redundant candidates in the pool were released, and their information and cervical samples were deleted or discarded under surveillance. The study protocol (Figure S2) was approved by the ethics committee of Ren Ji Hospital and approved by the participating medical centres.

The dose of the pelvic EBRT was 45–50.4 Gy, with a 1.8–2 Gy/fraction. Some patients were treated with concurrent or sequential therapy with a total lymph node boost dose of 57.5–65 Gy. The EBRT techniques included three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), or volume modulated radiotherapy (VMAT). In Arm 1 and Arm 2, 52 (82.5%) and 49 (86.0%) of the patients received concurrent platinum-based chemotherapy, and 11 (17.5%) and 8 (14.0%) of the patients received radiotherapy only. All the patients received treatment by using the MRI-guided ¹⁹²Ir HDR after-loading the therapy equipment (Micro-Selectron HDR V2), and the BT was performed after completion of the EBRT.
The implantation of the BT applicator and needle was performed as described below: the applicator was selected before the surgery, according to the disease conditions of patients. The tumor location, size, shape, para-uterine invasion, and relationship with surrounding organs were verified from the MRI images before and after the EBRT, as well as gynecological examination. The bowel preparation was performed on the day before the operation, and vaginal irrigation was performed on the day of operation. Combined intravenous and inhalation anesthesia was administered to the patients, then the patients were disinfected routinely, and the Foley urethral catheter was placed, with the balloon at the site of the vesical neck. The color ultrasound-assisted implantation of the applicator and needle was performed. The applicators used included the Utrecht interstitial Fletcher CT/MRI applicator set, interstitial ring CT/MRI applicator set, vaginal CT/MRI multi-channel applicator set, and self-made 3D-printed applicator. In patients that the para-uterine invasion has reached the pelvic wall, trans-perineal manual implantation was performed in addition to the above-mentioned applicators to meet the demands of dose distribution, and the depth of the needle was guided under the assistance of ultrasound. After the completion of the implantation, the applicator and needles were fixed. For one BT fraction in one application and two BT fractions every other day in one application, the fixation method of the applicator and needles remained the same. First, the needles were fixed on the applicator using the guiding tube, which was assembled on the applicator. Next, the applicator was filled with gauze for internal fixation, and then it was externally fixed on the patient's body with a T-shaped fixing belt. And then the rectum was pushed by the rectal pressure plate. The MRI was performed after the patient has been awakened. The application of analgesic drugs was decided based on the level of pain in patients. The analgesic drugs included were as follows: patient-controlled intravenous analgesia (PCIA), subcutaneous injection of opiates, and/or anti-inflammatory analgesics. The analgesic drugs were used in the recovery and treatment periods according to the requirements.
T2W MRI of each BT fraction was used for the delineation of target volume and OARs, as referred to in the GEC-ESTRO recommendations [4 (link)]. The high-risk clinical target volume (HR-CTV) was applied for the range of tumors that showed by the MRI and physical examinations following the EBRT, as well as the overall uterine cervix examination. The intermediate-risk clinical target volume (IR-CTV) was applied for the range of cervical cancer before the EBRT and for the extension of HR-CTV. The OARs included the bladder, small intestine, sigmoid colon, and rectum.
All the patients received the HDR-IGABT treatment at the 28 Gy/4f. The MRI was performed on the day when the operation has been completed. The patients in Arm 1 have received one BT fraction, and then the applicator or needles have been removed, while the patients in Arm 2 have received the first BT fraction. CT has been performed 16–24 h later to verify the locations of the applicator and needles. The second BT fraction was performed after confirmation.
All the patients received routine medical nursing, and stretch socks were worn to prevent deep venous thrombosis (DVT). Continuous electrocardiogram (ECG), blood pressure, and blood oxygen saturation were monitored during the waiting period. Antiemetics and anxiolytics were provided based on patient assessment. For the patients who received BT fractions every other day in one application, the segmental pressing massager was used in the waiting period, and corresponding nursing practices were also adopted during the waiting period to maintain the patients in the supine position. In addition, one doctor and two nurses were assigned for nursing and closely monitoring the continuous BT patients.