Study design
This study is a prospective, observational, single-center study. The study collected cervical cancer patients who signed the informed consent form in Guizhou Provincial People's Hospital and used plasma HPV-ctDNA to assess the MRD status of cervical cancer patients before and after concurrent chemoradiation therapy, and compared different MRD statuses before and after treatment and at 4, 6, 12, and 24 weeks after the end of treatment with the patient's efficacy and risk of recurrence. The target sample size is a sample of 30 patients. We hypothesize that MRD status changes significantly before, during, and after treatment and that MRD based on HPV-ctDNA assessment can accurately predict the efficacy and recurrence risk of patients with concurrent chemoradiotherapy for cervical cancer. The flowchart of the study is summarized in Figure 1. (The appropriate informed consent was provided by the relevant study personnel).
Figure 1. Flow chart for the study.
Primary endpoint
To evaluate the MRD status of patients with cervical cancer before and after simultaneous radiotherapy based on plasma HPV-ctDNA, to evaluate the efficacy and recurrence risk of cervical cancer after simultaneous radiotherapy based on MRD status, and to validate MRD as a dynamic prognostic indicator for cervical cancer.
Secondary endpoint
The secondary endpoint is that the changes in MRD status can assist clinical decision-making on whether to administer adjuvant chemotherapy, clarify the duration and benefit of adjuvant chemotherapy, and provide validation for clinical diagnosis and treatment. Based on the MRD status assessed by liquid biopsy HPV-ctDNA, combined with clinical prognostic indexes and imaging examinations, it can provide a new individualized assessment method and idea for the screening of highly sensitive cases for synchronous radiotherapy for locally advanced cervical cancer.
Inclusion Criteria
1. Age: ≥ 18 years, ≤ 75 years.
2. Histopathological confirmation of cervical cancer.
3. Imaging or PET/CT examination can be performed to understand the tumor situation and complete all follow-ups.
4. Measurable lesions before treatment.
5. Good physical condition: ECOG score 0-1 (or KPS score 70-100).
6. Estimated survival ≥ 6 months.
7. The baseline blood routine and biochemical indexes before chemoradiotherapy met the following criteria: hemoglobin ≥80g/L, absolute neutrophil count (ANC) ≥ 1.5×109/L, platelet ≥ 100×109/L, ALT and AST ≤ 2.5 times the upper limit of normal, and serum albumin ≥ 30g/L.
8. There are three preoperative items: if the patient is combined with syphilis, it is necessary to undergo expulsion therapy before treatment.
9. The patient has no history of allergy to rubber products.
10. Cardiopulmonary function is normal.
Exclusion criteria
1. Those who are allergic to rubber products.
2. Those who suffer from a severe acute infection that is not controlled or have purulent and chronic infection wounds that do not heal, active chronic hepatitis B, active tuberculosis, syphilis outbreak, AIDS, etc.
3. Patients with pre-existing severe heart disease, including congestive heart failure, uncontrollable high-risk arrhythmia, unstable angina, myocardial infarction, severe valvular heart disease, and intractable hypertension.
4. Those who suffer from neurological or psychiatric diseases or mental disorders that are not easy to control, have poor compliance, cannot cooperate and narrate treatment responses, have uncontrolled primary brain tumors or central nervous system metastases, and have obvious intracranial hypertension symptoms or neuropsychiatric symptoms.
5. Accompanied by malignant serous effusion.
6. History of severe enteritis and cystitis, bleeding, intestinal perforation, rectovaginal fistula, rectobladder fistula, etc.
7. Those who have participated in other clinical trials.
8. Other situations in which the investigator believes that the subject is not suitable to participate in this experiment.
Withdrawal Criteria
1. Subjects request to withdraw from the study.
2. Irreversible accidents occur.
3. Inability to conduct effective follow-up of the subjects.
4. The investigator considers that the subject must discontinue the study from a medical point of view.
5. Other unforeseen circumstances.
Termination criteria
1. The patient's condition worsens.
2. Occurrence of serious adverse events.
3. Poor patient compliance.
Collection of basic information
A questionnaire survey of basic information was conducted on the research subjects, including basic information (gender, age, height, weight), risk factors (smoking history, alcohol history, family history of tumor), clinical diagnosis report (B-ULTRASOUND, CT, MRI, etc.), pathological test report (tumor size, tumor stage, tumor location, tumor subtype, degree of differentiation, metastasis, invasion, immunohistochemical results, etc.), disease history (e.g., diabetes, Coronary heart disease, and hypertension, etc.), medication history, detailed imaging data (such as the maximum diameter and volume of the primary lesion), laboratory results such as SCC-Ag value and comprehensive medical history data, demographic information (such as income, education, smoking, alcohol consumption, etc.).
CCRT treatment options
1. Radiotherapy: including external beam radiotherapy (EBRT) and intracavitary brachytherapy (ICBT). The number of radiotherapy is 25~28 times, and the number of radiotherapy is adjusted according to the patient's condition. EBRT is 1.8~2.0 Gy 5 times a week for a total dose of 45~50 Gy and ICBT is 6.0 Gy for 5 times for a total dose of 30 Gy.
2. Concurrent chemotherapy: Single-agent cisplatin (40mg/m2, 1 time/week) based regimen is preferred for concurrent chemotherapy, and individualized adjustment is made according to tumor volume, pelvic lymph node condition, and patient's physical condition. During concurrent chemotherapy, patients undergo clinical examination and laboratory tests at least once a week and before each course of chemotherapy. Adequate hydration is required before and after concurrent chemotherapy.
Specimen collection and processing: Figure 2.
Figure 2. Sample collection timeline.
TP0=Pre-treatment, TP1=external irradiation, TP2=4 weeks after completion of internal irradiation treatment, TP3=6 weeks after completion of internal irradiation treatment, TP4=12 weeks after completion of internal irradiation treatment, TP5=24 weeks after completion of internal irradiation treatment.
Tissue sample collection
1. According to the needs of diagnosis and treatment, clinicians collect tissue samples of cervical cancer before treatment and during treatment (before the start of the first post-load), surgically excised soybean-sized tumor tissue, fixed by formalin, and place them in labeled cryopreservation tubes, stored at -80°C for subsequent testing. Record the time of tissue resection, surgical site, and other surgical information about the tissue sample.
2. Blood sample collection: Before treatment, during treatment (before the start of the first afterload), and 4 weeks, 6 weeks, 12 weeks, and 24 weeks after the end of treatment, 8-10 ml of elbow venous blood in enrolled patients with locally advanced cervical cancer was placed in a vacuum blood collection tube, the blood collection tube was gently inverted 5-6 times immediately, placed at 4 degrees for at least 30-45 min, 4 degrees at 1300 rpm centrifugation for 10 min plasma separation, serum and white blood cells were retained after treatment, and protease inhibitors were added to the serum immediately. The obtained samples were stored in a -80°C ultra-low temperature freezer for later testing. There are no special requirements for whether the subject is fasting or not.
ctDNA/MRD and HPV/MRD assays
Extraction of ctDNA
1. DNA extraction of tissues and leukocytes: DNA extraction of tissues and leukocytes was performed using the Qiamp DNA FFPE tissue kit. Plasma was extracted from cell-free DNA using the QIAsymphony DSP Circulating DNA Kit (Qiagen, Germany) according to the instructions of the extraction kit. The quality and quantity of cell-free DNA were examined using a NanoDrop 2000 ultra-microvolume spectrophotometer (ThermoFisher Scientific, USA) and a PicoGreendsDNA Quantification Kit (Life Technologies, USA).
2. Library construction and sequencing: Libraries were constructed using the NEBNext@ Ultram DNA Library Prep Kit for Illumina kit, sequencing adapters with ff unique identifiers were added to the DNA fragments according to the instructions, and DNA was sequenced using the IluminaHiseq 3000 platform with an average sequencing volume of 15 GB per sample.
Bioinformatics analysis
1. Sequencing data were in BCL format and converted to FASTQ (version: Illumina 1.8+) using bcf2fastq (v2.17.1.14, Illumina, Inc.) software. Trimmomatic was used for data quality control, and SAM files were generated using the MEM algorithm of BWA (0.7.12) and the default parameters aligned to the human reference genome (hg19, GRCh37 Genome Reference Consortium Human Reference 37). Convert the SAM file to a BAM file by Picard (1.119) and sort according to chromosomal coordinates. Use the Genome Analysis Toolkit (GATK, version 3.4-0) to optimize the local alignment of the alignment results.
2. SNVs/Indels/CNVs analysis: analysis of point mutations (SNVs) and indels (Indels) using VarScan2[5] (2.3.9) with a minimum detection frequency of 0.01 and a p-value of 0.05 to generate VCF results. THE VCF RESULTS WERE ANNOTATED USING ANNOVAR AND FURTHER MANUALLY CHECKED BY IGV. Copy number variation (CNVs) analysis was performed using ADTEx (1.0.4).
MRD-targeted gene sequencing
1. ctDNA extraction: ctDNA was extracted from plasma samples using a QIAGEN Circulating Nucleic Acid kit, the experimental operation process was referred to the instructions of the extraction kit, and the extracted ctDNA was directly purified and later set aside, and Qubit was used for quantification, with a total extraction amount of not less than 4ng.
2. Library construction and quality control: Separation of large and small fragments of plasma ctDNA, removal of interference of large DNA fragments, end repair of the screened DNA fragments, addition of A base terminus and Adaptor sequence, further purification of the DNA products with adapters, and increase the enrichment input through PCR amplification reaction.
3. Probe enrichment: For tumor driver genes, a DNA probe library hybridized to the target gene is designed, and the synthetic probe library is used to enrich the exome region corresponding to the target gene in the high-throughput sequencing library, and the 486panel probe of Nanjing Shihe Gene Biotechnology Co., Ltd. is currently used as the tumor-related gene capture method, and the sequencing length reaches 150bp.
4. Hands-on sequencing: The enriched library was sequenced using the Illumina Hiseq sequencing platform PE150 kit for high-throughput sequencing. The average sequencing depth of tissue samples is not less than 1000X, the average sequencing depth of cfDNA samples is not less than 5000X, and the average sequencing depth of whole blood control samples is not less than 100X.
Follow-up
All enrolled subjects will be followed up every 6 months, and if there are special circumstances, follow-up will be required at least once a year, and the follow-up will be terminated if the case dies. The follow-up was mainly to record the adjuvant treatment (treatment regimen, treatment duration, etc.), reexamination, postoperative recurrence, post-recurrence treatment, and survival of the subjects after concurrent chemoradiotherapy. Follow-up was mainly carried out in two forms: telephone follow-up and outpatient review. For lost-follow-up cases, the reason for loss to follow-up (e.g., empty phone number, wrong number, uncooperative subject, etc.) should be registered, and the last follow-up time should be recorded.
Sample size and statistical analysis
This is a prospective, single-arm safety and feasibility trial that will include a total of 30 subjects according to the relevant study[18, 19] and will not be subject to formal sample size measurement.
Allocation
Not applicable.
Masking
This is not a blinded study for either clinicians or patients.
Data collection methods
Datasets will be obtained from the corresponding author upon reasonable request. To conceal personal information, a two-digit participant number is used in the allocation order. Personal records of each patient will be retained by the investigator as raw data, including copies of informed consent forms, medical records, laboratory data, image data, patient diaries, and other records or notes. Screening, obtaining informed consent, enrolment, registration, and data collection will be conducted by the investigator or a qualified clinical research coordinator, and access to the eCRF will be granted only to registered investigators participating in this study. The results of the trial will be submitted to an international peer-reviewed journal and the main findings will be published in the journal.
Statistical methods
The data were statistically analyzed using SPSS27.0 software, and the measurements were expressed as mean ± standard deviation (`x±s), the overall rate and the constituent ratio data were tested by χ2 test, the Wilcoxon's rank test for the comparison of the data within the group was used for the paired samples, and the Kruskal-Wallis H test for the comparison of the data of the skewed distributions was used for the comparison of the data of the multiple independent samples, and the difference of p<0.05 was considered to be was statistically significant, and sensitivity, specificity and correctness were used for the evaluation of the two different detection methods.
Handling of missing values and outliers
In principle, all analyses will be performed without interpolating missing values or outliers. However, if missing values or outliers are identified before data locking that may significantly affect the results of the analyses, they will be statistically processed.
Monitoring
Data Security Monitoring
When 20% of the samples have been processed, the completeness of the data collection will be assessed and improvements will be made where necessary.
Safety Monitoring
If an SAE occurs in any patient, the content should be reported immediately to the appropriate institution's Institutional Review Board (IRB). If no SAE occurs, the results of the monitoring will be included in the final report. Any protocol violations, noncompliance, or unanticipated problems will also be reported to the IRB of the appropriate institution If more than 5 SAEs are reported at any institution, the entire investigator population will discuss the sustainability of the study and the need for possible termination or modification of the study. The results of this discussion will be reported to the IRB, and the Principal Investigator will provide compensation by relevant laws and regulations if a participant suffers bodily harm as a result of participation in this study.
Auditing
Data collection and protocol adherence will be monitored by appropriate personnel at 6-month intervals and may be conducted at other times if necessary.
Ethics
The study was approved by the Ethics Committee of Guizhou People's Hospital (2023-029).
The study is registered in ClinicalTrails.gov (NCT05950087).
Study Status
Patients should start on April 6, 2023, and are currently ongoing.