Routine Shallow-coverage Whole Genome Sequencing Testing of Liquid-based Cervical Smears for the Early Diagnosis of Ovarian Cancer

Overview

High-grade serous ovarian carcinoma (HGSOC) is the deadliest gynaecological tumour. The 5-year overall survival rate for advanced-stage disease is less than 30 per cent, due to diagnosis at an advanced stage. There is currently no validated screening test for ovarian cancer. According to the literature, the current hypothesis is that HGSOCs develop primarily from the distal part of the fallopian tube, which is anatomically close to the lower genital tract. Tumour cells have even been observed in cervical smears. Studies suggest that early diagnosis of HGSOC is possible non-invasively by detecting pathogenic variants of the TP53 gene in DNA extracted from cervical smears. Studies suggest that early diagnosis of HGSOC is possible using non-invasive methods by detecting pathogenic variants in the TP53 gene in deoxyribonucleic acid (DNA) extracted from cervical smears. In 2023, whole-genome sequencing (WGS) of DNA extracted from cervical smears to detect genomic instability successfully identified the presence of high-grade serous ovarian cancer up to nine years before diagnosis. This study demonstrates the feasibility of early diagnosis of ovarian cancer using genomic instability analysis via Shallow-coverage whole-genome sequencing (sWGS) on DNA obtained from cervical smears. This study aim to confirm the feasibility of diagnosing high-grade serous ovarian carcinoma by analysing genomic instability via sWGS on DNA extracted from liquid-based cervical smears, using a homogeneous series of samples from patients treated at the Toulouse University Cancer Institute-Oncopole (IUCT-Oncopole).

1. Cohort constitution Patients treated at the Toulouse University Cancer Institute-Oncopole (IUCT-Oncopole) for high-grade serous ovarian cancer (HGSOC) at any stage, and for whom an initial surgical procedure (diagnostic or cytoreductive) is planned as part of their treatment pathway, may be included. For these patients, tumour and healthy tissue samples are systematically collected (taken as part of the ovarian cancer care pathway) and a liquid-based cervical smear is performed (carried out to screen for concomitant cervical cancer). The liquid-based cytology technique, which has replaced the cervical smear used in retrospective studies, reduces the number of unsatisfactory samples, enables the detection of human papillomavirus (HPV) and allows molecular and cytological tests to be carried out using a single sample. Any abnormalities found in this sample may be considered specific to an ovarian origin, given the exclusion of patients with other cancers of the reproductive tract (in particular TP53-mutated endometrial carcinomas). The samples will be forwarded to the pathology department via the usual procedure. 2. Analysis of samples Tumour and healthy tissue samples are fixed in formalin and then embedded in paraffin. The diagnosis is made on the tumour sample using standard staining and immunohistochemical analysis. An assessment of the quantity of tumour material (cell density and tumour area) is carried out. Following extraction, the DNA is quantified and characterised by the molecular biology platform using the an assay kit from Thermo Fisher Scientific. Shallow-coverage whole-genome sequencing (sWGS) is routinely performed on the tumour sample with 2X coverage on platform to assess homologous recombination deficiency (HRD) status, thereby determining eligibility for PARP enzyme inhibitors prescription. The same procedure is used for healthy tissue as part of the study. The liquid-based cervical sample is processed according to the routine protocol as part of cervical cancer screening. The residual sample is centrifuged and DNA is extracted from the pellet to perform the sWGS technique, which is comparable to that carried out on tumour tissue. The residual samples - the block of healthy tissue, DNA extracted from the tumour tissue and cervical fluid - are stored. 3. Implementation and analysis using bioinformatics tools The sequences generated will be aligned to the GRCh38 reference genome using the bwa mem software, and duplicates will be removed using picard-tools. The open-source CPA8 pipelines and the adaptation of the genomic index (GI) for HGSOC published by the ONCOSARC team will be implemented in the available local environment. The comparative analysis will be based on sampling sites and patient characteristics (e.g. FIGO (International Federation of Gynaecology and Obstetrics) stage). The analysis will be carried out in collaboration with the bioinformatics team in the pathology department.