Detection of TP53 Clonal Variants in Papanicolaou Test Samples Collected up to 6 Years Prior to High-Grade Serous Epithelial Ovarian Cancer Diagnosis

Key Points Question Can clonal TP53 variants be detected in Papanicolaou tests performed several years before high-grade serous epithelial ovarian cancer (HGS-EOC) diagnosis? Findings This cohort study including 17 patients with HGS-EOC found that in 11 patients, tumor-specific TP53 variants were detected in Papanicolaou tests performed up to 6 years before the diagnosis of HGS-EOC. Meaning These findings suggest that very early diagnosis of HGS-EOC is potentially achievable and that further developments in highly sensitive molecular approaches could improve early diagnosis of HGS-EOC.


Introduction
High-grade serous epithelial ovarian cancer (HGS-EOC) is characterized by a clonal pathogenic variant in the TP53 (OMIM 191170) gene, which represents one of the early events in the etiopathogenetic process. 1,2 It has been demonstrated that the same TP53 clonal variant detected in the primary tumor site can also be detected in precancerous lesions in the Fallopian tube, known as serous tubal intraepithelial carcinomas. 3 Recently, early serous proliferations in the Fallopian tube were found to share the same TP53 variant identified in concurrent metastatic HGS-EOCs, even in the absence of a detectable serous tubal intraepithelial carcinomas. 4 A mathematical model based on a lesion-specific proliferation rate suggests that serous tubal intraepithelial carcinomas progression to carcinoma takes approximately 6 years. To our knowledge, experimental evidence in support of this theoretical prediction is lacking, 3 which hampers the potential development of a test for the early diagnosis of ovarian cancer. 3 Clonal pathogenic variants in the TP53 gene are suitable candidates to identify early steps in the neoplastic transformation toward HGS-EOC at the molecular level. Recently, several studies [5][6][7] have shown the feasibility of detecting somatic variants in DNA from endometrial and ovarian cancers retrieved from various types of vaginal samples collected at the time of diagnosis. The aim of this study was to explore the possibility of exploiting the Papanicolaou test conducted for cervical cancer screening years before diagnosis as a source of material to detect clonal variants in the TP53 gene as a basis to develop assays for the early diagnosis of HGS-EOC.

Methods
This study was approved by the ethics committee of San Gerardo Hospital, Monza, Italy. All participants provided signed informed consent, and participants did not receive any financial compensation. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.
Women with histologically confirmed stage II through IV HGS-EOC were selected from a collection of patients with HGS-EOC who underwent primary surgical treatment at the San Gerardo

Statistical Analysis
For selected continuous variables, mean and SD or median and interquartile range values and their corresponding measures of dispersion were provided. Statistical analyses were carried out with GraphPad Prism, version 8.4.2 (GraphPad).

Results
The Figure depicts the selection of HGS-EOC patients enrolled in the study, while Since we aimed to detect variants with very low allelic frequency, the relative abundance (RA)    Table 3).

Discussion
This cohort study found that TP53 clonal somatic variants found at the ovarian cancer site were detectable in the same patients' archival Papanicolaou tests performed up to 6 years before tumor diagnosis. Remarkably, for 2 of 3 patients for whom 2 or more archival Papanicolaou tests were available, the same clonal TP53 variant was confirmed in all samples. Moreover, although most of the TP53 variants were located in variant hot spots, they were not found in healthy women's samples, corroborating the etiopathogenetic role of selected TP53 variants in patients with HGS-EOC. To our 20 months before diagnosis. The discrepancy between these findings and our results is possibly due to technical issues, such as the likelihood that the DNA stability in liquid-based archival Papanicolaou tests was lower than that of our samples, which were brush-based and stored dry. These data suggest that it is possible to use the Papanicolaou test for early diagnosis of HGS-EOC.

Strengths and Limitations
This study has 2 main strengths. To our knowledge, this is the first investigation in which

Conclusions
This cohort study found specific variants in multiple Papanicolaou tests from the same patients conducted up to 6 years before the diagnosis of HGS-EOC. The identified variants were mostly located in variant hot spots. The development of a clinically and analytically accurate diagnostic test will require a large, longitudinal prospective study to be conducted with appropriate numbers of patients and healthy controls using standardized sampling procedures and highly sensitive NGS-based approaches to monitor the entire TP53 gene. Women harboring BRCA1 or BRCA2 germinal variants could be suitable candidates to be recruited into such a study, given their high risk of

JAMA Network Open | Oncology
Detection of TP53 Clonal Variants in Papanicolaou Test Samples Before HGS-EOC Diagnosis