High grade serous ovarian cancer (HGSC) has low survival partly due to the lack of methods for detection, diagnosis, and risk prediction. TP53 mutations, which drive HGSC, are found in gynecological tissues as the result of somatic evolution, but it is unknown whether an excess of mutations is linked to ovarian cancer. Here we investigate if TP53 mutation burden measured in uterine lavage, a minimally invasive gynecological liquid biopsy, can discriminate between patients with and without HGSC. We used ultradeep TP53 duplex sequencing (>15,000x duplex depth) to detect TP53 mutations in uterine lavage collected pre-operatively in 278 patients undergoing gynecological surgery for pelvic masses (average risk) or cancer risk-reduction (high risk). All lavages contained multiple TP53 mutant clones, which were used to quantify TP53 mutation burden frequency (MBF). Average risk patients with HGSC had significantly higher TP53 MBF independently of age and other risk factors (77% sensitivity, 89% specificity, AUC = 0.88). Excluding tumor TP53 clonal mutations from the lavage MBF calculation maintains this association, suggesting that it is the overall TP53 somatic mutation burden (rather than the discovery of the specific tumor driver mutation) that identifies HGSC. These results demonstrate that TP53 somatic mutations are common in uterine lavage but more abundant in patients with HGSC, highlighting a connection between TP53 somatic evolution and ovarian cancer. Uterine lavage offers a minimally invasive approach that could be valuable to identify patients with HGSC.

The advent of Next Generation Sequencing (NGS) technologies opened up the door to the study of somatic mutagenesis and selection through the analysis of a salient clone in a sample, as in the case of cancer. This was made possible by the development of an ecosystem of computational methods that enabled mutation calling, the study of mutational processes, and the quantification of positive selection, among other research avenues. Recently, the introduction of DNA duplex sequencing technologies has allowed the detection of somatic mutations that occur in one or few cells in a sample. This has unlocked the possibility to study the interplay between mutagenesis and selection on hundreds or thousands of clones per sample, providing access to understanding somatic evolution in scenarios that were not previously easy to access, such as normal tissues. The adoption of DNA duplex sequencing technologies has been hindered, among other reasons, by the lack of a complete ecosystem of computational tools that support end-to-end analysis from the sequencing raw data to the quantification of multiple aspects of mutagenesis and selection. To overcome this problem, we present DeepClone, a protocol that comprises an experimental DNA duplex sequencing library preparation solution, and two computational pipelines to readily identify somatic mutations, and carry out calculations of mutagenesis and selection in a cohort of samples. Find the code for the data processing steps here: https://github.com/bbglab/deepUMIcaller https://github.com/bbglab/deepCSA

Li-Fraumeni Syndrome (LFS) is caused by germline pathogenic variants in TP53 which predispose carriers to early onset cancer across multiple tissues. While genomically profiling those cancers has revealed factors contributing to their formation, little is understood about how LFS impacts clonal evolution in healthy tissues preceding cancer. Here, we use ultra-deep duplex sequencing (mean ~15,000× depth) to investigate somatic mutation and selection in a family carrying the germline TP53 p.R181H pathogenic variant and a cohort of non-carrier controls. In blood samples, the germline variant was associated with more mutations in a panel designed to capture genomewide mutagenesis, and with reduced positive selection on somatic TP53 mutations, despite confounding by chemotherapy treatment in one individual. DNMT3A and TET2 mutations were positively selected and GATA2 mutations were negatively selected across the cohort, independent of the p.R181H status. Extensive multi-tissue sampling of 22 non-cancerous and 6 cancerous samples was also performed at autopsy in one individual with LFS who succumbed to esophageal cancer. Cross-tissue analysis revealed excess mutations in sun-exposed skin, esophagus and chronically-inflamed stomach tissue, and highly parallel emergence of mutations in the p.R248 hotspot of TP53 across most (18/28) tissue samples. Most somatic TP53 mutations in LFS that could be assessed for phase arose on the chromosomal copy lacking the p.R181H variant. Our study reveals how the germline p.R181H variant reshapes baseline somatic mutation and selection in normal tissues and highlights the importance of understanding early somatic evolution in LFS prior to cancer development and treatment.

The advent of Next Generation Sequencing (NGS) technologies opened up the door to the study of somatic mutagenesis and selection through the analysis of a salient clone in a sample, as in the case of cancer. This was made possible by the development of an ecosystem of computational methods that enabled mutation calling, the study of mutational processes, and the quantification of positive selection, among other research avenues. Recently, the introduction of DNA duplex sequencing technologies has allowed the detection of somatic mutations that occur in one or few cells in a sample. This has unlocked the possibility to study the interplay between mutagenesis and selection on hundreds or thousands of clones per sample, providing access to understanding somatic evolution in scenarios that were not previously easy to access, such as normal tissues. The adoption of DNA duplex sequencing technologies has been hindered, among other reasons, by the lack of a complete ecosystem of computational tools that support end-to-end analysis from the sequencing raw data to the quantification of multiple aspects of mutagenesis and selection. To overcome this problem, we present DeepClone, a protocol that comprises an experimental DNA duplex sequencing library preparation solution, and two computational pipelines to readily identify somatic mutations, and carry out calculations of mutagenesis and selection in a cohort of samples. Find the code for the data processing steps here: https://github.com/bbglab/deepUMIcaller https://github.com/bbglab/deepCSA

TP53 mutations drive more than half of human cancers and are commonly found in normal tissues, where they clonally expand with age. Little is known, however, about how these clones influence cancer risk and how the tissue microenvironment might promote their expansion. This gap in knowledge is in part due to the small size of clones, which obscures their detection. Here, we used a multi-omics approach to characterize the TP53 clonal landscape and investigate the tissue microenvironment of the human fallopian tube, which is the site of origin of high-grade serous carcinoma (HGSC), an aggressive subtype of ovarian cancer almost exclusively driven by TP53 mutations. Our goal was to compare age-related TP53 clonal expansions in the fallopian tube with those identified in individuals with BRCA1, BRCA2, BRIP1, PALB2, or RAD51C/D germline mutations (germline mutation carriers) who are at a higher risk of developing HGSC. Human fallopian tube tissues were collected and macrodissected from 67 autopsy cases (average risk group, age: 0-91 years) and 132 prophylactic surgery cases (high-risk group, age: 32-69 years) to assess the TP53 clonal landscape using ultra-deep duplex sequencing (∼12,000x) of the fallopian tube. In addition, we used spatial transcriptomics to study the tissue microenvironment of 47 [RR1] distinct normal fallopian tube areas from 8 individuals (BRCA1: N = 2, BRCA2: N = 4; Non-carriers: N = 2) using the GeoMx Human Whole Transcriptome Atlas probe panel with segmentation separating epithelial and stromal regions of the fallopian tube. Duplex sequencing revealed TP53 mutations in the fallopian tubes of nearly all individuals, with more mutations, increased pathogenicity, and larger clones at older ages. TP53 pathogenic large clones were more abundant in the fallopian tube of germline carriers compared to similar age autopsy cases. Preliminary findings in our transcriptomic study showcased > 6000 transcripts detected across all fallopian tubes of individuals at high-risk of HGSC, though further analyses are underway to determine whether there are differences observed in the epithelial vs. stromal compartments of the fallopian tube. Our results demonstrate prevalent TP53 somatic evolution in the fallopian tube and an excess of pathogenic TP53 clonal expansions in germline carriers, providing a biological explanation for the increased risk of HGSC in these individuals. Coohleen Ann Coombes, Elizabeth U. Parker, Brendan F. Kohrn F. Kohrn, Jeanne Uy. Fredrickson, Shreya Suresh, Elena Latorre-Esteves, Emma Hazard, Melanie R. Dillon, Marc R. Radke, Anh P. Vo, Roseanne J. Gamboa, Katherine Lai, Emily Beirne, Bre A. Mills, Shreeram Akilesh, Ronit Katz, Barbara M. Norquist, Elizabeth M. Swisher, Rosa Ana Risques. Ultra-deep characterization of TP53 somatic evolution in the fallopian tube and its association with ovarian cancer risk [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr PR016.

Purpose:Long-standing mucosal inflammation is suspected to be one of the main drivers of colitis-associated colorectal cancer (CA-CRC), but far from all colitis patients develop cancer. Non-neoplastic mucosa located distant from neoplastic lesions may harbour early molecular events of carcinogenesis. We hypothesise that patients with UC who have progressed to neoplasia (progressors) exhibit a distinct molecular profile of mucosal inflammation from non-progressors. Patients and Methods:We performed transcriptomic profiling of 143 mucosal biopsies from non-neoplastic colonic segments of 14 UC progressors and 30 UC non-progressors using the Agilent SurePrint G3 human gene expression 60K microarray. Subsequently, we carried out gene set ontology analyses. In addition, we assessed lymphocyte infiltration to the mucosa of biopsies taken adjacent to biopsies used for transcriptomic analysis by immunohistochemistry. Results:Adjusting for molecular alterations associated with long disease duration, our findings revealed that UC Progressors' inflamed mucosa has a distinct gene expression profile of 529 significantly deregulated genes, eg LTB, CXCL13, CD19, C3 and CYP4F3. The profile was negatively enriched for biological processes (BPs) such as adaptive immune responses and complement system activity and positively enriched for processes related to detoxification. The negatively enriched BPs were supported by the presence of fewer infiltrating B cells and less lymphoid aggregates in the microenvironment of the inflamed mucosa of progressors. Conclusion:The inflamed colonic mucosa from UC patients who have progressed to dysplasia or cancer has a different gene expression profile than that of UC non-progressors, suggesting lower levels of lymphoid organ-initiating and immune cell-attracting signals and fewer infiltrating immune cells to the mucosa. Consequently, progressors may lack the ability to mount sufficient adaptive immune responses necessary to counteract driving forces of malignant transformation in UC.

Background TP53 mutations are the main drivers of aggressive, high-risk endometrial carcinomas commonly diagnosed in Black individuals. However, TP53 mutations have also been identified in benign, non-cancerous tissues. We sought to understand the TP53 mutational landscape in benign endometrium throughout the lifespan of Black and White individuals, accounting for structural socioeconomic context. Methods Ultra-sensitive TP53 mutation detection was performed with high-depth duplex sequencing (∼13,000×) in DNA extracted from histologically normal endometrium collected at autopsy (69 % of cases) or surgery (31 % of cases) from 83 individuals ages 0 to 81 (31 Black and 52 White, median age 35 years) without endometrial cancer. Histologically normal endometrium was also collected from 10 White individuals with endometrial cancer. Results We identified 266 coding TP53 mutations in the normal endometrium of individuals without endometrial cancer, 57 % of which were pathogenic. The number, pathogenicity, and size of TP53 mutant clones in normal endometrium increased with age. Multivariable models showed no significant association between race or socioeconomic metrics and TP53 mutation frequency in normal endometrium. An exploratory analysis on the histologically normal endometrium of White individuals with endometrial cancer identified the tumor mutations at low levels in the normal biopsy of 5 out of 6 cases. Conclusions Our study revealed prevalent TP53 somatic evolution in benign endometrium across human lifespan and no racial differences in this cohort of predominantly younger individuals. Future studies should consider the analysis of larger cohorts with older individuals to detect potential effects of racial disparities on TP53 somatic evolution later in life.

Men are at higher risk of several cancer types than women1. For bladder cancer the risk is four times higher for reasons that are not clear2. Smoking is also a principal risk factor for several tumour types, including bladder cancer3. As tumourigenesis is driven by somatic mutations, we wondered whether the landscape of clones in the normal bladder differs by sex and smoking history. Using ultradeep duplex DNA sequencing (approximately 5,000×), we identified thousands of clonal driver mutations in 16 genes across 79 normal bladder samples from 45 people. Men had significantly more truncating driver mutations in RBM10, CDKN1A and ARID1A than women, despite similar levels of non-protein-affecting mutations. This result indicates stronger positive selection on driver truncating mutations in these genes in the male urothelium. We also found activating TERT promoter mutations driving clonal expansions in the normal bladder that were associated strongly with age and smoking. These findings indicate that bladder cancer risk factors, such as sex and smoking, shape the clonal landscape of the normal urothelium. The high number of mutations identified by this approach offers a new strategy to study the functional effect of thousands of mutations in vivo-natural saturation mutagenesis-that can be extended to other human tissues.

Abstract High-grade serous carcinoma (HGSC), the most common and pervasive subtype of ovarian cancer originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. These lesions may evolve into carcinomas, eventually spreading to the ovary and peritoneum1,2. Recent research in non-cancerous tissues and gynecologic liquid biopsies of individuals with and without HGSC have demonstrated that somatic TP53 mutations accumulate and clonally expand with age3–9. However, TP53 somatic evolution has not been characterized in the normal fallopian tube epithelium. In addition, it is not known whether TP53 mutations are more abundant in individuals with germline BRCA1/2 mutations (BRCA carriers), who are at a high risk of HGSC10. The goals of this study were to characterize age-related TP53 somatic mutations in non-cancer fallopian tube epithelium across the human lifespan and to determine whether the TP53 mutational landscape is altered in BRCA carriers. Our cohort consisted of 1) autopsy cases, spanning the ages of 0-91 years, from individuals without ovarian cancer (N=46) and 2) prophylactic surgery cases from BRCA carriers and non-carriers (N=26, ongoing analyses). Fallopian tube tissues were collected, frozen, and macrodissected using a 1mm biopsy punch, enriching for the fallopian tube epithelium. DNA was extracted from each sample, the sequenced for TP53 using ultra-deep duplex sequencing (mean depth: ~12,000x). In the normal fallopian tube epithelium collected across human lifespan, we found that the number of unique TP53 mutations and the size of pathogenic clones increased with age, showcasing somatic evolution and clonal expansion of TP53 in non-cancerous fallopian tube epithelium with age. We also found that the number of TP53 mutations in hotspot codons increased with age, suggesting that cancer-like mutations are positively selected as individuals age. We compared the normal fallopian tube TP53 mutations with HGSC TP53 mutations and demonstrated that the types of mutations (e.g. indels, splice, nonsense, missense, and silent mutations) in the aging fallopian tube closely mirrored the mutations types observed in HGSC. Data analysis from BRCA carriers is ongoing, but based on preliminary data, we anticipate a higher burden of pathogenic TP53 mutations, indicating that predisposition to HGSC in BRCA carriers may be related to an excess of TP53 somatic evolution in the fallopian tube. Altogether, our research demonstrates that TP53 somatic evolution is a normal aging phenotype in the fallopian tube, with more and larger pathogenic clones occurring later in life. We expect to determine whether this effect is more prominent in BRCA carriers, which would provide a much needed biological explanation to understand the increased risk of HGSC in these patients, opening new venues for better cancer prediction and prevention. Citation Format: Coleen Ann Coombes, Elizabeth U. Parker, Brendan F. Kohrn, Jeanne Uy Fredrickson, Elena Latorre-Esteves, Emma Hazard, Shreya Suresh, Marc R. Radke, Anh P. Vo, Barbara S. Norquist, Rosana Risques. Characterizing somatic TP53 mutations in non-cancerous and high-risk fallopian tubes using ultra-deep sequencing [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B101.

Somatic TP53 mutations are prevalent in normal tissue but little is known about their association with cancer risk. Cervical liquid-based cytology (LBC), commonly known as Pap test, provides an accessible gynecological sample to test the value of TP53 somatic mutations as a biomarker for high-grade serous ovarian cancer (HGSC), a cancer type mostly driven by TP53 mutations. We used ultra-deep duplex sequencing to analyze TP53 mutations in LBC and blood samples from 70 individuals (30 with and 40 without HGSC) undergoing gynecologic surgery, 30 carrying BRCA1 or BRCA2 germline pathogenic variants (BRCApv). Only 30% of the tumor mutations were found in LBC samples. However, TP53 pathogenic mutations were identified in nearly all LBC and blood samples, with only 5.4% of mutations in LBC (20/368) also found in the corresponding blood sample. TP53 mutations were more abundant in LBC than in blood and increased with age in both sample types. BRCApv carriers with HGSC had more TP53 clonal expansions in LBC than BRCApv carriers without cancer. Our results show that, while not useful for direct cancer detection, LBC samples capture TP53 mutation burden in the gynecological tract, presenting potential value for cancer risk assessment in individuals at higher hereditary risk for ovarian cancer.

Abstract High-grade serous carcinoma (HGSC), the most common and pervasive subtype of ovarian cancer, originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. These lesions may evolve into carcinomas, eventually spreading to the ovary and peritoneum. Recent research in normal tissues and gynecologic liquid biopsies of individuals without HGSC has demonstrated that somatic TP53 mutations accumulate and clonally expand with age, suggesting that TP53 somatic evolution is part of the normal aging process​. To date, however, TP53 somatic evolution in the normal fallopian tube epithelium has not been characterized and it is unknown whether TP53mutations are more abundant in individuals with germline mutations in BRCA1 or BRCA2 (BRCA carriers) who are at a higher risk of developing HGSC​. In this study, we aimed to characterize age-related TP53 somatic mutations in the normal fallopian tube epithelium across the human lifespan and to determine whether the TP53 mutational landscape is altered in BRCA carriers. Our cohort consisted of 46 autopsy cases (age: 0-91 years) from individuals at low-risk of developing ovarian cancer and 48 prophylactic surgery cases (age: 32-69 years) from BRCA carriers. Fallopian tube tissues were collected, frozen, and macrodissected using a 1mm biopsy punch, enriching for the fallopian tube epithelium. DNA was extracted from each sample and sequenced for TP53 using ultra-deep duplex sequencing (mean depth: ~12,000x). Pathogenicity was determined using a machine learning algorithm (AlphaMissense) and a well-established predictor of TP53 pathogenicity (Seshat). Preliminary analyses in the normal fallopian tube epithelium collected across human lifespan demonstrated that the mutational profile (e.g. indels, splice, nonsense, missense, and silent mutations) in the aging fallopian tubes mirrored the mutational profile of HGSC. We also observed that the number of unique TP53 mutations and pathogenic clones increased with age, revealing TP53 somatic evolution. Additionally, the number of large TP53 mutant clones (>2 duplex mutant reads) increased exponentially with age, staring at age 30. When comparing the fallopian tubes of BRCA carriers to age-matched normal fallopian tubes, the percentage of pathogenic large clones in BRCA carriers was 22.8% vs. 6.0% in low-risk individuals (Fisher exact test, p < 0.0001), suggesting increased TP53 somatic evolution in the fallopian tubes of BRCA carriers. Altogether, our research demonstrates that TP53 somatic evolution is a normal aging phenotype in the fallopian tube, with more and larger pathogenic clones occurring later in life. Preliminary analyses in BRCA carriers indicate that somatic evolution is more prominent in these individuals, which may provide a biological explanation to the increased risk of HGSC and open new venues for better cancer prediction and prevention. Citation Format: CoohleenAnn Coombes, Brendan F. Kohrn, Jeanne U. Fredrickson, Elena Latorre-Esteves, Emma Hazard, Shreya Suresh, Marc R. Radke, Melanie Dillon, Anh P. Vo, Katherine Lai, Ronit Katz, Barbara M. Norquist, Elizabeth M. Swisher, Elizabeth U. Parker, Rosana Risques. IncreasedTP53somatic evolution in the fallopian tubes of individuals at high risk of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB416.

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

Abstract Background TP53 somatic mutations accumulate and clonally expand in histologically normal tissues with aging, however, little is known about this process and how it influences cancer development. Within endometrial carcinoma, TP53 mutations tend to be found in aggressive, non-endometrioid histologies associated with poor survival outcomes, more commonly affecting Black women. We seek to understand the TP53 mutational burden in benign endometrium by characterizing mutations acquired throughout a lifespan with the integration of social determinants of health in Black and White women. Methods Endometrial tissue was collected at autopsy or hysterectomy, confirmed with H&E staining, and macrodissected. Ultra-deep duplex sequencing (~10,000x) of isolated DNA was used to sequence TP53somatic mutations with high resolution. Each individual’s TP53 mutation frequency was calculated as the number of unique TP53 variants divided by the total duplex nucleotides sequenced. Mutations were classified as pathogenic if insertion-deletion, splice, or nonsense in addition to all pathogenic substitutions identified by the Seshat TP53 database. Socioeconomic metrics were acquired using zip codes and the 2021 US Census Bureau American Community Survey. Individuals with a history of endometriosis, prior malignancy, or prior chemotherapy were excluded. Results Benign endometrial tissue was collected from 69 individuals (24 Black, 45 White), including 22 aged 0-19 years, 25 aged 20-39 years, 17 aged 40-59 years, and 5 aged over 60 years. 174 TP53 variants were identified. Variant types included 66.7% missense (n=116), 13.2% insertion-deletion (n=23), 12.1% synonymous (n=21), 4.6% nonsense (n=8), and 3.4% splice (n=6). 102 TP53 mutations (58.6%) were pathogenic. TP53 mutation frequency increased linearly with age (Spearman correlation p<0.01). 12.6% of mutations were identified in more than one duplex mutant read (large clone) beginning at age 29 through age 81. The frequency of large TP53 mutant clones increased exponentially with age. In this limited dataset, there was no statistical association between socioeconomic metrics and the mutational burden of Black and White individuals. Conclusions The number and size of TP53 mutant clones in benign endometrium are associated with older age, indicating clonal evolution through life. Most somatic mutations identified were pathogenic substitutions or loss-of-function variants, confirming positive selection of cancer-like mutations. Additional data are needed to determine the role of socioeconomic factors in TP53 genomic burden. Citation Format: Eric Rios-Doria, Elizabeth U. Parker, Brendan F. Kohrn, Elena Latorre-Estevez, Jeanne Fredrickson, Elizabeth M. Swisher, Kemi M. Doll, Rosana Risques. TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A004.

Background Individuals with germline BRCA1 and BRCA2 pathogenic variants (BRCA carriers) are at high risk of developing high grade serous ovarian carcinoma (HGSC). HGSC is predominantly driven by TP53 mutations, but mutations in this gene are also commonly found in non-cancerous tissue as a feature of normal human aging. We hypothesized that HGSC predisposition in BRCA carriers may be related to increased TP53 somatic evolution, which could be detectable by ultra-deep sequencing of TP53 mutations in gynecological liquid biopsies. Methods Duplex sequencing was used to identify TP53 mutations with high sensitivity in peritoneal washes and cervical liquid-based cytology (LBC) collected at surgery from 60 individuals including BRCA1 and BRCA2 carriers, and non-carriers. TP53 mutation pathogenicity was compared across groups and with TP53 cancer mutations. Results TP53 mutations were more abundant in cervical LBC than in peritoneal washes but increased with age in both sample types. In peritoneal washes, but not in cervical LBC, pathogenic TP53 mutation burden was increased in BRCA1 carriers compared to non-carriers, independently of age. Five individuals shared identical pathogenic TP53 mutations in peritoneal washes and cervical LBC, but not in blood. Conclusions Ultra-deep sequencing of TP53 mutations in peritoneal washes collected at surgery reveals increased burden of pathogenic TP53 mutations in BRCA1 carriers. This excess of pathogenic TP53 mutations might be linked to the elevated risk of HGSC in these individuals. In some patients, concordant TP53 mutations were found in peritoneal washes and cervical LBCs, but the cell of origin remains unknown and deserves further investigation.

Supplementary Figure from Colorectal Cancer Is Associated with the Presence of Cancer Driver Mutations in Normal Colon