Secondary POLE Mutation Drives Metastasis in Hereditary Breast and Ovarian Cancer: Revealing Fallopian Tube Ultramutation

Introduction

Among DNA polymerases, DNA Polymerase Epsilon (POLE) is critical for correcting replication errors.¹ Somatic alterations in POLE's exonuclease domain occur in 7%-12% of uterine endometrial carcinomas, in 1%-2% of colorectal cancers, and sporadically in stomach cancer, glioblastoma, and breast cancer.^2-5 Associated P286R, S297F, and V411L mutations are frequently reported, resulting in a highly mutated state.⁵

Synchronous primary endometrial and ovarian cancers account for 10% of the cumulative cases of these diseases. Most studies have focused on synchronous primary endometrial and ovarian cancers with endometrioid histology; however, serous histology is less frequently reported and poorly understood. Furthermore, the genomics of synchronous primary endometrial and ovarian cancers in hereditary breast and ovarian cancer (HBOC) syndrome remains unclear.

Here, we present a case of a patient with HBOC syndrome harboring synchronous endometrial and fallopian tube tumors. Genomic analyses revealed that the fallopian tube cancer had developed a secondary POLE mutation during metastasis, generating an ultramutated genetic profile at the endometrial metastatic site. To our knowledge, this is the first report highlighting a novel aspect of ovarian cancer metastasis.

Case Study

Initial Diagnosis and Surgical Management

A 63-year-old female patient with a family history of cancer (her younger sister had died of breast cancer at age 33 years and her mother was diagnosed with breast cancer and later developed renal pelvis cancer) was referred to Keio University Hospital for further evaluation after a cervical or endometrial polyp (histologically confirmed as adenocarcinoma) was excised at a previous medical facility. Contrast-enhanced magnetic resonance imaging revealed a 35-mm tumor extending from the uterine cervix to the uterine corpus and an approximately 36-mm tumor in the right fallopian tube.

Histological findings did not conclusively indicate the origin of the uterine cervix or corpus tumor or whether the right fallopian tube tumor represented a synchronous primary cancer. Therefore, radical hysterectomy, bilateral salpingo-oophorectomy, para-aortic lymphadenectomy, and partial omentectomy were performed, treating the uterine tumor as primary cervical cancer.

Pathological Findings

Examinations revealed a prominent exophytic tumor (35 × 27 mm) extending from the uterine cervix to the uterine corpus, invading the myometrium. Atypical epithelial cells with hyperchromatic nuclei forming complex glandular, confluent tubular, solid, and papillary structures were observed. Regions of proliferating cells with bare nuclear forms and fine-to-granular chromatin were identified alongside pronounced neutrophil infiltration within the glandular lumens and stroma. The histological features indicated invasive carcinoma, with high-grade nuclear atypia and slit-like structures suggesting the differential diagnosis of high-grade serous carcinoma.

Immunohistochemical analysis of the uterine tumor revealed estrogen receptor (ER), progesterone receptor (PgR), and PAX8 expression, a Ki-67 index >90%, and a null p53 expression pattern. WT1 staining demonstrated distinct positive and negative expression regions, indicating heterogeneity (Figs 1A-1C). This supported the high-grade serous carcinoma diagnosis.

FIG 1.

Histopathological and immunohistochemical characterization of the uterine tumor. (A) HE staining showing high-grade serous carcinoma morphology. (B) p53 immunohistochemistry, exhibiting a null pattern. (C) WT1 immunohistochemistry, demonstrating regions of both positive and negative expression, indicating heterogeneity. (D) CD4 immunohistochemistry, highlighting moderate infiltration of CD4⁺ T cells. (E) CD8 immunohistochemistry, showing marked infiltration of CD8⁺ T cells. HE, hematoxylin and eosin.

In the right fallopian tube, a solid tumor (52 × 27 × 15 mm) was observed, composed of invasive, densely proliferative atypical epithelial cells with hyperchromatic nuclei arranged in papillary, tubular, and solid patterns. The fallopian tube epithelial lining exhibited regional atypia contiguous with the invasive carcinoma. The invasive carcinoma was positive for ER, PgR, and WT1 with a null p53 pattern and Ki-67 index >90% (Figs 2A-2C). No evidence of metastasis was found in the left adnexa, omentum, or lymph nodes.

FIG 2.

Histopathological and immunohistochemical characterization of the fallopian tube tumor. (A) HE staining showing invasive carcinoma with papillary, tubular, and solid patterns. (B) p53 immunohistochemistry, exhibiting a null pattern. (C) WT1 immunohistochemistry, demonstrating diffuse positive expression. (D) CD4 immunohistochemistry, highlighting limited infiltration of CD4⁺ T cells. (E) CD8 immunohistochemistry, showing sparse infiltration of CD8⁺ T cells. HE, hematoxylin and eosin.

Histopathological evaluation revealed serous tubal intraepithelial carcinoma (STIC) within the fallopian tube epithelium. The lesion comprised atypical epithelial cells displaying significant nuclear atypia, null p53 pattern, and Ki-67 proliferation index of 60%-70% (Figs 3A-3C).

FIG 3.

Histopathological and immunohistochemical characterization of the STIC. (A) HE staining showing atypical epithelial cells within the fallopian tube epithelium. (B) p53 immunohistochemistry, exhibiting a null pattern. (C) Ki-67 immunohistochemistry, demonstrating a proliferation index of 60%-70%. STIC, serous tubal intraepithelial carcinoma.

Immunohistochemical analysis revealed significant differences in the immune microenvironment between the primary fallopian tube tumor and the metastatic uterine tumors with POLE mutations. The metastatic uterine tumors showed a marked increase in CD8⁺ T-cell infiltration and a moderate increase in CD4⁺ T-cell count than the fallopian tube tumor (Figs 1D-1E, 2D-2E). This is consistent with the well-characterized immunogenic phenotype of POLE-ultramutated cancers, underscoring their biological relevance for immunotherapeutic strategies.

Genomic Analysis

To elucidate the evolutionary relationship between the fallopian tube and uterine tumors, we conducted a comprehensive genomic analysis using gene panel sequencing. Four tumor regions were examined using next-generation sequencing: the STIC, fallopian tube tumor, and WT1-positive and WT1-negative uterine tumor regions. The mutational landscape, illustrated as a heat map (Fig 4), highlighted shared and unique mutations.

FIG 4.

Mutational spectrum of four samples. This heat map presents the mutational profiles, TMB, and MSI status of four regions: STIC, fallopian tube cancer, and uterine tumors (WT1-positive and WT1-negative). MSI, microsatellite instability; TMB, tumor mutational burden.

This genomic analysis identified a spectrum of mutations indicative of a shared clonal origin. Pathogenic variants shared among all regions included BRCA2 p.E49*, p.S1385*, TP53 p.E51*, ENO1 p.S373*, and SETD2 p.E1886*. The fallopian tube tumor and WT1-negative uterine tumor shared FANCD2 p.E634* and PIK3R1 p.S147*. The fallopian tube tumor and WT1-positive uterine tumor shared PBRM1 p.S27* and APC p.S713*.

POLE exonuclease domain mutations (EDMs; p.S297F) were identified in the WT1-positive and WT1-negative regions of uterine tumors, but not in the fallopian tube tumor. Although the fallopian tube tumor exhibited a low tumor mutational burden (8 mutations/mb), the uterine tumors were ultramutated (126 and 185 mutations/mb in the WT1-positive and WT1-negative regions, respectively). High similarity (0.9) was observed in mutation signature 10, supporting the ultramutated phenotype associated with POLE EDMs.

Germline Findings

BRCA2 variants, c.145G>T (p.E49*) and c.4154C>A (p.S1385*), classified as pathogenic in the ClinVar database, were identified in all sites. Germline testing on BRACAnalysis CDx system (Myriad Genetics, Salt Lake City, UT) confirmed the BRCA2 variant NM_000059.4 (BRCA2): c.145G>T (p.E49*), whereas the BRCA2 variant c.4154C>A (p.S1385*) was somatic. This identification confirmed HBOC syndrome diagnosis.

Discussion

Here, we elucidated the role of POLE EDMs in the evolution and metastatic behavior of fallopian tube cancers within the context of HBOC, based on genomic profiling. The discovery of secondary POLE EDMs, particularly p.S297F, which emerged during metastatic progression and was present in the metastatic uterine tumors, provides insight into fallopian tube cancer metastasis. Our hypothetical model of cancer development includes STIC to fallopian tube cancer development and POLE mutation emergence leading to the ultramutated phenotype in the metastatic uterine tumors (Fig 5). S297F is an invariant residue in Pol1, Pold, and T4 polymerases and is a part of the POL1-proofreading active site. Mice with amino acid changes in these polymerases display increased mutagenesis and tumor development,⁶ underscoring the functional importance of p.S297F in cancer progression and ultramutated state.

FIG 5.

Conceptual model of cancer development and progression. The schematic diagram depicts the progression from TP53 mutations in the fallopian tube epithelium, leading to the formation of STIC and subsequent development of fallopian tube cancer. During the metastatic process, the acquisition of POLE mutations generates an ultramutated phenotype, contributing to significant histological and genomic heterogeneity in the uterine tumors.

POLE mutation may drive other passenger mutations causing varied cancer pathologies. This genomic heterogeneity likely resulted in genetic variability in individual tumor cells, contributing to heterogeneous WT1 expression. The metastatic uterine tumors harboring POLE mutations exhibited ultramutated phenotypes and demonstrated higher CD4 and CD8 expression than the primary fallopian tube tumor. This suggests an altered immune microenvironment at the metastatic site, highlighting the potential for immunotherapeutic strategies targeting these ultramutated tumors. Beyond immunotherapy, DNA damage response–targeted agents, such as ataxia telangiectasia and Rad3-related inhibitors, may also hold therapeutic potential in POLE-mutated cancers, although clinical evidence remains limited.

Our findings challenge the traditional understanding that synchronous primary tumors in endometrial and ovarian cancers are separate entities. Massively parallel sequencing, recently used to define the clonal relationship between endometrial and ovarian tumors, often reveals single tumors with metastasis rather than independent primary tumors.^7-10 These studies frequently identify the endometrium as the primary origin. Although our genomic profiling similarly revealed shared clonal mutations between the uterine corpus and fallopian tube tumors, supporting a metastatic relationship, our findings uniquely indicate the fallopian tube was the primary site. Although uterus-to-fallopian tube metastasis is possible, the presence of STIC and the restriction of POLE mutations to the uterine tumors strongly support the fallopian tube as the primary site, with subsequent uterine spread. This underscores the importance of molecular profiling in differentiating between synchronous primary tumors and metastatic disease, particularly in cases with overlapping histology.

Historically, synchronous primary tumors in endometrial and ovarian cancers were classified based on histopathological and morphological criteria, which are inadequate for accurately distinguishing metastatic tumors from dual-primary tumors. Our results highlight the limitations of traditional methods, especially when the histological presentation is atypical or heterogeneous. Both the fallopian tube and uterine tumors in this case exhibited high-grade serous carcinoma histological subtypes, a common phenotype in HBOC. The patient's HBOC 2ground likely predisposed her to high-grade serous carcinoma in the fallopian tube—a recognized primary site in HBOC-related cancers. This genetic predisposition combined with secondary POLE EDM development during metastatic progression represents a rare but significant tumor evolution pattern. Whether secondary POLE mutations during metastatic progression are specific to HBOC remains unclear. To our knowledge, this is the first report describing this phenomenon. Additional clinical studies are required to determine its prevalence in HBOC and non-HBOC settings.

In conclusion, our findings highlight the critical role of POLE mutations in HBOC tumor progression and metastatic behavior. These results provide insight into tumor evolution and suggest potential avenues for personalized therapeutic strategies, including immunotherapy. This case demonstrates the importance of integrating genomic data and clinical context to accurately diagnose and manage synchronous tumors and emphasizes the need for further research to refine diagnostic frameworks and expand their clinical applications in similar cases.

Methods

Ethical Considerations

The patient provided informed consent before study initiation. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Keio University (approval numbers: 20070081, 20231104).

Next-Generation Sequencing–Based Cancer-Gene Panel Test

In January 2022, we launched Rapid-Neo, an in-house clinical sequencing system. All genome sequencing procedures were performed at our hospital as previously described, with some modifications. The next-generation sequencing panel test was used to analyze tumor DNA for genomic alterations in 216 cancer-related genes. It required five undyed 10-μm pathology specimens from formalin-fixed, paraffin-embedded tissues. Owing to the small size, DNA was extracted from the STIC region using laser microdissection. A minimum of 50 ng DNA was extracted. DNA libraries were prepared for genome sequencing if the DNA integrity was >2.0. Targeted capture sequencing of the 216 cancer-related genes was performed using a next-generation sequencer (NextSeq 550; Illumina, San Diego, CA).

Confirmation of Germline Variants

Germline BRCA1/2 testing was performed on blood samples using the BRACAnalysis CDx system (Myriad Genetics, Salt Lake City, UT).

DATA SHARING STATEMENT

The data supporting the findings of this study are not available, as the study participant did not consent to public sharing of the data. The underlying code for this study is not publicly available for proprietary reasons.

AUTHOR CONTRIBUTIONS

Conception and design: Mitsuyo Jisaka, Kohei Nakamura

Financial support: Mitsuyo Jisaka, Kohei Nakamura

Administrative support: Mitsuyo Jisaka

Provision of study materials or patients: Mitsuyo Jisaka, Takashi Iwata, Kenta Masuda

Collection and assembly of data: Mitsuyo Jisaka, Tatsuyuki Chiyoda, Takashi Iwata, Kenta Masuda, Ryutaro Kawano, Sayaka Funata, Reika Takamatsu, Wataru Yamagami

Data analysis and interpretation: Mitsuyo Jisaka, Tatsuyuki Chiyoda, Hiroshi Nishihara

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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