Dawn R. Cochrane

Targeted and Shallow Whole-Genome Sequencing Identifies Therapeutic Opportunities in p53abn Endometrial Cancers

Abstract Purpose: Shallow whole-genome sequencing (sWGS) can detect copy-number (CN) aberrations. In high-grade serous ovarian cancer (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers to identify additional prognostic stratification and therapeutic opportunities. Experimental Design: sWGS and targeted panel sequencing was performed on formalin-fixed, paraffin-embedded p53abn endometrial cancers. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. Results: In 187 p53abn endometrial cancers, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. Twenty-two percent of potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with a high ploidy state, and CCNE1, ERBB2, and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. Twenty-eight percent of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. Thirty-four percent of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on IHC, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations. Conclusions: sWGS and targeted sequencing identified therapeutic opportunities in 75% of patients with p53abn endometrial cancer. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn endometrial cancers.

The unique metabolome of clear cell ovarian carcinoma

AbstractClear cell ovarian carcinoma (CCOC) is an aggressive malignancy affecting younger women. Despite ovarian cancer subtypes having diverse molecular and clinical characteristics, the mainstay of treatment for advanced stage disease remains cytotoxic chemotherapy. Late stage CCOC is resistant to conventional chemotherapy, which means a suboptimal outcome for patients affected. Despite detailed genomic, epigenomic, transcriptomic, and proteomic characterisation, subtype‐specific treatment for CCOC has shown little progress. The unique glycogen accumulation defining CCOC suggests altered metabolic pathway activity and dependency. This study presents the first metabolomic landscape of ovarian cancer subtypes, including 42 CCOC, 20 high‐grade serous and 21 endometrioid ovarian carcinomas, together comprising the three most common ovarian carcinoma subtypes. We describe a distinct metabolomic landscape of CCOC compared with other ovarian cancer subtypes, including alterations in energy utilisation and cysteine metabolism. In addition, we identify CCOC‐specific alterations in metabolic pathways including serine biosynthesis and ROS‐associated pathways that could serve as potential therapeutic targets. Our study provides the first in‐depth study into the metabolome of ovarian cancers and a rich resource to support ongoing research efforts to identify subtype‐specific therapeutic targets that could improve the dismal outcome for patients with this devastating malignancy. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Activated immune infiltrates expand opportunities for targeted therapy in p53‐abnormal endometrial carcinoma

AbstractTumor protein p53 mutated/abnormal (p53abn) endometrial carcinomas account for over 50% of deaths but comprise only 15% of all endometrial carcinomas. Most patients show limited response to standard‐of‐care chemotherapy with or without radiotherapy, and only a minority of cases are amenable to targeted therapies like poly‐ADP ribose polymerase (PARP) inhibitors and HER2‐directed therapies. Recent immunotherapy clinical trials have demonstrated remarkable efficacy, not only in mismatch repair deficient (MMRd) tumors but also in a subset of mismatch repair‐proficient (MMRp) tumors. However, the immune microenvironment and its relationship to other therapeutic targets in MMRp endometrial carcinoma remains poorly understood. Here, we characterize the immune microenvironment of p53abn endometrial carcinoma, the most clinically aggressive subtype of MMRp endometrial carcinoma, and correlate antitumor immune signatures with other targetable alterations. We accrued 256 treatment‐naïve p53abn endometrial carcinomas and systemically profiled T‐cell, B‐cell, myeloid, and tumor‐cell populations with multiplex immunofluorescence to assess the tissue localization and functional status of immune cells. Shallow whole‐genome sequencing was performed on a subset of 126 cases. Patterns of immune infiltration were compared to survival outcomes and mutational signatures. Mixture modeling divided p53abn endometrial carcinoma into tumor‐infiltrating lymphocyte (TIL)‐rich and TIL‐poor subsets. Over 50% of tumors were TIL‐rich. TIL‐rich cases overexpressed targetable immune evasion molecules and were associated with longer overall and disease‐specific survival in multivariate analysis. This effect was particularly pronounced in advanced stage disease and in patients who did not receive adjuvant chemotherapy. TIL did not associate with homologous recombination deficient mutational signatures or HER2 amplification. Our findings demonstrate a biological rationale for immunotherapy in a substantial subset of patients with p53abn endometrial cancer and may help inform combination therapies with immune checkpoint inhibition, PARP inhibitors, and anti‐HER2 agents. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Papillary and ductal patterns of mesonephric‐like adenocarcinomas are often overlooked: a retrospective revaluation of over 1000 endometrial carcinomas

AimsMesonephric‐like adenocarcinoma (MLA) of the endometrium is often a diagnostic challenge, due to its morphological resemblance to other more common Müllerian neoplasms. This study aimed to retrospectively identify overlooked MLA in a large endometrial carcinoma cohort, using a combination of immunohistochemistry (IHC), morphology and KRAS sequencing.Methods and resultsIHC was conducted on 1094 endometrial carcinomas, identifying 16 potential MLA cases based on GATA3+ and/or TTF1+ and ER− staining patterns, which subsequently underwent detailed histological review, KRAS sequencing and ProMisE molecular classification. Of the IHC screen‐positive cases, one was positive for both GATA3 and TTF1, nine were positive for GATA3 only and six were positive for TTF1 only. All IHC screen‐positive cases were POLE wild‐type. All five tumours in the NSMP category showed morphological features of MLA, while the three MMRd and eight p53abn tumours did not show MLA morphology. The five cases diagnosed as MLA on review were all originally diagnosed as low‐grade endometrioid adenocarcinoma probably because of rare morphological patterns, being predominantly papillary or ductal. Four of the five cases harboured a KRAS mutation.ConclusionThis study highlights the importance of a comprehensive diagnostic approach for accurately identifying endometrial MLA and for pathologists to be aware of papillary and ductal patterns in endometrial carcinoma assessment. Further exploration into the molecular landscape of MLA is essential for refining diagnostic criteria and developing targeted therapies.

Single cell transcriptomes of normal endometrial derived organoids uncover novel cell type markers and cryptic differentiation of primary tumours

AbstractEndometrial carcinoma, the most common gynaecological cancer, develops from endometrial epithelium which is composed of secretory and ciliated cells. Pathologic classification is unreliable and there is a need for prognostic tools. We used single cell sequencing to study organoid model systems derived from normal endometrial endometrium to discover novel markers specific for endometrial ciliated or secretory cells. A marker of secretory cells (MPST) and several markers of ciliated cells (FAM92B, WDR16, and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. We performed single cell sequencing on endometrial and ovarian tumours and found both secretory‐like and ciliated‐like tumour cells. We found that ciliated cell markers (DYDC2, CTH, FOXJ1, and p73) and the secretory cell marker MPST were expressed in endometrial tumours and positively correlated with disease‐specific and overall survival of endometrial cancer patients. These findings suggest that expression of differentiation markers in tumours correlates with less aggressive disease, as would be expected for tumours that retain differentiation capacity, albeit cryptic in the case of ciliated cells. These markers could be used to improve the risk stratification of endometrial cancer patients, thereby improving their management. We further assessed whether consideration of MPST expression could refine the ProMiSE molecular classification system for endometrial tumours. We found that higher expression levels of MPST could be used to refine stratification of three of the four ProMiSE molecular subgroups, and that any level of MPST expression was able to significantly refine risk stratification of the copy number high subgroup which has the worst prognosis. Taken together, this shows that single cell sequencing of putative cells of origin has the potential to uncover novel biomarkers that could be used to guide management of cancers. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Changes in the tumour microenvironment mark the transition from serous borderline tumour to low‐grade serous carcinoma

AbstractLow‐grade serous ovarian carcinoma (LGSC) is a rare and lethal subtype of ovarian cancer. LGSC is pathologically, biologically, and clinically distinct from the more common high‐grade serous ovarian carcinoma (HGSC). LGSC arises from serous borderline ovarian tumours (SBTs). The mechanism of transformation for SBTs to LGSC remains poorly understood. To better understand the biology of LGSC, we performed whole proteome profiling of formalin‐fixed, paraffin‐embedded tissue blocks of LGSC (n = 11), HGSC (n = 19), and SBTs (n = 26). We identified that the whole proteome is able to distinguish between histotypes of the ovarian epithelial tumours. Proteins associated with the tumour microenvironment were differentially expressed between LGSC and SBTs. Fibroblast activation protein (FAP), a protein expressed in cancer‐associated fibroblasts, is the most differentially abundant protein in LGSC compared with SBT. Multiplex immunohistochemistry (IHC) for immune markers (CD20, CD79a, CD3, CD8, and CD68) was performed to determine the presence of B cells, T cells, and macrophages. The LGSC FAP+ stroma was associated with greater abundance of Tregs and M2 macrophages, features not present in SBTs. Our proteomics cohort reveals that there are changes in the tumour microenvironment in LGSC compared with its putative precursor lesion, SBT. These changes suggest that the tumour microenvironment provides a supportive environment for LGSC tumourigenesis and progression. Thus, targeting the tumour microenvironment of LGSC may be a viable therapeutic strategy. © 2024 The Pathological Society of Great Britain and Ireland.

Adult‐type granulosa cell tumor of the ovary: a FOXL2‐centric disease

AbstractAdult‐type granulosa cell tumors (aGCTs) account for 90% of malignant ovarian sex cord‐stromal tumors and 2–5% of all ovarian cancers. These tumors are usually diagnosed at an early stage and are treated with surgery. However, one‐third of patients relapse between 4 and 8 years after initial diagnosis, and there are currently no effective treatments other than surgery for these relapsed patients. As the majority of aGCTs (>95%) harbor a somatic mutation in FOXL2 (c.C402G; p.C134W), the aim of this study was to identify genetic mutations besides FOXL2 C402G in aGCTs that could explain the clinical diversity of this disease. Whole‐genome sequencing of 10 aGCTs and their matched normal blood was performed to identify somatic mutations. From this analysis, a custom amplicon‐based panel was designed to sequence 39 genes of interest in a validation cohort of 83 aGCTs collected internationally. KMT2D inactivating mutations were present in 10 of 93 aGCTs (10.8%), and the frequency of these mutations was similar between primary and recurrent aGCTs. Inactivating mutations, including a splice site mutation in candidate tumor suppressor WNK2 and nonsense mutations in PIK3R1 and NLRC5, were identified at a low frequency in our cohort. Missense mutations were identified in cell cycle‐related genes TP53, CDKN2D, and CDK1. From these data, we conclude that aGCTs are comparatively a homogeneous group of tumors that arise from a limited set of genetic events and are characterized by the FOXL2 C402G mutation. Secondary mutations occur in a subset of patients but do not explain the diverse clinical behavior of this disease. As the FOXL2 C402G mutation remains the main driver of this disease, progress in the development of therapeutics for aGCT would likely come from understanding the functional consequences of the FOXL2 C402G mutation.

FOXL2 in adult‐type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

AbstractA recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult‐type granulosa cell tumours (AGCTs). In contrast, various loss‐of‐function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain‐ or change‐of‐function mutation with altered DNA‐binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA‐seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

STING pathway expression in low‐grade serous carcinoma of the ovary: an unexpected therapeutic opportunity?

AbstractOvarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype‐specific treatment modalities, especially for women with widespread and chemo‐resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS–STING pathway that mediates innate immune defence against infectious DNA‐containing pathogens and also detects tumour‐derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low‐grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high‐grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low‐grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

The proteome of clear cell ovarian carcinoma

AbstractClear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late‐stage CCOC is not responsive to gold‐standard chemotherapy and results in suboptimal outcomes for patients. In‐depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.

Proteomic analysis uncovers biological diversity in molecularly defined endometrial carcinomas

While endometrial cancer has an overall favorable prognosis, some patients have poor outcomes and may benefit from further refinements of the current classification systems. Molecular classification stratifies endometrial cancer patients into four prognostic subtypes: POLEmut, MMRd (mismatch repair deficient), p53abn, and NSMP (no specific molecular profile), where patients with POLEmut have the best prognosis and p53abn has the worst prognosis. We used proteomic profiling to assess if additional prognostic or predictive information could be identified across or within molecular subtypes. Global proteome profiling of formalin fixed, paraffin embedded samples, that had clinicopathologic and outcome data, was performed on 184 endometrial cancers encompassing all four molecular subtypes, including replicate samples of the same tumor, and both biopsy and final hysterectomy specimens. To ensure representation of each subtype, we profiled an approximately equal distribution in the 148 unique tumors; 34 (23%) POLEmut, 40 (27%) MMRd, 35 (24%) p53abn and 39 (26%) NSMP, rather than the population-based distributions. There was high reproducibility in the proteomic profiles of intra-tumor replicate samples, and between matched biopsy and hysterectomy tumor samples. Consensus clustering identified four clusters with different prognosis, named 'Adhesion', 'Immune', 'Proliferation', and 'Metabolic' based on the functional characteristics of the enriched proteins. We associated protein expression features with common mutations, molecular subtype, and outcomes. These results demonstrate the biologic diversity within endometrial cancers, both between and within molecular subtypes, and provide candidate features for functional and clinical investigation.

Oral contraceptive use is associated with a reduction in the physical size of fallopian tube p53 signatures

Oral contraceptives reduce ovarian cancer risk, but the mechanism of risk reduction is not understood. We examined whether oral contraceptive pill (OCP) use influences p53 signatures, which are putative early fallopian tube precursors for high-grade serous ovarian carcinomas. For this retrospective cohort (n = 250) of subjects aged over 50 years who had fallopian tubes removed at the time of a benign gynecologic procedure, we used health records to identify 72 patients who used OCPs for at least 5 years and 178 subjects with no history of OCP use. Immunohistochemistry for p53 was performed on all fallopian tube sections, with 8 individuals removed for lack of identifiable tissue. p53 Signatures were identified and stratified based on size. Logistic regressions were run to estimate the association between OCP use and p53 lesion and lesion size. There was no difference in the occurrence of p53 lesions with 20 of 70 of OCP users (28.6%) and 57 of 172 of those with no history of OCP use (33.1%). Subjects who used OCPs were more likely to have a small lesion (OR 1.98, 95% CI 1.03 to 3.83) and had decreased risk of having a medium/large lesion (OR 0.38, 95% CI 0.18 to 0.79). A total of 2 serous tubal intraepithelial lesions and 2 serous tubal intraepithelial carcinomas were identified in OCP-naive patients, whereas none were found in those with a history of OCP use. OCP exposure was associated with a shift toward smaller p53 lesion size but was not found to be associated with a difference in the number of p53 lesions between OCP-exposed and unexposed patients. Future research should examine whether OCP use reduces proliferation and clonal expansion of p53 signature lesions toward higher risk precursors and, eventually, cancer. There were no serous tubal intraepithelial lesions and serous tubal intraepithelial carcinomas in patients with OCP exposure.