C Blake Gilks

Prognostic values of molecular subtypes and SWI/SNF protein expression in de‐differentiated/undifferentiated endometrial carcinoma

AimsClassification and risk stratification of endometrial carcinoma (EC) has transitioned from histopathological features to molecular classification, e.g. the ProMisE classifier, identifying four prognostic subtypes: POLE mutant (POLEmut) with almost no recurrence or disease‐specific death events, mismatch repair deficient (MMRd) and no specific molecular profile (NSMP), with intermediate outcome and p53 abnormal (p53abn) with poor outcomes. However, the applicability of molecular classification is unclear in rare but aggressive histotypes of EC, e.g. de‐differentiated and undifferentiated endometrial cancers (DD/UDEC). Here, we aim to assembled a cohort of DD/UDEC from a single institution and analysed the prognostic significance of ProMisE molecular subtypes and the expression of SWItch/sucrose non‐fermentable (SWI/SNF) chromatin remodelling complex members, previously implicated in the pathogenesis of DD/UDEC.Methods and resultsWe accrued 88 DD/UDEC cases, assessed POLE status by Sanger sequencing and performed immunohistochemistry for p53, mismatch repair and SWI/SNF proteins on the tissue microarrays assembled. Assignment of molecular subtypes was possible in 80 tumours; POLE sequencing failed in the remaining eight cases. There were 12 (15%) POLEmut, 44 (55%) MMRd, 14 (17.5%) p53abn and 10 (12.5%) NSMP DD/UDEC. POLEmut DD/UDECs had excellent outcomes, but the other three molecular subtypes all had poor outcomes, with no significant differences among them. The loss of one or more SWI/SNF proteins [AT‐rich interactive domain‐containing protein 1A (ARID1A), ARID1B, SWI/SNF‐related, matrix‐associated, actin‐dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), SMARCA2], observed in 66% (55 of 83) cases, was not of prognostic significance.ConclusionsThese results indicate that all molecular subtypes of DD/UDEC except POLEmut behave in an aggressive fashion. Further study is needed to determine whether these molecular alterations can be targeted with adjuvant therapy, in order to improve outcomes of patients with DD/UDEC.

Assessing the impact of deep‐learning assistance on the histopathological diagnosis of serous tubal intraepithelial carcinoma (STIC) in fallopian tubes

AbstractIn recent years, it has become clear that artificial intelligence (AI) models can achieve high accuracy in specific pathology‐related tasks. An example is our deep‐learning model, designed to automatically detect serous tubal intraepithelial carcinoma (STIC), the precursor lesion to high‐grade serous ovarian carcinoma, found in the fallopian tube. However, the standalone performance of a model is insufficient to determine its value in the diagnostic setting. To evaluate the impact of the use of this model on pathologists' performance, we set up a fully crossed multireader, multicase study, in which 26 participants, from 11 countries, reviewed 100 digitalized H&E‐stained slides of fallopian tubes (30 cases/70 controls) with and without AI assistance, with a washout period between the sessions. We evaluated the effect of the deep‐learning model on accuracy, slide review time and (subjectively perceived) diagnostic certainty, using mixed‐models analysis. With AI assistance, we found a significant increase in accuracy (p < 0.01) whereby the average sensitivity increased from 82% to 93%. Further, there was a significant 44 s (32%) reduction in slide review time (p < 0.01). The level of certainty that the participants felt versus their own assessment also significantly increased, by 0.24 on a 10‐point scale (p < 0.01). In conclusion, we found that, in a diverse group of pathologists and pathology residents, AI support resulted in a significant improvement in the accuracy of STIC diagnosis and was coupled with a substantial reduction in slide review time. This model has the potential to provide meaningful support to pathologists in the diagnosis of STIC, ultimately streamlining and optimizing the overall diagnostic process.

Recurrence rates and patterns of recurrence in stage IA p53abn endometrial cancer with and without myometrial invasion

Optimal management of patients with stage IA p53abn endometrial cancer without myoinvasion, classified as intermediate risk in the 2020 European Society of Gynaecological Oncology, European Society for Radiotherapy and Oncology, and European Society of Pathology (ESGO-ESTRO-ESP) guidelines, and the 2022 European Society of Medical Oncology (ESMO) guidelines, is currently unclear. Practice varies from surgery alone to adjuvant radiation±chemotherapy. Our aim was to assess the risk of disease recurrence in patients with stage IA p53abn endometrial cancer without myoinvasion compared with stage IA with myoinvasion (<50%). Stage IA p53abn endometrial cancers were identified from retrospective cohorts. Cases were segregated into stage IA with no myoinvasion, including (1) tumor restricted to a polyp, (2) residual endometrial tumor, and (3) no residual tumor in hysterectomy specimen, versus stage IA p53abn with myoinvasion (<50%), with treatment and outcomes assessed. There were 65 stage IA p53abn endometrial cancers with no myoinvasion (22 polyp confined, 38 residual endometrial tumor, 2 no residual in hysterectomy specimen, 3 not specified) and 97 with myoinvasion. There was no difference in survival outcomes in patients with stage IA without myoinvasion (16% of patients recurred, 19% if there was residual endometrial disease) compared with stage IA with myoinvasion (17%). The risk of recurrence was lowest in patients with stage IA p53abn endometrial cancer without myoinvasion treated with chemotherapy±radiation (8%). Most recurrences in patients with stage IA without myoinvasion were distant (89%), with no isolated vaginal vault recurrences, and all except one distant recurrence occurred in patients who had not received adjuvant chemotherapy. The recurrence rate in patients with stage IA p53abn endometrial cancer without myoinvasion was 16%, highest in the setting of residual endometrial disease (19%), and exceeding the threshold where adjuvant therapy is often considered. The high frequency of distant recurrences observed may support chemotherapy as part of the treatment regimen.

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.

FIGO 2023 endometrial cancer staging: too much, too soon?

An updated International Federation of Gynecology and Obstetrics (FIGO) staging system for endometrial carcinoma was introduced in June 2023. The new system represents a significant departure from traditional endometrial and other gynecological carcinoma staging systems which are agnostic of parameters such as tumor type, tumor grade, lymphovascular space invasion, and molecular alterations. The updated system, which incorporates all of these 'non-anatomical' parameters, is an attempt to make staging more personalized and relevant to patient prognostication and management, and to align with the European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) risk stratification. Herein, we present a critical review of the new staging system and discuss its advantages and disadvantages. The authors propose that the new FIGO staging system should be first appraised at a multi-institutional and global level with the input of all relevant societies (gynecology, pathology, gynecologic oncology, medical oncology, radiation oncology) to understand the impact, scope, and supporting evidence of the proposed changes. Such a process is fundamental to produce a robust system that pathologists and treating clinicians can adopt.

Mesonephric‐like adenocarcinoma harbours characteristic copy number variations and a distinct DNA methylation signature closely related to mesonephric adenocarcinoma of the cervix

Abstract Mesonephric‐like adenocarcinoma (MLA) of the female genital tract is an uncommon histotype that can arise in both the endometrium and the ovary. The exact cell of origin and histogenesis currently remain unknown. Here, we investigated whole genome DNA methylation patterns and copy number variations (CNVs) in a series of MLAs in the context of a large cohort of various gynaecological carcinoma types. CNV analysis of 19 MLAs uncovered gains of chromosomes 1q (18/19, 95%), 10 (15/19, 79%), 12 (14/19, 74%), and 2 (10/19, 53%), as well as loss of chromosome 1p (7/19, 37%). Gains of chromosomes 1q, 10, and 12 were also identified in the majority of mesonephric adenocarcinomas of the uterine cervix (MAs) as well as subsets of endometrioid carcinomas (ECs) and low‐grade serous carcinomas of the ovary (LGSCs) but only in a minority of serous carcinomas of the uterine corpus (USCs), clear cell carcinomas (CCCs), and tubo‐ovarian high‐grade serous carcinomas (HGSCs). While losses of chromosome 1p together with gains of chromosome 1q were also identified in both MA and LGSC, gains of chromosome 2 were almost exclusively identified in MLA and MA. Unsupervised hierarchical clustering and t‐SNE analysis of DNA methylation data (Illumina EPIC array) identified a co‐clustering for MLAs and MAs, which was distinct from clusters of ECs, USCs, CCCs, LGSCs, and HGSCs. Group‐wise comparisons confirmed a close epigenetic relationship between MLA and MA. These findings, in conjunction with the established histological and immunophenotypical overlap, suggest bona fide mesonephric differentiation, and support a more precise terminology of mesonephric‐type adenocarcinoma instead of MLA in these tumours. © 2023 The Authors. The Journal of Pathology published by John Wiley &amp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clinical Behavior and Molecular Landscape of Stage I p53-Abnormal Low-Grade Endometrioid Endometrial Carcinomas

Abstract Purpose: The clinical significance of the p53-abnormal (p53abn) molecular subtype in stage I low-grade endometrioid endometrial carcinoma (EEC) is debated. We aimed to review pathologic and molecular characteristics, and outcomes of stage I low-grade p53abn EEC in a large international cohort. Experimental Design: Previously diagnosed stage I p53abn EC (POLE–wild-type, mismatch repair–proficient) low-grade EEC from Canadian retrospective cohorts and PORTEC-1&amp;2 trials were included. Pathology review was performed by six expert gynecologic pathologists blinded to p53 status. IHC profiling, next-generation sequencing, and shallow whole-genome sequencing was performed. Kaplan–Meier method was used for survival analysis. Results: We identified 55 stage I p53abn low-grade EEC among 3,387 cases (2.5%). On pathology review, 17 cases (31%) were not diagnosed as low-grade EEC by any pathologists, whereas 26 cases (47%) were diagnosed as low-grade EEC by at least three pathologists. The IHC and molecular profile of the latter cases were consistent with low-grade EEC morphology (ER/PR positivity, patchy p16 expression, PIK3CA and PTEN mutations) but they also showed features of p53abn EC (TP53 mutations, many copy-number alterations). These cases had a clinically relevant risk of disease recurrence (5-year recurrence-free survival 77%), with pelvic and/or distant recurrences observed in 12% of the patients. Conclusions: A subset of p53abn EC is morphologically low-grade EEC and exhibit genomic instability. Even for stage I disease, p53abn low-grade EEC are at substantial risk of disease recurrence. These findings highlight the clinical relevance of universal p53-testing, even in low-grade EEC, to identify women at increased risk of recurrence.

Co-existent endometrial and ovarian carcinoma: molecular and pathological features define low risk entity

Most co-existent endometrial and ovarian carcinomas are clonally related and exhibit an indolent disease course. Pathologic assignment and clinical management of this entity vary greatly. The International Federation of Gynecology and Obstetrics (FIGO) 2023 endometrial carcinoma staging/risk stratification system introduced a new substage for co-existent endometrial and ovarian carcinomas that meet strict pathologic criteria (stage IA3, distinct from IIIA1). Our aim was to validate if FIGO IA3 identifies a subset of co-existent endometrial and ovarian carcinomas at very low risk of recurrence and determine whether further refinement, through molecular features and expanded ovarian pathologic criteria, could improve prognostic discernment and direct more patients for consideration of de-escalation. Clinicopathologic, molecular, and outcome data were collected on patients with co-existent endometrial and ovarian carcinoma, extracted from pathology archives and molecularly classified endometrial carcinoma cohorts. Among the 154 co-existent endometrial and ovarian carcinoma patients, higher recurrence rates were observed with the p53abn (2/6, 33%), mismatch repair deficiency (MMRd) (7/34, 21%) or no specific molecular profile (NSMP) estrogen receptor (ER) negative-low (2/15, 13%) molecular sub-types, compared with patients with POLEmut or NSMP ER strong positive tumors. Thirty-two patients met FIGO IA3 criteria, with one recurrence and death event (MMRd). Eliminating patients with adverse molecular features (p53abn or MMRd endometrium or ovary, or NSMP ER negative-low endometrium) and expanding criteria to include any POLEmut or cases with bilateral ovarian involvement, intra- or pre-operative ovarian rupture, or ovarian surface involvement significantly improved risk stratification (p = .008) and added 48 co-existent endometrial and ovarian carcinoma patients (>2-fold increase) with no recurrence events (mean follow-up: 6 years). There was 91% concordance of molecular sub-type assignment between endometrial and ovarian tumors. FIGO IA3 criteria identify a subset of co-existent endometrial and ovarian carcinomas with excellent outcomes. However, incorporating molecular features into the definition enables greater prognostic discernment and supports the inclusion of patients with a broader range of pathologic features with indolent disease (increased from 20% to 49% of the cohort, 0 recurrences) who may be candidates for treatment de-escalation.

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 &amp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Subclonal p53 immunostaining in the diagnosis of endometrial carcinoma molecular subtype

AimsThe significance of subclonal expression of p53 (abrupt transition from wild‐type to mutant‐pattern staining) is not well understood, and the arbitrary diagnostic cut‐off of 10% between NSMP and p53abn molecular subtypes of endometrial carcinoma (EC) has not been critically assessed. Our aim was to characterise subclonal p53 and discrepant p53 expression/TP53 sequencing results in EC and assess their clinical significance.Methods and resultsSubclonal p53 immuostaining on whole sections from 957 ECs was recorded. Agreement between TP53 mutational assessment and p53 immunostaining was evaluated. Subclonal p53 IHC staining was seen in 4.0% (38 of 957) of cases, with 23 of 957 (2.4%) showing mutant‐pattern p53 staining in ≥10% of tumour cells. It was most commonly seen in POLEmut (nine of 65, 14%) and MMRd (13 of 274, 4.7%) EC (‘multiple classifier’ ECs), where subclonal p53 staining does not impact the molecular subtype diagnosis. Excluding POLEmut and MMRd EC, 11 of 957 (1.1%) showed ≥10% subclonal p53 from which four patients died of disease, while there were no deaths due to disease in the five patients with &lt;10% mutant‐pattern p53 staining. Agreement between p53 immunostaining and TP53 sequencing was 92.6%; most of the discrepant results were in the ultramutated POLEmut or hypermutated MMRd ECs. In NSMP and p53abn EC the agreement between IHC and sequencing was 95.8%.ConclusionsSubclonal p53 staining ≥10% is present in only 1.1% of EC after excluding ‘multiple classifier’ ECs. The cut‐off of ≥10% subclonal p53 staining identified patients at increased risk of dying from EC, supporting its use to diagnose p53abn molecular subtype.

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.

Practical guidance for assessing and reporting lymphovascular space invasion (LVSI) in endometrial carcinoma

Lymphovascular space invasion (LVSI) is an important prognostic parameter in endometrial carcinoma (EC) and has gained increasing interest in recent years due to an expanding body of evidence of its independent prognostic value, especially when the presence of LVSI is quantified. A key strength of LVSI as a prognostic factor is that it can be detected on routine microscopic examination, without ancillary tests, and thus can be used in low‐resource settings. A weakness, however, is the lack of uniformly applied criteria for assessment and quantification of LVSI, resulting in interobserver variation in diagnosis. This is confounded by artefacts and other morphological features that may mimic LVSI (commonly referred to as pseudo‐LVSI). Despite these issues, multiple studies have shown that LVSI is strongly associated with lymph node (LN) metastasis and is an independent risk factor for LN recurrence and distant metastasis. Consequently, the presence of substantial/extensive LVSI has become an important consideration in formulating adjuvant treatment recommendations in patients with EC, and this has been incorporated in the recent International Federation of Gynecology and Obstetrics (FIGO) 2023 staging system. Herein, we review the current literature on LVSI in EC and discuss its role as a prognostic marker, the reproducibility of LVSI assessment and distinction between LVSI and its mimics. We provide illustrations of key diagnostic features and discuss the two‐tiered (none/focal versus substantial) system of LVSI classification. This work is intended to provide guidance to practising pathologists and unify the approach towards LVSI assessment in EC.

Validation and clinical performance of a single test, DNA based endometrial cancer molecular classifier

We have previously shown that DNA based, single test molecular classification by next generation sequencing (NGS) (Proactive Molecular risk classifier for Endometrial cancer (ProMisE) NGS) is highly concordant with the original ProMisE classifier and maintains prognostic value in endometrial cancer. Our aim was to validate ProMisE NGS in an independent cohort and assess the performance of ProMisE NGS in real world clinical practice to address if there were any practical challenges or learning points for implementation. We evaluated DNA extracted from an external research cohort of 211 endometrial cancer cases diagnosed in 2016 from Germany, Switzerland, and Austria, across seven European centers, comparing standard molecular classification (NGS for A total of 545 endometrial cancers were compared. Prognostic differences in progression free, disease specific, and overall survival between the four molecular subtypes were observed for the NGS classifier, recapitulating the survival curves of original ProMisE. In 28 of 545 (5%) discordant cases (8/211 (4%) in the validation set, 20/334 (6%) in the real world cohort), molecular subtype was able to be definitively assigned in all, based on review of the histopathological features and/or additional immunohistochemistry. DNA based molecular classification identified twice as many 'multiple classifier' endometrial cancers; 37 of 545 (7%) compared with 20 of 545 (4%) with original ProMisE. External validation confirmed that single test, DNA based molecular classification was highly concordant (95%) with original ProMisE classification, with prognostic value maintained, representing an acceptable alternative for clinical practice. Careful consideration of reasons for discordance and knowledge of how to correctly assign multiple classifier endometrial cancers is imperative for implementation.

The spectrum of oestrogen receptor expression in endometrial carcinomas of no specific molecular profile

AimsDecreased oestrogen receptor (ER) expression is a marker of poor prognosis in endometrial carcinomas (EC) of no specific molecular profile (NSMP), but the optimal cut‐off to separate high‐risk ‘low ER’ versus low‐risk ‘high ER’ expression has not been defined. Here we characterised the distribution of ER staining in a cohort of ECs.Methods and resultsBiopsy specimens from 120 cases of NSMP EC were stained for ER and assigned an Allred score. In 66 additional cases ER staining of matched biopsy and hysterectomy were compared. Twelve of 120 tumours had an Allred score of 0–3, including three endometrioid carcinomas (EEA) (one G1, two G3), four clear cell carcinomas (CCC), two mesonephric‐like adenocarcinoma (MLA) and one each of: gastric‐type adenocarcinoma, carcinosarcoma and endometrial carcinoma NOS. Three had Allred scores of 4–5: two MLA and one high‐grade carcinoma with yolk sac differentiation. Five had Allred scores of 6: four EEA (one G1, one G2, two G3) and one mixed clear cell and endometrioid carcinoma. The remaining 100 tumours with Allred scores ≥ 7 were all EEA (66 G1, 28 G2, five G3 and one grade unknown). Comparing the biopsy versus hysterectomy ER staining (n = 66), the results were within a single Allred score point, except two cases with strong diffuse expression in the biopsy (Allred 8) and moderate expression in the hysterectomy (Allred 5).ConclusionsMost NSMP ECs (&gt; 80%) show high ER expression (Allred score ≥ 7). All non‐endometrioid carcinomas and a few endometrioid carcinomas had lower ER expression (Allred score ≤ 6) or were completely negative.

p53 and ovarian carcinoma survival: an Ovarian Tumor Tissue Analysis consortium study

AbstractOur objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high‐grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi‐institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36–3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11–2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

Endometrial carcinoma: molecular subtypes, precursors and the role of pathology in early diagnosis

AbstractEndometrial carcinoma (EC) is classified into a wide range of morphological variants; this list has expanded over the past decade with the inclusion of mesonephric‐like and dedifferentiated carcinoma as EC variants in the fifth edition of the WHO Classification of Female Genital Tumours, and recognition that carcinosarcoma is a biphasic carcinoma rather than a sarcoma. Each EC variant has distinct molecular abnormalities, including TCGA‐based molecular subtypes, allowing further subclassification and adding complexity. In contrast to this rapid progress in understanding EC, there are only two recognized EC precursor lesions: endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) and serous intraepithelial carcinoma, a situation that has not changed for many years. Diagnosis of EC precursors is a cornerstone of surgical pathology practice, with early diagnosis contributing to the relatively favorable prognosis of EC. In this review we relate the precursor lesions to each of the EC morphological variants and molecular subtypes, discuss how successful early diagnosis is for each variant/molecular subtype and how it might be improved, and identify knowledge gaps where there is insufficient understanding of EC histogenesis. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

Performance of a HER2 testing algorithm specific for p53‐abnormal endometrial cancer

AimsHuman epidermal growth factor receptor 2 (HER2) amplification in endometrial cancer (EC) is almost completely confined to the p53‐abnormal (p53abn) molecular subtype and independent of histological subtype. HER2 testing should therefore be molecular subtype‐directed. However, the most optimal approach for HER2 testing in EC has not been fully established. Therefore, we developed an EC‐specific HER2 immunohistochemistry (IHC) scoring method and evaluated its reproducibility and performance to establish an optimal diagnostic HER2 testing algorithm for p53abn EC.Methods and resultsHER2 IHC slides of 78 p53abn EC were scored by six gynaecopathologists according to predefined EC‐specific IHC scoring criteria. Interobserver agreement was calculated using Fleiss’ kappa and the first‐order agreement coefficient (AC1). The consensus IHC score was compared with HER2 dual in‐situ hybridisation (DISH) results. Sensitivity and specificity were calculated. A substantial interobserver agreement was found using three‐ or two‐tiered scoring [κ = 0.675, 95% confidence interval (CI) = 0.633–0.717; AC1 = 0.723, 95% CI = 0.643–0.804 and κ = 0.771, 95% CI = 0.714–0.828; AC1 = 0.774, 95% CI = 0.684–0.865, respectively]. Sensitivity and specificity for the identification of HER2‐positive EC was 100 and 97%, respectively, using a HER2 testing algorithm that recommends DISH in all cases with moderate membranous staining in &gt;10% of the tumour (IHC+). Performing DISH on all IHC‐2+ and ‐3+ cases yields a sensitivity and specificity of 100%.ConclusionsOur EC‐specific HER2 IHC scoring method is reproducible. A screening strategy based on IHC scoring on all cases with subsequent DISH testing on IHC‐2+/‐3+ cases has perfect test accuracy for identifying HER2‐positive EC.

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 (&gt;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.

Validated biomarker assays confirm that ARID1A loss is confounded with MMR deficiency, CD8 + TIL infiltration, and provides no independent prognostic value in endometriosis‐associated ovarian carcinomas

Abstract ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis‐associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8 + tumour‐infiltrating lymphocytes (CD8 + TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8 + TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients ( p  = 0.012) and was associated with MMRd ( p  &lt; 0.001) and the presence of CD8 + TILs ( p  = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss‐of‐function mutation as a result of small indels ( p  = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8 + TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8 + TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss‐of‐function mutations in endometriosis‐associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

Molecular subclassification of vulvar squamous cell carcinoma: reproducibility and prognostic significance of a novel surgical technique

Vulvar squamous cell carcinoma is subclassified into three prognostically relevant groups: (i) human papillomavirus (HPV) associated, (ii) HPV independent p53 abnormal (mutant pattern), and (iii) HPV independent p53 wild type. Immunohistochemistry for p16 and p53 serve as surrogates for HPV viral integration and In this retrospective cohort study, 68 cases treated by vulvar field resection were identified from the Leipzig School of Radical Pelvic Surgery. Immunohistochemistry for p16 and p53 was performed at three different institutions and evaluated independently by seven pathologists and two trainees. Tumors were classified into one of four groups: HPV associated, HPV independent p53 wild type, HPV independent p53 abnormal, and indeterminate. Selected cases were further interrogated by (HPV RNA in situ hybridization, Final subclassification yielded 22 (32.4%) HPV associated, 41 (60.3%) HPV independent p53 abnormal, and 5 (7.3%) HPV independent p53 wild type tumors. Interobserver agreement (overall Fleiss' kappa statistic) for the four category classification was 0.74. No statistically significant differences in clinical outcomes between HPV associated and HPV independent vulvar squamous cell carcinoma were observed. Interobserver reproducibility of vulvar squamous cell carcinoma subclassification based on p16 and p53 immunohistochemistry may support routine use in clinical practice. Vulvar field resection surgery showed no significant difference in clinical outcomes when stratified based on HPV status.

Comparison of p53 immunohistochemical staining in differentiated vulvar intraepithelial neoplasia (dVIN) with that in inflammatory dermatoses and benign squamous lesions in the vulva

AimsDifferentiated vulvar intraepithelial neoplasia (dVIN), the precursor lesion to human papillomavirus‐independent vulvar squamous cell carcinoma (VSCC), can be difficult to distinguish from vulvar inflammatory dermatoses. Our goal was to determine if p53 could be a useful biomarker for dVIN, by characterizing p53 percentage, intensity and patterns of staining in dVIN and its histological mimics.Methods and resultsWe studied p53 immunohistochemical staining patterns in 16 dVIN cases and 46 vulvar non‐neoplastic squamous lesions [12 lichen sclerosus (LS); seven lichen simplex chronicus; three lichen planus (LP); six psoriasis; 13 spongiotic dermatitis (SPO); and five candidiasis]. dVIN cases were adjacent to a p16‐negative invasive VSCC in resection specimens. All dVIN cases showed null‐type or moderate to strong uniform p53 staining in &gt;70% of basal cells, with moderate to strong continuous parabasal staining extending to two‐thirds of the epidermis. This was in contrast to weak or weak to moderate patchy p53 staining in the majority of other lesions. Moderate to strong and increased basal p53 staining (≥70%) was also observed in a subset of LS cases (5/12, 42%), LP cases (1/3, 33%), and SPO cases (36%, 4/11); however, in all categories, this was limited to the basal layer, and any staining in the parabasal layers was patchy.ConclusionStrong and uniform p53 staining of basal cells, extending into the parabasal layers, and a complete absence of staining (null type) is useful in distinguishing dVIN from other mimics in the vulva. p53 staining of lesser intensity or quantity, particularly basal overexpression only, overlaps with that in vulvar inflammatory lesions.

Performance of the pattern‐based interpretation of p53 immunohistochemistry as a surrogate for TP53 mutations in vulvar squamous cell carcinoma

AimsThe most commonly mutated gene in vulvar squamous cell carcinoma (VSCC) is TP53 and its prognostic value, particularly in HPV‐independent VSCC, is uncertain. In other tumours, p53 immunohistochemistry (IHC) is an excellent surrogate marker for TP53 mutations. In order to study this in VSCC, we assigned six p53 IHC patterns into two final classes: ‘wild‐type’ or ‘mutant’. We determined the performance and interobserver variability of this pattern‐based p53 IHC approach.Methods and resultsTwo experienced gynaecological pathologists scored the predefined p53 IHC patterns of 59 VSCC, independently and blinded for molecular data. Agreement was calculated by Cohen's kappa. All disagreements regarding p53 IHC patterns were resolved by a consensus meeting. After DNA isolation, the presence of pathogenic TP53 variants was determined by next‐generation sequencing (NGS). Sensitivity, specificity and accuracy of p53 IHC as a surrogate marker for TP53 mutation status were calculated. Initial p53 IHC pattern interpretation showed substantial agreement between both observers (k = 0.71, P &lt; 0.001). After consensus, 18 cases (30.5%) were assigned a final p53 IHC class as TP53 wild‐type and 41 cases (69.5%) as mutant. The accuracy between the p53 IHC class and TP53 mutation status, after the consensus meeting, was 96.6%. Moreover, the sensitivity and specificity were high 95.3% [95% confidence interval (CI) = 82.9–99.1% and 100% (95% CI = 75.9–100%)].ConclusionsPattern‐based p53 IHC classification is highly reproducible among experienced gynaecological pathologists and accurately reflects TP53 mutations in VSCC. This approach to p53 IHC interpretation offers guidance and provides necessary clarity for resolving the proposed prognostic relevance of final p53 IHC class within HPV‐independent VSCC.

Vulval squamous cell carcinoma and its precursors

Vulval squamous cell carcinoma (VSCC) can arise through two distinct pathways [human papillomavirus (HPV)‐associated and HPV‐independent], and these VSCC variants are recognised as different disease entities on the basis of different aetiologies, morphological features, molecular events during oncogenesis, precursor lesions, prognosis, and response to treatment. The precursor of HPV‐associated VSCC, variously referred to as high‐grade squamous intraepithelial lesion (HSIL) [vulvar intraepithelial neoplasia (VIN) 2/3] or usual‐type VIN, is morphologically identical to the more common HSIL (cervical intraepithelial neoplasia 2/3) of the cervix. The precursor lesions of HPV‐independent VSCC include differentiated VIN, differentiated exophytic vulvar intraepithelial lesion, and vulvar acanthosis with altered differentiation; these have been under‐recognised by pathologists in the past, leading to delays in treatment. This review will discuss the recent advances in diagnostic surgical pathology of VSCC and its precursors, and how these diagnoses can impact on patient management.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

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.