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

AIMS: Decreased 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 RESULTS: Biopsy 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). CONCLUSIONS: Most NSMP ECs (> 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.

Endometrial Carcinosarcomas are Almost Exclusively of p53abn Molecular Subtype After Exclusion of Mimics.

SUMMARY: Our aim was to assess the molecular subtype(s) and perform a detailed morphologic review of tumors diagnosed as carcinosarcoma in a population-based cohort. Forty-one carcinosarcomas were identified from a cohort of 973 endometrial carcinomas diagnosed in 2016. We assessed immunostaining and sequencing data and undertook expert pathology reviews of these cases as well as all subsequently diagnosed (post-2016) carcinosarcomas of no specific molecular profile (NSMP) molecular subtype (n=3) from our institutions. In the 2016 cohort, 37 of the 41 carcinosarcomas (91.2%) were p53abn, 2 (4.9%) were NSMP, and 1 each (2.4%) were POLE mut and mismatch repair deficiency molecular subtypes, respectively. Of the 4 non-p53abn tumors on review, both NSMP tumors were corded and hyalinized (CHEC) pattern endometrioid carcinoma, the mismatch repair deficiency tumor was a grade 1 endometrioid carcinoma with reactive stromal proliferation, and the POLE mut tumor was grade 3 endometrioid carcinoma with spindle cell growth, that is, none were confirmed to be carcinosarcoma on review. We found 11 additional cases among the 37 p53abn tumors that were not confirmed to be carcinosarcoma on the review (3 undifferentiated or dedifferentiated carcinomas, 5 carcinomas with CHEC features, 2 carcinomas showing prominent reactive spindle cell stroma, and 1 adenosarcoma). In the review of institutional cases reported as NSMP carcinosarcoma after 2016, 3 were identified (1 adenosarcoma and 2 mesonephric-like adenocarcinoma on review). In this series, all confirmed endometrial carcinosarcomas were p53abn. The finding of any other molecular subtype in a carcinosarcoma warrants pathology review to exclude mimics.

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

OBJECTIVES: 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%). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

OBJECTIVES: 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. METHODS: 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 POLE status, immunohistochemistry for mismatch repair and p53) with ProMisE NGS (NGS for POLE and TP53, microsatellite instability assay) for concordance metrics and Kaplan-Meier survival statistics across molecular subtypes. In parallel, we assessed all patients who had undergone a new NGS based molecular classification test (n=334) comparing molecular subtype assignment with the original ProMisE classifier. RESULTS: 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. CONCLUSION: 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.

Classification of Vulvar Squamous Cell Carcinoma and Precursor Lesions by p16 and p53 Immunohistochemistry: Considerations, Caveats, and an Algorithmic Approach.

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV-/p53wt), or HPV-independent/p53 abnormal (HPV-/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Noncomplementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH and were classified into the HPV+ group, whereas 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH, and harbored TP53 mutations (1 splice site, 4 missense); they were classified as HPV-/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns: 115 (80.4%) HPV-/p53abn and 27 (18.9%) HPV-/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH and was negative for TP53 mutations-HPV+ category. The use of IHC also led to revised diagnoses-HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that "double-positive" p16/p53 should be classified as HPV-/p53abn. We propose an algorithm that will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

p53-Abnormal "Fields of Dysplasia" in Human Papillomavirus-Independent Vulvar Squamous Cell Carcinoma Impacts Margins and Recurrence Risk.

Abnormal p53 (p53abn) immunohistochemical (IHC) staining patterns can be found in vulvar squamous cell carcinoma (VSCC) and differentiated vulvar intraepithelial neoplasia (dVIN). They can also be found in the adjacent skin that shows morphology that falls short of the traditional diagnostic threshold for dVIN. Vulvectomy specimens containing human papillomavirus-independent p53abn VSCC with margins originally reported as negative for invasive and in situ disease were identified. Sections showing the closest approach by invasive or in situ neoplasia to margins were stained with p53 IHC stains. We evaluated the following: (1) detection of morphologically occult p53abn in situ neoplasia, (2) rates of margin status change after p53 IHC staining, and (3) effect of p53abn IHC staining at margins on the 2-year local recurrence rates. Seventy-three human papillomavirus-independent p53abn VSCCs were included. Half (35/73, 48%) had documented an in situ lesion in the original report. The use of p53 IHC staining identified 21 additional cases (29%) with the p53abn in situ lesions that were originally unrecognized. The histology of in situ lesions in the p53abn "field" varied and became more subtle (morphologically occult) farther away from the VSCC. Fifteen (21%) cases had a morphologically occult and previously unrecognized p53abn in situ lesion present at a resection margin, which conferred an increased risk of local recurrence (5/7 [71.4%] vs 6/22 [27.3%], P = .036). The p53abn in situ lesions at a margin were confirmed to have TP53 mutations by sequencing. p53 IHC staining identified morphologically occult p53abn in situ lesions surrounding human papillomavirus-independent VSCC. p53abn IHC staining at a margin was associated with a 3-fold increased risk of local recurrence.

Grade and Estrogen Receptor Expression Identify a Subset of No Specific Molecular Profile Endometrial Carcinomas at a Very Low Risk of Disease-Specific Death.

Endometrial carcinoma (EC) can be divided into 4 prognostic molecular subtypes, and no specific molecular profile (NSMP) type is the most commonly occurring type (∼50%). Although described as having an intermediate to favorable prognosis, this subtype encompasses pathologically and molecularly diverse tumors. We aimed to identify factors associated with outcomes within the NSMP ECs that might be used to stratify prognosis and direct treatment. Clinicopathologic, immunohistochemical, and genetic features of a large series of NSMP EC were used to identify parameters that could identify the subset associated with a very favorable outcome (disease-specific death rate <5% at 5 years, termed low-risk NSMP). A total of 1110 NSMP ECs were profiled. In a univariate analysis, stage, grade, lymphovascular invasion, estrogen receptor (ER) and progesterone receptor (PR) expression, L1CAM overexpression, and mutations in PIK3CA were associated with disease-specific survival. Two critical features, grade and ER expression, identified a low-risk NSMP subset (grade 1-2, ER-positive [>1%], 84% of cases), which showed a 5-year disease-specific death rate of 1.6% across all stages and 1.4% within stage I. The remaining cases (high-risk NSMPs, grade 3, and/or ER-negative status) were responsible for most of the disease-specific deaths (disease-specific death rate at 5 years, 22.9%; hazard ratio compared with that of low-risk NSMPs: 16.3; 95% CI, 8.4-31.7). Within NSMP EC, the low-risk and high-risk categories were of prognostic significance independent of the stage on a multivariate analysis. Low-grade and ER-positive NSMP ECs are a homogeneous low-risk group associated with an exceptionally favorable prognosis in which de-escalation and/or endocrine therapy strategies can be applied. Grade 3 and/or ER-negative status identifies a high-risk NSMP subset, including rare high-grade histotypes (eg, clear cell, dedifferentiated, and mesonephric-like), responsible for most NSMP-related deaths. Subclassification of NSMPs allows for the category of low-risk EC molecular subtypes to be dramatically expanded because it now includes both POLEmut and the much more common low-risk NSMP EC.

Subclonal p53 immunostaining in the diagnosis of endometrial carcinoma molecular subtype.

AIMS: The 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 RESULTS: Subclonal 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 <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%. CONCLUSIONS: Subclonal 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.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool.

OBJECTIVES: Despite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. METHODS: DNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. RESULTS: The new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. CONCLUSION: ProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Molecular subtype stratified outcomes according to adjuvant therapy in endometrial cancer.

OBJECTIVES: Recent data support the predictive implications of molecular subtype assignment in endometrial cancer (EC). Our objective was to retrospectively assess clinical outcomes according to adjuvant treatment received within EC molecular subtypes. METHODS: Clinical outcomes (disease-specific and progression-free survival DSS/PFS) of EC patients from a single institution and population-based cohorts that had undergone molecular classification were assessed with respect to adjuvant therapy received and 2016 ESMO risk group. RESULTS: 2472 ECs were assessed; 184 (7.4%) POLEmut, 638 (25.8%) MMRd, 1223 (49.5%) NSMP and 427 (17.3%) p53abn. N = 774 (34.6%) of the cohort were ESMO 2016 high risk and 109 (4.8%) were advanced or metastatic. In patients with MMRd EC, assessed across and within stage, there was no observed benefit in DSS or PFS with the addition of chemotherapy +/- radiation compared to radiation alone in ESMO high risk (p = 0.694) or ESMO high, advanced, metastatic risk groups combined (p = 0.852). In patients with p53abn EC, adjuvant chemotherapy given with radiation was associated with significantly longer DSS compared to radiation alone in ESMO high risk (p = 0.007) and ESMO high, advanced and metastatic risk groups combined (p = 0.015), even when restricted to stage I disease (p < 0.001) and when compared in serous vs. non-serous histotypes (p = 0.009). CONCLUSIONS: Adjuvant chemotherapy is associated with more favorable outcomes for patients with p53abn EC, including stage I disease and non-serous histotypes, but does not appear to add benefit within MMRd ECs for any stage of disease, consistent with PORTEC-3 molecular subanalysis. Prospective trials, assessing treatment efficacy within molecular subtype are needed, however these 'real-world' data should be considered when discussing adjuvant treatment with patients.

Molecular Profiling of Endometrial Cancer From TCGA to Clinical Practice.

Molecular classification provides an objective, reproducible framework for categorization of endometrial cancers (ECs), informing prognosis and selection of therapy. Currently, the uptake of molecular classification, integration in to EC management algorithms, and enrollment in molecular subtype-specific clinical trials lags behind what it could be. Access to molecular testing is not uniform, and subsequent management (surgical, adjuvant therapy) is unacceptably variable. We are in the midst of a critical landscape change in this disease site, with increasing emphasis on the integration of molecular features in EC care that can potentially improve standard of care globally. This article summarizes the rationale for molecular classification of ECs, strategies for implementation in low and high resource settings, and actionable opportunities based on this information.

Targeted Molecular Testing in Endometrial Carcinoma: Validation of a Clinically Driven Selective ProMisE Testing Protocol.

Incorporation of molecular classification into clinicopathologic assessment of endometrial carcinoma (EC) improves risk stratification. Four EC molecular subtypes, as identified by The Cancer Genome Atlas, can be diagnosed through a validated algorithm Pro active M olecular R is k Classifier for E ndometrial Cancer (ProMisE) using p53 and mismatch repair (MMR) protein immunohistochemistry (IHC), and DNA polymerase epsilon ( POLE) mutational testing. Cost and access are major barriers to universal testing, particularly POLE analysis. We assessed a selective ProMisE algorithm (ProMisE-S): p53 and MMR IHC on all EC's with POLE testing restricted to those with abnormal MMR or p53 IHC (to identify POLEmut EC with secondary abnormalities in MMR and/or p53) and those with high-grade or non-endometrioid morphology, stage >IA or presence of lymphovascular space invasion (so as to avoid testing on the lowest risk tumors). We retrospectively compared the known ProMisE molecular classification to ProMisE-S in 912 EC. We defined a group of "very low-risk" EC (G1/G2, endometrioid, MMR-proficient, p53 wild-type, stage IA, no lymphovascular space invasion) in whom POLE testing will not impact on patient care; using ProMisE-S, POLE testing would not be required in 55% of biopsies and 38% of all EC's, after evaluation of the hysterectomy specimen, in a population-based cohort. "Very low-risk" endometrioid EC with unknown POLE status showed excellent clinical outcomes. Fifteen of 166 (9%) of all p53abn EC showed G1/G2 endometrioid morphology, supporting the potential value of universal p53 IHC. The addition of molecular testing changed the risk category in 89/896 (10%) EC's. In routine practice, POLE testing could be further restricted to only those patients in whom this would alter adjuvant therapy recommendations.

Molecular classification in endometrial cancer: Opportunities for precision oncology in a changing landscape.

Endometrial carcinoma (EC) classification and risk stratification have undergone a global transformation in the last decade, shifting from a reliance on poorly reproducible histomorphological parameters such as grade and histotype, toward a molecular classification that is consistent and biologically informative. Molecular classification enables reliable categorization of ECs, provides prognostic information, and is now beginning to drive clinical management, including surgery and adjuvant therapy. Within this framework, we now have the ability to further refine both the prognostic and predictive value of molecular classification. As we move toward the routine implementation of this classification system as a stratification tool for research, clinical trials, and patient care, it is imperative that access to these tests be equitable. Furthermore, continued education will be critical for patients and providers to understand the value that this molecular information provides.

Variability in endometrial carcinoma pathology practice: opportunities for improvement with molecular classification.

We assessed the landscape of diagnostic pathology practice and how molecular classification could potentially impact management of patients with endometrial cancer by collecting patient samples, clinicopathologic data, and patient outcomes from EC patients diagnosed in 2016 at 10 Canadian tertiary cancer centers and 19 community hospitals. ProMisE molecular subtype (POLEmut, MMRd, p53abn, No Specific Molecular Profile (NSMP)) was assigned retrospectively. 1357 patients were fully evaluable including 85 POLEmut (6.3%), 380 MMRd (28.0%), 643 NSMP (47.4%), and 249 p53abn ECs (18.3%). Immunohistochemistry (IHC) for MMR proteins was undertaken at the time of primary diagnosis in 2016 in only 42% of the cohort (570/1357; range 3.5-95.4%/center). p53 IHC had only been performed in 21.1% of the cohort (286/1357; range 10.1-41.9%/center). Thus, based on the retrospective molecular subtype assignment, 54.7% (208/380) of MMRd EC had not been tested with MMR IHC (or MSI) and 48.2% (120/249) of p53abn ECs were not tested with p53 IHC in 2016. Molecular subtype diversity within histotypes was profound; most serous carcinomas were p53abn (91.4%), but only 129/249 (51.8%) p53abn EC were serous. Low-grade (Gr1-2) endometrioid carcinomas were mostly NSMP (589/954, 61.7%) but included all molecular subtypes, including p53abn (19/954, 2.0%). Molecular subtype was significantly associated with clinical outcomes (p < 0.001) even in patients with stage I disease (OS p = 0.006, DSS p < 0.001, PFS p < 0.001). Assessment of national pathologic practice in 2016 shows highly variable use of MMR and p53 IHC and demonstrates significant opportunities to improve and standardize biomarker reporting. Inconsistent, non-reflexive IHC resulted in missed opportunities for Hereditary Cancer Program referral and Lynch Syndrome diagnosis, and missed potential therapeutic implications (e.g., chemotherapy in p53abn EC, immune blockade for MMRd EC). Routine integration of molecular subtyping into practice can improve the consistency of EC pathology assessment and classification.

Endometrial carcinoma molecular subtype correlates with the presence of lymph node metastases.

BACKGROUND: The role of lymph node assessment/dissection (LND) in endometrial cancer (EC) has been debated for decades, with significant practice variation between centers. Molecular classification of EC provides prognostic information and can be accurately performed on preoperative endometrial biopsies. We assessed the association between molecular subtype and lymph node metastases (LNM) in order to determine if this tool could be used to stratify surgical decision making. METHODS: All EC patients undergoing primary staging surgery with planned complete pelvic +/- para-aortic LND from a single institution in the 2015 calendar year were identified, with clinicopathological and outcome data assessed in the context of retrospectively assigned molecular classification. RESULTS: 172 patients were included. Molecular classification of the total cohort showed 21 POLEmut (12.2%), 47 MMRd (27.3%), 74 NSMP (43.1%), and 30 p53abn (17.4%) ECs. Complete pelvic +/- para-aortic LND was performed in 171 of 172 patients, and LNM were found in 31/171 (18.1%). This included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). LNM were pelvic only in 83.9%, and pelvic plus para-aortic in 16.1%. There were no isolated para-aortic LNM. Molecular subtype was significantly associated with LNM (p = 0.004). There was a strong association between the presence of LNM and p53abn EC (nodal involvement in 44.8% of cases), with LNM detected in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. On multivariate analysis, molecular subtype and preoperative CA 125 > 25 were significantly associated with LNM (p = 0.021 and p = 0.022 respectively) but preoperative grade and histotype were not (p = 0.24). CONCLUSION: EC molecular subtype is significantly associated with the presence of LNM. As molecular classification can be obtained on preoperative diagnostic specimens, this information can be used to guide surgical treatment planning and may reduce the cost and morbidity of unnecessary lymph node staging in EC care.

Variation in practice in endometrial cancer and potential for improved care and equity through molecular classification.

OBJECTIVES: We measured the variation in practice across all aspects of endometrial cancer (EC) management and assessed the potential impact of implementation of molecular classification. METHODS: Centers from across Canada provided representative tumor samples and clinical data, including preoperative workup, operative management, hereditary cancer program (HCP) referrals, adjuvant therapy, surveillance and outcomes, for all EC patients diagnosed in 2016. Tumors were classified into the four ProMisE molecular subtypes. RESULTS: A total of 1336 fully evaluable EC patients were identified from 10 tertiary cancer centers (TC; n = 1022) and 19 community centers (CC; n = 314). Variation of surgical practice across TCs was profound (14-100%) for lymphadenectomy (LND) (mean 57% Gr1/2, 82% Gr3) and omental sampling (20% Gr1/2, 79% Gr3). Preoperative CT scans were inconsistently obtained (mean 32% Gr1/2, 51% Gr3) and use of adjuvant chemo or chemoRT in high risk EC ranged from 0-55% and 64-100%, respectively. Molecular subtyping was performed retrospectively and identified 6% POLEmut, 28% MMRd, 48% NSMP and 18% p53abn ECs, and was significantly associated with survival. Within patients retrospectively diagnosed with MMRd EC only 22% had been referred to HCP. Of patients with p53abn EC, LND and omental sampling was not performed in 21% and 23% respectively, and 41% received no chemotherapy. Comparison of management in 2016 with current 2020 ESGO/ESTRO/ESP guidelines identified at least 26 and 95 patients that would have been directed to less or more adjuvant therapy, respectively (10% of cohort). CONCLUSION: Molecular classification has the potential to mitigate the profound variation in practice demonstrated in current EC care, enabling reproducible risk assessment, guiding treatment and reducing health care disparities.

Significance of p53 and presence of differentiated vulvar intra-epithelial neoplasia (dVIN) at resection margin in early stage human papillomavirus-independent vulvar squamous cell carcinoma.

OBJECTIVE: Vulvar squamous cell carcinoma and in situ lesions can be stratified by human papillomavirus (HPV) and TP53 status into prognostic risk groups using p16 and p53 immunohistochemistry. We assessed the significance of vulvar squamous cell carcinoma resection margin positivity for either differentiated vulvar intra-epithelial neoplasia (dVIN) or abnormal p53 immunohistochemistry, and other pathologic variables, in a cohort of patients with HPV-independent (HPV-I) p53 abnormal (p53abn) vulvar squamous cell carcinomas. METHODS: Patients with stage I-II HPV-I p53abn vulvar squamous cell carcinoma with negative invasive margins who did not receive adjuvant radiation from a single institution were included. Tumors underwent margin reassessment using p53 immunohistochemistry. Cases were segregated into (1) morphologic dVIN at margin; or (2) abnormal p53 immunohistochemistry staining at margin without morphologic dVIN (p53abn immunohistochemistry); or (3) margins negative by morphology and p53 immunohistochemistry. Clinicopathologic/outcome data were collected. RESULTS: A total of 51 patients were evaluated: (1) 12 with dVIN on margin; (2) 12 with p53abn immunohistochemistry on margin without morphologic dVIN; and (3) 27 with margins negative for morphologic dVIN and p53abn immunohistochemistry. The recurrence rate for patients with dVIN or p53abn immunohistochemistry on the margin was equally high at 75% each, compared with 33% with margins negative for morphologic dVIN and p53abn immunohistochemistry (p=0.009). On multivariate analysis, positive in situ margins maintained an association with disease recurrence (p=0.03) whereas invasive margin distance (radial and deep), lymphovascular invasion, and tumor size did not. CONCLUSIONS: Patients with stage I-II HPV-I vulvar squamous cell carcinoma with margins positive for either dVIN or p53abn immunohistochemistry without morphologic dVIN showed increased disease recurrence, regardless of invasive margin distance. These findings show that p53 immunohistochemistry is a useful adjunct for evaluating margin status in HPV-I vulvar squamous cell carcinoma and may support repeat excision for positive in situ margins (dVIN or p53abn immunohistochemistry).

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

OBJECTIVES: 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 TP53 mutational status. We assessed the reproducibility of the subclassification based on p16 and p53 immunohistochemistry and evaluated the prognostic significance of vulvar squamous cell carcinoma molecular subgroups in a patient cohort treated by vulvar field resection surgery. METHODS: 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, TP53 sequencing). RESULTS: 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. CONCLUSION: 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.

A Scoping Review of Treatment Outcome Measures for Vulvar Intraepithelial Neoplasia.

OBJECTIVE: The goal of this study is to identify a list of clinician-reported outcome measures (CROMs) and patient-reported outcome measures (PROMs) through a review of published studies reporting on any therapeutic interventions for vulvar intraepithelial neoplasia (VIN). MATERIALS AND METHODS: A systematic search of published studies reporting on any therapeutic interventions for VIN was performed on MEDLINE, Embase, Cochrane Database, PsychInfo, and CINAHL from inception to September 20, 2021, based on predetermined study selection criteria. Data were extracted and analyzed by 2 authors independently using Covidence software. RESULTS: Thirty two of 2386 studies identified met study selection criteria. None of the 32 studies provided an explicit definition of VIN treatment "success." The most common CROM was "clinical response to treatment." The most common scale used to measure this outcome was "complete response/partial response/no response"; however, 17 of 23 studies (73.9%) did not define these values. Laboratory CROMs were reported in 12/32 (37.5%) studies. Patient-reported outcome measures were reported in only 10 of 32 studies(31.3%) -the most common PROM was "symptoms." Only 2 of 32 studies measured PROMs related to "quality of life" domains. Adverse events/treatment-related adverse effects were reported in 24 of 32 studies (75%), although 71% of studies provided no details on how these data were collected. CONCLUSIONS: There is a large variation in outcome measures, instruments, and scales used for any clinician-reported treatment outcome such as "clinical response." Most studies do not include patient-reported outcome measures assessing quality of life domains. A Core Outcome Set for the treatment of VIN is needed to improve the quality of VIN research.

A Systematic Review of Risk Factors for Development, Recurrence, and Progression of Vulvar Intraepithelial Neoplasia.

OBJECTIVE: Vulvar intraepithelial neoplasia (VIN) is a premalignant condition with high recurrence rates despite treatment. Vulvar intraepithelial neoplasia develops through separate etiologic pathways relative to the presence or absence of human papillomavirus (HPV) and TP53 mutations. This systematic review was conducted (1) to identify historical risk factors for the development, recurrence, and progression of VIN and (2) to critique these risk factors in the context of advances made in the stratification of VIN based on HPV or TP53 status. MATERIALS AND METHODS: A systematic search was performed on MEDLINE, Embase, Cochrane Database, PsychInfo, and CINAHL from inception to July 5, 2021. Three gynecologic oncologists independently evaluated the eligibility of studies based on predetermined inclusion and exclusion criteria, abstracted data, and then analyzed the relevant data. RESULTS: A total of 1,969 studies (involving 6,983 patients) were identified. Twenty-nine studies met inclusion criteria. The quality of evidence was low; primarily level 2b (Oxford Centre for Evidence-Based Medicine). Risk factors associated with the development of VIN include: smoking and coexisting vulvar dermatoses. Risk factors associated with recurrence include: smoking, multifocal disease, and positive surgical margins. Recent studies identified the presence of differentiated VIN/TP53 mutation as the most significant risk factor for both VIN recurrence and malignant progression. CONCLUSIONS: The current body of evidence consists primarily of small retrospective observational studies. Well-designed retrospective case-control series and/or prospective observational studies are urgently needed. Ideally, future studies will collect standardized data regarding associated risk factors and stratify women with VIN based on HPV and TP53 status.