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

Low-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.

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.

Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer.

Importance: The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear. Objective: To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy. Design, Setting, and Participants: Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy. Exposure: TIL abundance in breast tissue from resected primary tumors. Main Outcomes and Measures: The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center. Results: This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6). Conclusions and Relevance: In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

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.

Ki67 assessment protocol as an integral biomarker for avoiding radiotherapy in the LUMINA breast cancer trial.

AIMS: The LUMINA trial demonstrated a very low local recurrence rate in women ≥55 years with low-risk luminal A breast cancer (defined as grade I-II, T1N0, hormone receptor positive, HER2 negative and Ki67 index ≤13.25%) treated with breast-conserving surgery and endocrine therapy (but no other systemic therapy), supporting the safe omission of radiation in these women. Here we describe the protocol for Ki67 assessment, the companion diagnostic used to guide omission of adjuvant radiotherapy. METHODS: Ki67 immunohistochemistry was performed on full-face sections at one of three regional labs. Pathologists trained in the International Ki67 in Breast Cancer Working Group (IKWG) method demarcated tumour areas on scanned slides and scored 100 nuclei from each of at least five randomly selected 1-mm fields. For cases with high Ki67 heterogeneity, further virtual cores were selected and scored in order to confidently assign a case as luminal A (≤13.25%) or B (>13.25%). Interlaboratory variability was assessed through an annual quality assurance programme during the study period. RESULTS: From the quality assurance programme, the mean Ki67 index across all cases/labs was 13%. The observed intraclass correlation coefficient (ICC) and kappa statistics were ≥0.9 and ≥0.7, respectively, indicating a substantial level of agreement. Median scoring time was 4 min per case. The IKWG-recommended scoring method, performed directly from slides, requiring up to four scored fields, is concordant with the LUMINA scoring method (ICC ≥ 0.9). CONCLUSION: Ki67 is a practical, reproducible, and inexpensive biomarker that can identify low-risk luminal A breast cancers as potential candidates for radiation de-escalation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01791829.

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.

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.

The proteome of clear cell ovarian carcinoma.

Clear 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.

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.

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 < 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.

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.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

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).

Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis.

BACKGROUND: Endometrial cancers (ECs) with somatic mutations in DNA polymerase epsilon (POLE) are characterized by unfavorable pathological features, which prompt adjuvant treatment. Paradoxically, women with POLE-mutated EC have outstanding clinical outcomes, and this raises concerns of overtreatment. The authors investigated whether favorable outcomes were independent of treatment. METHODS: A PubMed search for POLE and endometrial was restricted to articles published between March 1, 2012, and March 1, 2018, that provided individual patient data (IPD), adjuvant treatment, and survival. Following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) reporting guidelines for IPD, the authors used univariate and multivariate one-stage meta-analyses with mixed effects Cox models (random effects for study cohorts) to infer the associations of treatment, traditional prognostic factors, and outcome, which was defined as the time from first diagnosis to any adverse event (progression/recurrence or death from EC). RESULTS: Three hundred fifty-nine women with POLE-mutated EC were identified; 294 (82%) had pathogenic mutations. Worse outcomes were demonstrated in patients with nonpathogenic POLE mutations (hazard ratio, 3.42; 95% confidence interval, 1.47-7.58; log-rank P < .01). Except for stage (P < .01), traditional prognosticators were not associated with progression/recurrence or death from disease. Adverse events were rare (11 progressions/recurrences and 3 disease-specific deaths). Salvage rates in patients who experienced recurrence were high and sustained, with 8 of 11 alive without evidence of disease (range, 5.5-14.2 years). Adjuvant treatment was not associated with outcome. CONCLUSIONS: Clinical outcomes for ECs with pathogenic POLE mutations are not associated with most traditional risk parameters, and patients do not appear to benefit from adjuvant therapy. The observed low rates of recurrence/progression and the high and sustained salvage rates raise the possibility of safely de-escalating treatment for these patients. LAY SUMMARY: Ten percent of all endometrial cancers have mutations in the DNA repair gene DNA polymerase epsilon (POLE). Women who have endometrial cancers with true POLE mutations experience almost no recurrences or deaths from their cancer even when their tumors appear to have very unfavorable characteristics. Additional therapy (radiation and chemotherapy) does not appear to improve outcomes for women with POLE-mutated endometrial cancer, and this supports the move to less therapy and less associated toxicity. Diligent classification of endometrial cancers by molecular features provides valuable information to inform prognosis and to direct treatment/no treatment.

A clinical calculator to predict disease outcomes in women with triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by substantial risks of early disease recurrence and mortality. We constructed and validated clinical calculators for predicting recurrence-free survival (RFS) and overall survival (OS) for TNBC. METHODS: Data from 605 women with centrally confirmed TNBC who underwent primary breast cancer surgery at Mayo Clinic during 1985-2012 were used to train risk models. Variables included age, menopausal status, tumor size, nodal status, Nottingham grade, surgery type, adjuvant radiation therapy, adjuvant chemotherapy, Ki67, stromal tumor-infiltrating lymphocytes (sTIL) score, and neutrophil-to-lymphocyte ratio (NLR). Final models were internally validated for calibration and discrimination using ten-fold cross-validation and compared with their base-model counterparts which include only tumor size and nodal status. Independent external validation was performed using data from 478 patients diagnosed with stage II/III invasive TNBC during 1986-1992 in the British Columbia Breast Cancer Outcomes Unit database. RESULTS: Final RFS and OS models were well calibrated and associated with C-indices of 0.72 and 0.73, as compared with 0.64 and 0.62 of the base models (p < 0.001). In external validation, the discriminant ability of the final models was comparable to the base models (C-index: 0.59-0.61). The RFS model demonstrated greater accuracy than the base model both overall and within patient subgroups, but the advantages of the OS model were less profound. CONCLUSIONS: This TNBC clinical calculator can be used to predict patient outcomes and may aid physician's communication with TNBC patients regarding their long-term disease outlook and planning treatment strategies.

Molecular characterization of invasive and in situ squamous neoplasia of the vulva and implications for morphologic diagnosis and outcome.

Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC) is an aggressive clinical entity. Current diagnostic guidelines for premalignant lesions are ambiguous, and their molecular profile and progression events are still unclear. We selected 75 samples, from 40 patients, including 33 VSCC, 8 verrucous carcinomas (VC), 13 differentiated-type vulvar intraepithelial neoplasia (dVIN), 11 suspicious for dVIN (?dVIN), 6 differentiated exophytic vulvar intraepithelial lesions (DE-VIL), 2 vulvar acanthosis with altered differentiation (VAAD), and 2 usual-type vulvar intraepithelial neoplasia (uVIN/HSIL). Invasive and precursor lesions were matched in 29 cases. Clinical information, p16 immunohistochemistry, and mutation analysis were performed on all lesions. All dVIN, ?dVIN, DE-VIL, and VAAD were p16 negative, all uVIN/HSIL were p16 positive. In the HPV-independent group, mutations were identified in 6 genes: TP53 (n = 40), PIK3CA (n = 20), HRAS (n = 12), MET (n = 5), PTEN (n = 4), and BRAF (n = 1). TP53 mutations occurred in 73% (22/30) VSCC, 85% (11/13) dVIN, 70% (7/10) ?dVIN and no VC (0/8), DE-VIL (0/6) nor VAAD (0/2). Basal atypia was the only reliable feature of TP53 mutations. ?dVIN lesions that were non-acanthotic and atypical but obscured by inflammation, all harbored TP53 mutations. In lesions without TP53 mutations, PIK3CA (50% VC, 33% DE-VIL, 100% VAAD, 40% VSCC) and HRAS (63% VC, 33% DE-VIL, 0% VAAD, 20% VSCC) mutations were found. Mutational progression from in situ to invasive was seen (7/26, 27%) and usually involved TP53 (4/26, 15%). Cases with TP53 and PIK3CA co-mutations had the worse clinical outcomes (p < 0.001). We recommend testing for p53 in all HPV-independent lesions suspicious for dVIN, even in the presence of marked inflammation or non-acanthotic skin, particularly when close to a margin. VC, VAAD, and DE-VIL, were almost never mutated for TP53, but instead often harbored PIK3CA and HRAS mutations. In VSCC, combined TP53 and PIK3CA mutations may inform prognosis.