Biomarkers in the Diagnosis of Endometrial Precancers. Molecular Characteristics, Candidate Immunohistochemical Markers, and Promising Results of Three-Marker Panel: Current Status and Future Directions.

Endometrial carcinoma stands as the most prevalent gynecological cancer and the fourth most common cancer affecting women. The incidence of endometrial cancer has been steadily increasing over the past decade, posing a significant threat to public health. The early detection of its precancers remains a critical and evolving concern to reduce mortality associated with endometrial carcinoma. In the last decade, our understanding of endometrial carcinoma and its precancers has advanced through systematic investigations into the molecular genetics of endometrial carcinoma and its precancers. In this review, we focus on advances in precancers associated with the endometrioid subtype, by far the most common histologic variant of endometrial adenocarcinoma. Recent investigations have led to the identification of new biomarkers, and the proposed incorporation of these biomarkers or biomarker panels into the diagnostic framework of endometrial carcinoma precancers. Here, we review these recent advances and their relevance to the histopathologic diagnosis of endometrial carcinoma precancers.

Predicting Progestin Therapy Response With PTEN, PAX2, and ß-Catenin in Patients With Endometrioid Precancer.

This study investigates the predictive value of biomarkers PTEN, PAX2, and ß-catenin for therapeutic outcomes in patients with atypical endometrial hyperplasia or endometrioid intraepithelial neoplasia undergoing progestin therapy. In a retrospective study of 128 patients, we analyzed a total of 351 endometrial biopsy samples and categorized outcomes into responders (absence of residual disease) and nonresponders (presence of residual disease). We found aberrant biomarker expression in pretreatment cases: 48% for PTEN, 65% for PAX2, and 36% for ß-catenin. Approximately 77.3% of patients responded to progestin treatment, with nonresponders showing significantly higher initial PTEN loss (75.86% vs 39.79%, P < 0.001). Nonresponders also demonstrated significant PTEN loss (53.33% vs 20.55%, P < 0.001), PAX2 loss (57.33% vs 41.22%, P < 0.05), and ß-catenin nuclear staining (53.45% vs 27.91%, P < 0.01) in follow-up samples. In addition, nonresponders exhibited lower recovery of intact PTEN and PAX2, along with higher ß-catenin aberrancy in cases initially showing normal ß-catenin levels. We conclude that persistent aberrant PTEN and PAX2 expression, coupled with emerging aberrant ß-catenin in follow-ups, indicates a greater likelihood of treatment failure. Conversely, the absence of these aberrations suggests successful progestin therapy. Our findings highlight the utility of this 3-marker panel in assessing residual disease status and predicting progestin treatment outcomes, thus offering critical insights for patient management.

Expression of B7-H3 and TIM-3 in gastric-type endocervical adenocarcinoma: prevalence, association with PD-L1 expression, and prognostic significance.

Gastric-type endocervical adenocarcinoma (GEA) is the second most common subtype of endocervical adenocarcinoma and has a poor prognosis. Anti-programmed death-1 and anti-programmed death-ligand 1 (PD-L1) inhibitors have emerged as a major treatment option for GEA; however, data on the expression of other immune checkpoints in GEA are limited. We analyzed the expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and B7 homolog 3 protein (B7-H3) in 58 GEA and investigated their prognostic significance as well as association with PD-L1 expression and other known prognostic factors. Applying the tumor proportion score (TPS) with a cutoff of 1%, B7-H3 and TIM-3 were present in 48.3% and 17.2% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, TIM-3 expression was present in 70.7% of cases. Moreover, the expression of three checkpoints (B7-H3, TIM-3, and PD-L1) was incompletely overlapping. Patients with B7-H3 positive tumors (by TPS) or TIM-3 positive tumors (by TPS) had significantly worse recurrence-free survival (RFS) and overall survival (OS) (log-rank). Using CPS, patients with TIM-3 positive tumors showed significantly worse RFS (log-rank). Similarly, B7-H3 positivity (by TPS) and TIM-3 positivity (by TPS) were associated with worse RFS and OS in univariate analysis. TIM-3 positivity (by CPS) was associated with worse RFS in univariate analysis and the final Cox multivariate analysis. In conclusion, our results show that (1) B7-H3 and TIM-3 are frequently expressed in GEA and their expression overlaps incompletely with PD-L1; and (2) both B7-H3 and TIM-3 are independent negative prognostic markers in GEA.

Utility of a PAX2, PTEN, and ß-catenin Panel in the Diagnosis of Atypical Hyperplasia/Endometrioid Intraepithelial Neoplasia in Endometrial Polyps.

The diagnosis of atypical hyperplasia/endometrioid intraepithelial neoplasm (AH/EIN) within endometrial polyps (EMPs) often poses a diagnostic conundrum. Our previous studies demonstrated that a panel of immunohistochemical (IHC) markers consisting of PAX2, PTEN, and ß-catenin can be effectively utilized for the identification of AH/EIN. A total of 105 AH/EIN within EMP were analyzed using the 3-marker panel. We also evaluated these cases for the presence of morules. Benign EMP (n=90) and AH/EIN unassociated with polyp (n=111) served as controls. Aberrant expression of PAX2, PTEN, or ß-catenin was observed in AH/EIN in EMP in 64.8%, 39.0%, and 61.9% of cases, respectively. At least 1 IHC marker was abnormal in 92.4% of cases. Overall, 60% of AH/EIN in EMP demonstrated abnormal results for≥2 IHC markers. The prevalence of PAX2 aberrancy was significantly lower in AH/EIN in EMP than in nonpolyp AH/EIN (64.8% vs. 81.1%, P =0.007), but higher than in benign EMP (64.8% vs. 14.4%, P <0.00001). The prevalence of ß-catenin aberrancy was significantly higher in AH/EIN in EMP than in nonpolyp AH/EIN (61.9% vs. 47.7%, P =0.037). All control benign EMP demonstrated normal expression of PTEN and ß-catenin. Morules were present in 38.1% of AH/EIN in EMP versus 24.3% in nonpolyp AH/EIN, and absent in benign EMP. A strong positive association was found between ß-catenin and morules (Φ=0.64). Overall, 90% cases of atypical polypoid adenomyoma (n=6) and mucinous papillary proliferation (n=4) showed IHC marker aberrancy. In conclusion, the 3-marker IHC panel (PAX2, PTEN, and ß-catenin) is (1) a useful tool in the diagnosis of AH/EIN in EMP; (2) PAX2 loss should be interpreted with caution and in combination with morphology and other markers.

ß-catenin, Pax2, and Pten Panel Identifies Precancers Among Histologically Subdiagnostic Endometrial Lesions.

Despite refinements in histologic criteria for the diagnosis of endometrioid precancers, many challenging cases are encountered in daily practice, creating diagnostic uncertainty and suboptimal patient management. Recently, an immunohistochemical 3-marker panel consisting of ß-catenin, Pax2, and Pten was identified as a useful diagnostic adjunct. However, previous studies focused either on cancers or diagnostically unambiguous precancers, leaving questions about the applicability and utility of the panel in endometria with architectural features near or below the threshold of accepted histologic criteria for endometrioid precancers. Here, in a retrospective study of 90 patients, we evaluated the performance of the 3-marker panel. Notably, the panel detected a subset of disordered proliferative endometria (8/44, 18%), nonatypical hyperplasias (19/40, 48%), and cases with ambiguous features (3/6, 50%) with aberrancy for ≥1 markers. Marker-aberrant cases were more likely to progress to endometrioid precancer or cancer ( P =0.0002). Patterns of marker aberrancy in the index and progressor cases from individual patients provided evidence for origin in a common precursor, and next-generation sequencing of the progressor cases rationalized marker aberrancy for ß-catenin and Pten. The results unequivocally demonstrate that some lesions that do not approach current histologic thresholds are bona fide neoplastic precursors with clinically-relevant driver events that can be detected by the 3-marker panel. The findings provide further validation for the diagnostic utility of the panel in clinical practice and its application in difficult or ambiguous cases.

Aggressive High-grade Uterine Sarcoma Harboring MEIS1-NCOA2 Fusion and Amplification of Multiple 12q13-15 Genes: A Case Report With Morphologic, Immunohistochemical, and Molecular Analysis.

MEIS1-NCOA1/2 fusions are recently described gene rearrangements found in rare sarcomas, mainly involving the genitourinary and gynecologic tracts, with 3 cases reported in the uterine corpus. Although local recurrence was very common, no death has been reported, and some investigators consider these sarcomas low grade. Amplification of genes located at the 12q13-15 locus, especially MDM2 , is the hallmark genetic abnormality in well-differentiated and dedifferentiated liposarcoma of the soft tissue. Some uterine tumors have also been reported to harbor MDM2 amplification, including a proportion of Müllerian adenosarcomas, BCOR fusion-positive high-grade endometrial stromal sarcoma, BCORL1 -altered high-grade endometrial stromal sarcoma, rare JAZF1 fusion-positive low-grade endometrial stromal sarcoma, rare undifferentiated uterine sarcoma, and a single case of MEIS1-NCOA2 fusion sarcoma. Here, we report a case of high-grade MEIS1-NCOA2 fusion uterine sarcoma which also harbored amplification of multiple 12q13-15 genes, including MDM2 , CDK4 , MDM4 , and FRS2 , that exhibited aggressive clinical course leading to patient's death within 2 yr of the initial diagnosis. To the best of our knowledge, this is the first documented case of fatal MEIS1-NCOA2 fusion uterine sarcoma, and the second case of MEIS1-NCOA2 fusion uterine sarcoma that also harbors MDM2 amplification.

Morules But Not Squamous Differentiation are a Reliable Indicator of CTNNB1 (ß-catenin) Mutations in Endometrial Carcinoma and Precancers.

Although collectively regarded as "squamous differentiation (SD)" in endometrial endometrioid carcinoma (EEC) and atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN), morules (often referred to as "squamous morules") and true SD may represent two distinct phenomena. Here, we explored the distinction between morules versus SD and investigated the association of morules and SD with CTNNB1 mutations. A total of 270 cases of EEC and AH/EIN were studied, including EEC with (n=36) or without (n=36) morules and AH/EIN with (n=80) or without (n=118) morules. Cases were analyzed by immunohistochemistry and selected cases (n=20) by targeted next-generation sequencing panel. Near-perfect agreement was found between morules and glandular ß-catenin nuclear staining in AH/EIN and EEC. A strong positive association was found between morules and glandular ß-catenin nuclear staining ( P <0.0001, Φ=0.59 in AH/EIN; P <0.0001, Φ=0.85 in EEC). There was no association between (1) morules and glandular PAX2 or PTEN aberrant expression or (2) SD and aberrant expression of ß-catenin, PAX2 or PTEN (Φ=0.09, ß-catenin; Φ=0.16, PAX2; Φ=0.13, PTEN). CTNNB1 mutations were identified in all 20 selected morule-containing cases (100%). Next-generation sequencing was performed on 2 (preprogestin and postprogestin treatment) biopsies from 1 patient, revealing identical mutational profile in morules and glands. In conclusion, (1) SD and morules are distinct biological phenomena; (2) the presence of morules, but not SD, is a reliable indicator of CTNNB1 mutations in EEC and AH/EIN. Our findings demonstrate that SD and morules are distinct biological phenomena. Since morules but not SD are associated with ß-catenin mutations, the distinction is clinically relevant and should be included in diagnostic reports.

Prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 expression in endometrial serous carcinoma.

Endometrial serous carcinoma (ESC) is an aggressive type of endometrial carcinoma with a poor prognosis. Immune checkpoint blockade has evolved as a novel treatment option for endometrial cancers; however, data on expression of immune checkpoints that may be potential targets for immunotherapy in ESC are limited. We analyzed the prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 immune checkpoints in 99 ESC and evaluated their correlation with CD8 + tumor infiltrating lymphocytes. Applying the tumor proportion score (TPS) with a cutoff of 1%, PD-L1, TIM-3 and B7-H3 expression was present in 17%, 10% and 93% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, PD-L1, TIM-3 and B7-H3 expression was present in 63%, 67% and 94% of cases, respectively. Expression of these markers was largely independent of one another. PD-L1 correlated with higher CD8 + T-cell density when evaluated by either TPS (p = 0.02) or CPS (p < 0.0001). TIM-3 correlated with CD8 + T-cell density when evaluated by CPS (p < 0.0001). PD-L1 positivity was associated with improved overall survival (p = 0.038) when applying CPS. No association between PD-L1 expression and survival was found using TPS, and there was no association between TIM-3 or B7-H3 positivity and survival by either TPS or CPS. Using TPS, PD-L1 correlated with a higher tumor stage but not with survival, whereas the converse was true when PD-L1 was evaluated by CPS, suggesting that PD-L1 expression in immune cells correlates with prognosis and is independent of tumor stage. In conclusion, PD-L1, TIM-3 and B7-H3 may be potential therapeutic targets in selected patients with ESC. Further investigation of their roles as predictive biomarkers is needed.

Endometrial polyps are non-neoplastic but harbor epithelial mutations in endometrial cancer drivers at low allelic frequencies.

Endometrial polyps (EMPs) are common exophytic masses associated with abnormal uterine bleeding and infertility. Unlike normal endometrium, which is cyclically shed, EMPs persist over ovulatory cycles and after the menopause. Despite their usual classification as benign entities, EMPs are paradoxically associated with endometrial carcinomas of diverse histologic subtypes, which frequently arise within EMPs. The etiology and potential origins of EMPs as clonally-derived neoplasms are uncertain, but previous investigations suggested that EMPs are neoplasms of stromal origin driven by recurring chromosomal rearrangements. To better define benign EMPs at the molecular genetic level, we analyzed individual EMPs from 31 women who underwent hysterectomy for benign indications. The 31 EMPs were subjected to comprehensive genomic profiling by exome sequencing of a large panel of tumor-related genes including oncogenes, tumor suppressors, and chromosomal translocation partners. There were no recurring chromosomal rearrangements, and copy-number analyses did not reveal evidence of significant chromosome-level events. Surprisingly, there was a high incidence of single nucleotide variants corresponding to classic oncogenic drivers (i.e., definitive cancer drivers). The spectrum of known oncogenic driver events matched that of endometrial cancers more closely than any other common cancer. Further analyses including laser-capture microdissection showed that these mutations were present in the epithelial compartment at low allelic frequencies. These results establish a link between EMPs and the acquisition of endometrial cancer driver mutations. Based on these findings, we propose a model where the association between EMPs and endometrial cancer is explained by the age-related accumulation of endometrial cancer drivers in a protected environment that-unlike normal endometrium-is not subject to cyclical shedding.

Low grade endometrial endometrioid adenocarcinoma: A review and update with emphasis on morphologic variants, mimics, immunohistochemical and molecular features.

Endometrioid adenocarcinoma (ECa) may feature a number of morphologic variations that can pose diagnostic challenge. The purpose of this review and update is to examine the spectrum of morphologic variants and mimics of low-grade (FIGO grades 1 and 2) ECa, with a focus on histologic, immunohistochemical, and molecular features that may inform diagnosis and treatment. In addition to ECa of usual type, variants with unique cytologic and/or architectural features presented include the following: 1) ECa with mucinous differentiation of conventional (Müllerian) type; 2) ECa with squamous differentiation; 3) ECa with morular metaplasia; 4) ECa with patterns resembling cervical transformation zone tissue and/or microglandular hyperplasia; 5) ECa with cytoplasmic clearing; 6) ECa with papillation, including villoglandular variant of ECa, ECa with small nonvillous papillae, and ECa with a "low-grade serous"-like component or surface changes mimicking ovarian serous borderline tumor; 7) corded and hyalinized variant of ECa; 8) ECa with spindled epithelial cells; 9) ECa with sex cord-like pattern; and 10) ECa with other unusual cytologic and associated features. For each variant, relevant differential diagnoses and diagnostic strategies are discussed. The most clinically significant distinctions come into play in the differential diagnosis between low-grade ECa and one of its high-grade mimics. In this setting, the most fundamental tool in the pathologist's diagnostic arsenal is recognition of the low-grade cytologic features typical of low-grade ECa. Circumspect evaluation of cytologic features, complemented by an awareness of the morphologic spectrum, an appropriate battery of immunohistochemical stains when needed, and mindfulness of the clinical scenario, should guide the pathologist to the correct histotype in even challenging cases.

Human Papillomavirus‒Positive and ‒Negative Vulvar Squamous Cell Carcinoma Are Biologically but Not Clinically Distinct.

Vulvar squamous cell carcinoma pathogenesis is traditionally defined by the presence or absence of human papillomavirus (HPV), but the definition of these groups and their molecular characteristics remain ambiguous across studies. In this study, we present a retrospective cohort analysis of 36 patients with invasive vulvar squamous cell carcinoma where HPV status was determined using RNA in situ hybridization and PCR. Clinical annotation, p16 immunohistochemistry, PD-L1 immunohistochemistry, HPV16 circular E7 RNA detection, and RNA sequencing of the cases were performed. A combination of in situ hybridization and PCR identified 20 cases (55.6%) as HPV positive. HPV status did not impact overall survival (hazard ratio: 1.36, 95% confidence interval = 0.307-6.037, P = 0.6857) or progression-free survival (hazard ratio: 1.12, 95% confidence interval = 0.388-3.22, P = 0.8367), and no significant clinical differences were found between the groups. PD-L1 expression did not correlate with HPV status, but increased expression of PD-L1 correlated with worse overall survival. Transcriptomic analyses (n = 23) revealed distinct groups, defined by HPV status, with multiple differentially expressed genes previously implicated in HPV-induced cancers. HPV-positive tumors showed higher global expression of endogenous circular RNAs, including several circular RNAs that have previously been implicated in the pathogenesis of other cancers.

Navigating through perplex morphologic changes after exogenous hormone usage.

Clinical application of exogenous hormone as a method of contraception and/or treatment of various gynecologic disorders is exceedingly common. Unfortunately, the concurrent use of these agents also complicates the interpretation of pathology specimens. Various studies have shown that morphologic changes induced by hormonal therapies are present in both non-neoplastic and neoplastic tissues within the women's reproductive tract. It is important to understand the exogenous hormone induced morphologic changes, as it helps the pathologists make the accurate diagnosis, and in turn, guide clinicians to make optimal clinical decisions. In this review, we summarize the morphologic changes in both neoplastic and non-neoplastic endometrial, cervical, and myometrial surgical specimens after hormonal therapies, particularly after progestin treatment. In the endometrium, particularly in the scenario of progestin-treated atypical endometrial hyperplasia/endometrioid intraepithelial neoplasia (AEH/EIN), there is notoriously poor interobserver agreement and difficulty in assessing for the residual disease. We summarize current literature and propose our recommended approach in assessing these challenging endometrial biopsies, including a diagnostic algorism, the use of PAX-2, PTEN, beta-catenin immunohistochemistry panel, as well as consistency in diagnostic wording of the report. In the cervix, progestin makes dysplastic lesions appear metaplastic, thus high-grade squamous dysplastic lesions may be easily missed. Within the myometrium, lesions such as adenomyosis may show various degree of decidualization, while smooth muscle neoplasms may show apoplectic changes, and stromal lesions including endometrial stromal sarcoma may show more eosinophilic cytoplasm. All such changes may pose more or less diagnostic challenges in our daily practice. However, most are readily recognizable when we understand particular hormone related scenarios.

Reliable Identification of Endometrial Precancers Through Combined Pax2, ß-Catenin, and Pten Immunohistochemistry.

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably ß-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, ß-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), ß-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or ß-catenin. A focused panel of only 3 markers (Pax2, Pten, and ß-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

Retroperitoneal Sclerosing Angiomyolipoma with Long-Term Follow up: A Case Report with Unique Clinicopathologic and Genomic Profile.

Sclerosing angiomyolipoma (sAML) is a rare variant of the perivascular epithelioid tumors exhibiting distinct morphology with extensive stromal hyalinization, which makes it challenging to recognize. It often lacks an adipose tissue component and melanocytic markers may be expressed only focally, further posing a diagnostic challenge. Here, we report a case of sAML of the left pararenal retroperitoneum in a 52-year-old woman with 92 months of clinical follow up and discuss the histologic features, immunoprofile, molecular alterations, and differential diagnoses that can aid in the diagnosis of this unique and rare entity.

Programmed death-ligand 1 expression in human papillomavirus-independent cervical adenocarcinoma and its prognostic significance.

AIMS: In the 2020 World Health Organization classification of female genital tumours, endocervical adenocarcinomas (ECAs) are subclassified into human papillomavirus (HPV)-associated (HPVA) and HPV-independent (HPVI) groups on the basis of their distinct aetiologies and clinical behaviours. The aim of this study was to investigate programmed death-ligand 1 (PD-L1) expression and its prognostic value in HPVI ECA and HPVA ECA, and compare these between the two entities. METHODS AND RESULTS: A total of 93 ECAs accessioned between 2013 and 2020 were selected for further analysis, including 48 usual-type HPVA ECAs and 45 HPVI ECAs. Then, we evaluated PD-L1 expression in whole tissue sections of these cases by using the tumour proportion score (TPS) and the combined positive score (CPS). Heterogeneous PD-L1 expression was observed in both HPVI ECAs and usual-type HPVA ECAs. However, no significant difference in PD-L1 expression was seen among different histological types of ECA when either the CPS or the TPS was used. Gastric-type ECA (GEA) was associated with higher clinical stage (P = 0.001), worse progression-free survival (PFS) (P = 0.008) and worse overall survival (OS) (P = 0.02) than usual-type HPVA ECA and non-GEA HPVI ECA. When the TPS was used, PD-L1-positive GEA was associated with significantly worse PFS (P = 0.03) and OS (P = 0.015) than PD-L1-negative GEA. CONCLUSIONS: Our data show frequent PD-L1 expression in HPVI ECAs, supporting the potential role of the programmed cell death protein 1/PD-L1 pathway as a therapeutic target for these tumours. Our data also support PD-L1 as a negative prognostic marker associated with a potentially unfavourable outcome for GEAs.

Clinical and Histopathologic Features Can Help Target Immunohistochemical Stain Use in the Diagnosis of Viral Esophagitis.

OBJECTIVES: Herpes simplex virus (HSV) and cytomegalovirus (CMV) immunohistochemical stains (IHC) are frequently applied on esophageal biopsies. Our aims were to identify IHC use patterns in viral esophagitis (VE), and clinicopathologic features of VE that could guide IHC use. METHODS: We included 58 VE cases and 60 controls, defined as patients with negative HSV/CMV IHC between January 2006 and July 2017. Biopsies were reviewed and histologic features and clinical data recorded. RESULTS: Thirteen cases required IHC for diagnosis. IHC was performed in 13 HSV and 5 CMV cases where diagnostic viral inclusions were present. VE patients were more likely to have endoscopic ulcer (P=0.002) and be immunocompromised (P<0.001). Pretest clinical concern for VE was common (P=0.006). Histologically, VE patients were more likely to have ulcer (P=0.004), ulcer exudate rich in neutrophils and histiocytes (P=0.001), neutrophils in squamous mucosa (P<0.001), histiocyte aggregates >15 (P<0.001) and spongiosis (P<0.001). Controls had frequent eosinophils, alone (P=0.008) or admixed with other inflammatory cells (P<0.0001). CONCLUSIONS: IHC is used in VE biopsies despite definite viral inclusions on hematoxylin and eosin and in patients without concerning histology or clinical concern for VE. History, endoscopic findings, and histology can be used to better target IHC use in VE.

T Cell Receptor Repertoires Acquired via Routine Pap Testing May Help Refine Cervical Cancer and Precancer Risk Estimates.

Cervical cancer is the fourth most common cancer and fourth leading cause of cancer death among women worldwide. In low Human Development Index settings, it ranks second. Screening and surveillance involve the cytology-based Papanicolaou (Pap) test and testing for high-risk human papillomavirus (hrHPV). The Pap test has low sensitivity to detect precursor lesions, while a single hrHPV test cannot distinguish a persistent infection from one that the immune system will naturally clear. Furthermore, among women who are hrHPV-positive and progress to high-grade cervical lesions, testing cannot identify the ~20% who would progress to cancer if not treated. Thus, reliable detection and treatment of cancers and precancers requires routine screening followed by frequent surveillance among those with past abnormal or positive results. The consequence is overtreatment, with its associated risks and complications, in screened populations and an increased risk of cancer in under-screened populations. Methods to improve cervical cancer risk assessment, particularly assays to predict regression of precursor lesions or clearance of hrHPV infection, would benefit both populations. Here we show that women who have lower risk results on follow-up testing relative to index testing have evidence of enhanced T cell clonal expansion in the index cervical cytology sample compared to women who persist with higher risk results from index to follow-up. We further show that a machine learning classifier based on the index sample T cells predicts this transition to lower risk with 95% accuracy (19/20) by leave-one-out cross-validation. Using T cell receptor deep sequencing and machine learning, we identified a biophysicochemical motif in the complementarity-determining region 3 of T cell receptor ß chains whose presence predicts this transition. While these results must still be tested on an independent cohort in a prospective study, they suggest that this approach could improve cervical cancer screening by helping distinguish women likely to spontaneously regress from those at elevated risk of progression to cancer. The advancement of such a strategy could reduce surveillance frequency and overtreatment in screened populations and improve the delivery of screening to under-screened populations.

Significance of Oil-Red-O positive macrophages in bronchoalveolar lavage in diagnosing E-cigarettes or vaping product use-associated lung injury: A case series.

BACKGROUND: Lipid-laden macrophages detected by Oil-Red-O (ORO) stain in fresh bronchoalveolar lavage (BAL) specimens have been proposed as a potential diagnostic marker for E-cigarettes or vaping product use-associated lung injury (EVALI). However, studies are few, and the sensitivity and specificity of the test have not been thoroughly investigated. METHODS: We performed ORO stain on fresh BAL specimens from six confirmed EVALI and 36 non-EVALI patients. After semi-quantitative analysis, the sensitivity and specificity of ORO-positive macrophages (OPM) for detection of EVALI were calculated. RESULTS: No significant difference in cytomorphology or raw macrophage count was observed between EVALI and non-EVALI groups (49% vs 55% of all nucleated cells). However, with ORO stain, all EVALI specimens (6/6) showed a high percentage (≥50% of all macrophages) of OPM (mean 87%), and large (≥25% of host macrophage nuclear size) lipid droplets (mean 42%), while the majority of non-EVALI specimens showed a low percentage of OPM (32/36, mean 10%), and small lipid droplets (34/36, mean 6%). The differences between the two groups in both high OPM and large lipid droplet rates are statistically significant (P < .0001 for both comparisons). The combined sensitivity and specificity of high OPM and large lipid droplets for diagnosing EVALI were 100% and 94%, respectively. CONCLUSION: In BAL specimens obtained from patients with clinically suspected EVALI, a high percentage of OPM with large lipid droplets showed high sensitivity and specificity for the diagnosis of EVALI and may serve as a potentially useful tool in the evaluation of vaping-related lung injury, improving diagnostic accuracy.

Serial genomic analysis of endometrium supports the existence of histologically indistinct endometrial cancer precursors.

The endometrium is unique as an accessible anatomic location that can be repeatedly biopsied and where diagnostic biopsies do not extirpate neoplastic lesions. We exploited these features to retrospectively characterize serial genomic alterations along the precancer/cancer continuum in individual women. Cases were selected based on (1) endometrial cancer diagnosis/hysterectomy and (2) preceding serial endometrial biopsies including for some patients an early biopsy before a precancer histologic diagnosis. A comprehensive panel was designed for endometrial cancer genes. Formalin-fixed, paraffin-embedded specimens for each cancer, preceding biopsies, and matched germline samples were subjected to barcoded high-throughput sequencing to identify mutations and track their origin and allelic frequency progression. In total, 92 samples from 21 patients were analyzed, providing an opportunity for new insights into early endometrial cancer progression. Definitive invasive endometrial cancers exhibited expected mutational spectra, and canonical driver mutations were detectable in preceding biopsies. Notably, ≥1 cancer mutations were detected prior to the histopathologic diagnosis of an endometrial precancer in the majority of patients. In 18/21 cases, ≥1 mutations were confirmed by abnormal protein levels or subcellular localization by immunohistochemistry, confirming genomic data and providing unique views of histologic correlates. In 19 control endometria, mutation counts were lower, with a lack of canonical endometrial cancer hotspot mutations. Our study documents the existence of endometrial lesions that are histologically indistinct but are bona fide endometrial cancer precursors. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Irreversible Electroporation for Renal Ablation Does Not Cause Significant Injury to Adjacent Ureter or Bowel in a Porcine Model.

Objective: To evaluate the safety of irreversible electroporation (IRE) for renal ablation adjacent to the ureter or bowel. Materials and Methods: Six adult pigs each underwent bilateral IRE of the kidney. To simulate adjacence, the left proximal ureter and duodenum were secured onto the left and right kidney capsule, respectively. Two IRE probes were placed into the renal parenchyma and configured to bridge the ureter and bowel. Therapeutic IRE was delivered at 2000 V/cm for 70 pulses in both forward and reverse polarity. The animal was survived and euthanized at 1, 3, or 14 days. Histopathology was obtained for all potentially injured bowel and ureteral segments. Retrograde pyelogram (RPG) was performed on each left-sided ureter. Results: Histologic analysis of the ureter identified reactive changes at the level of the periureteral adipose tissue, which progressed from acute inflammation on day 1 to focal fibrosis by day 14. Urothelial mucosa and surrounding smooth muscle layers were unaffected at all time points. RPGs did not show any abnormalities in all specimens. Histologic analysis of the bowel demonstrated acute inflammation in the serosa and subserosal tissue on day 1. Three days after IRE, inflammation and crypt abscesses were focally present in the deep aspects of the bowel mucosa. Inflammation in the mucosal layer resolved 14 days after IRE. Conclusions: In a porcine model of renal IRE, no significant injury was apparent after intentional ablation adjacent to the ureter and bowel. IRE may be a safe alternative to thermal ablation for tumors near the ureter or bowel.