American Journal of Clinical Pathology

Clinical Validation of the Onclarity Assay After Assay Migration to the High-Throughput COR Instrument Using SurePath Screening Samples From the Danish Cervical Cancer Screening Program

Abstract Objectives This study presents the clinical assessment of the Onclarity HPV Assay (Becton Dickinson) on the novel COR high-throughput instrument (Becton Dickinson) using the international guidelines in a routine setting. Methods Screening samples collected in BD SurePath from women aged 30 years and older were used in this validation. Noninferiority of the Onclarity HPV Assay on the COR instrument (Onclarity-COR) was assessed with the comparator assay glycoprotein 5–positive (GP5+)/6+ enzyme immunoassay (GP-EIA) for clinical sensitivity on 122 cervical intraepithelial neoplasia 2 and greater samples. Specificity was assessed using 887 samples with twice-normal cytology. Inter- and intralaboratory reproducibility analysis was assessed using 525 samples. Finally, a time-and-motion study was performed to evaluate COR instrument performance characteristics. Results The Onclarity-COR was noninferior to the GP-EIA for both sensitivity (P = .0016) and specificity (P < .0001). The intralaboratory reproducibility was 98.3% (κ = 0.96), and interlaboratory agreement was 98.5 % (κ = 0.96). The daily hands-on time for the COR instrument was 58 minutes, and walk-away time was 7 hours, 2 minutes per 8-hour day shift. Conclusions The Onclarity-COR instrument fulfills international validation criteria on sensitivity, specificity, and laboratory reproducibility. The Onclarity assay’s extended genotyping capability, together with its high-throughput characteristics, makes the COR instrument an excellent candidate for use in human papillomavirus primary cervical cancer screening.

Papanicolaou test interpretation utilizing the Hologic Genius Digital Diagnostics System vs manual glass slide review: A retrospective study of 596 cases

Abstract Objective Advances in digital pathology and artificial intelligence have the potential to enhance cytopathology practice. The objective of this study was to compare the performance of the Hologic Genius Digital Diagnostics System with manual glass slide review for Papanicolaou test interpretation. Methods Three cytologists retrospectively reviewed 596 Papanicolaou tests using both the Genius Digital Diagnostics System and manual review, with a 4-week washout period between reviews. The study set consisted of 299 Papanicolaou tests originally interpreted as negative for intraepithelial lesion or malignancy and 297 tests originally interpreted as atypical squamous cells of undetermined significance or above (ASC-US+). Cases interpreted as ASC-US+, reactive/repair, or endometrial cells in a woman older than 45 years of age were additionally reviewed by 1 of 5 cytopathologists. Concordance was calculated between each method and the original cytologic interpretation (reference standard). Sensitivity and specificity for detection of high-grade disease were determined for each method. Cytologist review time per case was recorded. Results Digital interpretation was concordant with the original interpretation in 578 of 596 (97%) cases, while manual interpretation was concordant with the original interpretation in 577 of 596 (97%) cases. Digital review had higher sensitivity for detection of high-grade disease than manual review did (100% vs 86%) but was less specific (93% vs 98%). The average digital review time per case was statistically significantly shorter than manual review time (194.5 seconds vs 485.0 seconds, P < .001). Conclusions Papanicolaou test interpretation using the Genius Digital Diagnostics System is noninferior to manual review. Digital review had higher sensitivity for detection of high-grade disease and statistically significantly reduced screening time.

AmpFire HPV and ScreenFire RS HPV validation trial

Abstract Objectives The human papillomavirus (HPV) screening assays from Atila Biosystems, including the new AmpFire (14 type) and ScreenFire RS (13 type), were subjected to a series of validation tests. Methods We used a set of samples from the Chinese Multi-Site Screening Trial (previously tested with cobas 4800 and the next-generation SeqHPV) to satisfy Meijer’s criteria for clinical end-point validation. We selected 556 self-collected specimens composed of 273 high-risk HPV (hrHPV) positives and 283 hrHPV negatives on the cobas 4800 and SeqHPV. Of the 273 hrHPV-positive cases, 108 had a disease end point of cervical intraepithelial neoplasia grade 2 (CIN2) or higher, including 47 with cervical intraepithelial neoplasia grade 3 (CIN3+) or higher. We simulated the VALGENT framework for inter- and intralaboratory validation and evaluated the new 4-channel risk-stratified ScreenFire assay in a hierarchal fashion. Results Both AmpFire and ScreenFire detected 106 (98.1%) of 108 cases with CIN2 or higher, with specificities of 56.7% and 58.1%, respectively. Intralaboratory concordance for 2 runs of AmpFire and ScreenFire was 95.13% and 96.03%, respectively, for overall hrHPV types and 99.10% and 99.46%, respectively, for HPV 16. The interlaboratory concordance of AmpFire and ScreenFire was 93.68% and 94.04% for overall hrHPV and 98.92% and 99.28%, respectively, for HPV 16. Other genotype correlation percentages were similarly high, with κs ranging from 0.86 to 0.94. The ScreenFire RS assay demonstrated excellent “genotype-specific concordance” when evaluated for “clinical guidance” in a hierarchal fashion (noting only the highest risk channel) with both the cobas 4800 and SeqHPV for less than CIN2, CIN2, and CIN3 or higher. Conclusions The excellent intra- and interlaboratory reproducibility and the established clinical performance, together with the platforms’ simplicity, make these assays particularly applicable to low-resource settings.

Significance of concurrent HPV testing with unsatisfactory Papanicolaou test for prediction of follow-up HPV, Papanicolaou test, and biopsy results

Abstract Objectives Approximately 1% to 2% of routine cytologic specimens collected for Papanicolaou testing are unsatisfactory for evaluation. The American Society for Colposcopy and Cervical Pathology 2019 guidelines recommend repeat testing within 2 to 4 months of an unsatisfactory Papanicolaou test (UPT) result. Methods We evaluated the utility of follow-up Papanicolaou testing, human papillomavirus (HPV) testing, and biopsy in 258 cases of UPTs. Results High-risk HPV testing was positive in 17.4% (n = 45) and negative in 82.6% (n = 213) of cases at the time of initial UPT; 8.1% (n = 21) of cases had discordant HPV test results. Similarly, 3.8% (n = 8) of initially HPV-negative cases were reported to be HPV-positive on follow-up; 28.9% (n = 13) of initially HPV-positive cases were reported to be HPV negative on follow-up. In total, 27.1% (n = 70) of cases underwent biopsy. Biopsies with significant findings were present in 40% (n = 12) of HPV-positive cases and 7.5% (n = 3) of HPV-negative cases. Low-grade squamous intraepithelial lesion (LSIL) (low-grade cervical intraepithelial neoplasia [CIN-1]) was the most significant finding in all 3 HPV-negative biopsies; 58.3% (n = 7) of HPV-positive biopsies showed LSIL (CIN-1), 13.3% (n = 4) showed HSIL (high-grade CIN), and 3.3% (n = 1) showed invasive carcinoma. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of concurrent HPV testing at the time of UPT for predicting follow-up HPV test result within 1 year of initial UPT are 80.0%, 94.0%, 71.1%, and 96.2%, respectively. The sensitivity, specificity, PPV, and NPV of initial HPV test results for predicting follow-up Papanicolaou test results are 67.7%, 89.7%, 48.8%, and 95.0%, respectively. Results Concurrent HPV testing in the setting of UPT can be a sensitive tool for predicting follow-up HPV status and significant findings of squamous intraepithelial lesions on follow-up Papanicolaou tests and biopsy.

Cotesting in Cervical Cancer Screening

Classifying Anal Intraepithelial Neoplasia 2 Based on LAST Recommendations

Abstract Objectives: The Lower Anogenital Squamous Terminology (LAST) recommendations classify human papillomavirus–associated squamous lesions into low- and high-grade squamous intraepithelial lesions (LSILs/HSILs). Our study aimed to assess interobserver agreement among 6 experienced pathologists in assigning 40 anal lesions previously diagnosed as anal intraepithelial neoplasia 2 (AIN 2) to either HSIL or non-HSIL categories. Methods: Agreement based on photomicrographs of H&E alone or H&E plus p16 immunohistochemistry was calculated using κ coefficients. Results: Agreement was fair based on H&E alone (κ = 0.42; 95% confidence interval [CI], 0.34-0.52). Adding p16 improved agreement to moderate (κ = 0.55; 95% CI, 0.54-0.62). On final diagnosis, 21 cases (53%) had unanimous diagnoses, and 19 (47%) were divided. When designating p16 results as positive or negative, agreement was excellent (κ = 0.92; 95% CI, 0.83-0.95). Among variables (staining location, extent, and intensity), staining of the basal/parabasal layers was a consistent feature in cases with consensus for positive results (20/20). Of the 67 H&E diagnoses with conflicting p16 results, participants modified 32 (48%), downgrading 23 HSILs and upgrading 9 non-HSILs. Conclusions: Although p16 increased interobserver agreement, disagreement remained considerable regarding intermediate lesions. p16 expression, particularly if negative, can reduce unwarranted HSIL diagnoses and unnecessary treatment.

Underrecognized Patterns of High-Grade Squamous Intraepithelial Lesion on ThinPrep Preparations

Pathologic sampling of the omentum for neoplasms that involve the female genital tract: A retrospective analysis of 1055 cases

Abstract Objective We sought to assess the number of blocks that should routinely be submitted for microscopic examination of omentectomy specimens associated with neoplasms that involve the female genital tract. Methods Clinicopathologic data were retrospectively reviewed in 1055 cases wherein the omentum was resected for possible gynecologic cancer staging. We investigated any associations between the microscopic positivity rate (MPR) and the number of blocks submitted, block groups (categorized as 1-2 blocks, 3-4 blocks, 5-6 blocks, and >6 blocks), and block to size ratio (the number of blocks submitted to the widest specimen dimension, classified as approximate deciles). Results Of the 1055 cases we studied, 536 (50.8%) were grossly normal, and 519 (49.2%) were abnormal. Within the grossly normal group, there were no statistically significant differences in MPR between the block groups and between cases with 1, 2, 3, 4, 5, or 6 blocks submitted (P > .50 for all pairwise comparisons). Cochran-Armitage tests for trend did not show any linear trend between increasing block groups (P = .88) or increasing block to size ratios (P = .39) and MPR; a binomial logistic regression analysis confirmed that neither block groups (odds ratio, 1.144 [95% CI, 0.794-1.648]; P = .47) nor block to size ratio (odds ratio, 1.022 [95% CI, 0.770-1.358]; P = .88) showed a statistically significant linear relation to MPR. For diffusely or multifocally abnormal cases, the highest MPR (95.5%) was reached at the 1 to 2 blocks group level, and MPR did not statistically significantly increase with higher levels of sampling. Conclusions Submitting 1 to 2 block sections of the omentum in the studied setting results in an MPR that is not statistically significantly lower than the MPR associated with higher levels of sampling.

Pilomatrix-like breast carcinoma: A mammary analog of pilomatrix-like high-grade endometrioid carcinoma (PiMHEC)

Abstract Objectives To describe what is, to our knowledge, the first recognized case of a triple-negative breast carcinoma (TNBC) with a PiMHEC-like phenotype. Pilomatrix-like high-grade endometrioid carcinoma (PiMHEC) is a high-grade carcinoma with divergent differentiation resembling cutaneous pilomatrix carcinoma that was recently described in the endometrium and ovary. For reference, pertinent features of PiMHEC include (1) high-grade basaloid to squamoid morphology with the presence of ghost cells; (2) only focal p63 and/or p40 expression despite a squamoid appearance; (3) CTNNB1 mutation, accompanied by diffusely aberrant β-catenin expression and LEF1 and/or CDX2 expression; and (4) loss of site-specific markers (ie, PAX8, ER). Methods Here we report the histologic, immunophenotypic and molecular genetic features of a case of a triple-negative breast carcinoma (TNBC) with a PiMHEC-like phenotype. Results The tumor developed immediately adjacent to a HER2+, androgen receptor (AR)+, GATA3+ conventional grade 3 invasive ductal carcinoma (IDC) with only membranous β-catenin expression. The PiMHEC-like component had all of the above-noted morphologic and immunophenotypic features of endometrial PiMHEC but with loss of GATA3 and AR rather than PAX8 and ER. Molecular analysis performed on both tumor components demonstrated a shared TP53 point mutation and an exon 3 CTNNB1 mutation restricted to the PiMHEC-like component, implying a clonal relationship with secondary acquisition of CTNNB1. Following neoadjuvant chemotherapy, the HER2+ conventional component had completely resolved, but the PiMHEC-like component had very little response. Conclusions This case demonstrates that a PiMHEC-like phenotype may be seen as a form of TNBC that can develop from conventional IDC, with loss of site-specific biomarkers, acquisition of CTNNB1 mutation, and resistance to conventional chemotherapy.

Presence of 10 transformation zone cells in endocervical curettage should not be required for adequacy

Abstract Objectives In another publication, we concluded endocervical curettage (ECC) should have a minimum number of squamous cells for adequacy, similar to the requirements for adequate cervical Papanicolaou smears. Here, we investigate if also, similar to cervical Papanicolaou smears, the presence of at least 10 cells from the endocervical/transformation zone (EC/TZ) in ECCs should be used as a quality assurance measure or if, instead, at least 10 EC/TZ cells should be part of the adequacy criteria for ECC, with an emphasis on diagnosis of at least high-grade squamous dysplasia (HGD). Methods All patients with at least HGD diagnosed on an excisional biopsy specimen (loop electrosurgical excision procedure [LEEP]) from May 1, 2018, to December 31, 2019, and an ECC in the preceding 6 months at our institution were included. Number of EC/TZ cells present in ECCs was counted visually and categorized as less than or greater than 10 TZ cells. A χ2 test was used to evaluate the proportion of ECCs with and without HGD and the presence or absence of at least 10 EC/TZ cells. Given our recent work encouraging at least 1000 squamous cells in an ECC to be considered adequate, we also evaluated only ECCs with greater than 1000 squamous cells with and without HGD and the presence or absence of at least 10 EC/TZ cells. P value was <.05. Results Fifty-one LEEPs with HGD and a preceding ECC in the previous 6 months were identified. Of the 51 ECCs, 6 had fewer than 10 EC/TZ cells and 45 had at least 10 EC/TZ cells. A similar proportion of the ECCs with HGD had at least 10 EC/TZ cells as those without HGD (93% vs 86%, P = .53). Using only ECCs with greater than 1000 squamous cells, we still found no statistical difference in the proportion of ECCs with HGD having greater than 10 EC/TZ cells compared to those without HGD (91% vs 100%, P = .49). Conclusions We found that the presence of at least 10 EC/TZ cells does not increase the likelihood of finding HGD in an ECC performed in the 6 months prior to a LEEP with HGD. Similar to the use of the TZ component in cervical Papanicolaou smears, the presence or absence of at least 10 TZ cells in an ECC should only be considered a quality assurance measure and not be used as a criterion for adequacy of the specimen.

Metastatic Neoplasms Involving the Stomach

Abstract Objectives Metastatic neoplasms involving the stomach are rare and diagnostically challenging if clinical history of malignancy is absent or unavailable. This study was designed to identify the tumors that most frequently metastasize to the stomach and the morphologic features that can provide clues to investigate the possibility of metastasis and predict the primary sites. Methods All patients with metastatic neoplasms involving the stomach were included in the study. The H&E- and immunohistochemical-stained slides were reviewed, and all clinical, endoscopic, and radiologic information was recorded. Results One hundred fifty patients, including 84 (56%) women and 66 (44%) men (mean age, 64 years), were identified. Gastric metastases were the initial presentation in 15% cases. Epithelial tumors (73.3%) comprised the largest group, followed by melanoma (20.6%), sarcomas (4%), germ cell tumors (1.3%), and hematolymphoid neoplasms (0.7%). Lobular breast carcinoma was the most common neoplasm overall in women, while in men, it was melanoma. Solid/diffuse growth pattern (75%) was more common compared with glandular morphology. The solid/diffuse category included lobular breast carcinoma (21.3%), melanoma (20.6%), and renal cell carcinoma (10.6%), while the glandular category was dominated by gynecologic serous carcinomas (7.3%) with papillary/micropapillary architecture. Conclusions Metastatic neoplasms should be considered in the differential diagnosis of gastric neoplasms, particularly those with a diffuse/solid growth pattern. Glandular neoplasms are difficult to differentiate from gastric primaries except for Müllerian neoplasms, which frequently show a papillary/micropapillary architecture.

What Constitutes Optimal Cervical Screening for Young Women Ages 21 to 29 Years?

Increased High-Risk Human Papillomavirus Viral Load Is Associated With Immunosuppressed Microenvironment and Predicts a Worse Long-Term Survival in Cervical Cancer Patients

Abstract Objectives To evaluate the correlation between tumor-infiltrating lymphocytes (TILs) and the viral load of high-risk human papillomavirus (HR-HPV) in cervical cancer patients. Methods A total of 62 cervical cancer patients were recruited during 1993-1994 and assigned into four groups treated with radiotherapy alone or radiotherapy combined with chemotherapy and/or thermotherapy. Ki67+ tumor cells, CD4+, CD8+, FoxP3+, OX40+ and granzyme B+ TILs were detected by immunohistochemistry. The viral load of HR-HPV in biopsy tissues before therapy was detected by in situ hybridization. Results The patients with high HPV viral load showed a significantly lower 15-year survival rate and an advanced International Federation of Gynecology and Obstetrics (FIGO) stage and increased recurrence rate. The distribution of Ki67+ tumor cells, FoxP3+ TILs, and CD8+/FoxP3+ ratio was obviously different between low and high HPV viral load groups. A worse clinical outcome was also implicated with increased HPV viral load tested by Cox regression analysis. Conclusions Patients with increased HR-HPV viral load tend to be resistant to therapy with decreased immune surveillance in the immune microenvironment. Thus, HR-HPV viral load would influence the local immune microenvironment, and then further affect the survival of cervical cancer patients.

Human Papillomavirus (HPV) 16 and 18/45 Genotyping-Directed Follow-up of Women With Messenger RNA HPV-Positive, Cytology-Negative Cervical Screening Test Results

Abstract Objectives In this study, we sought to correlate genotype test results for human papillomavirus (HPV) types 16, 18, and 45 with histopathologic follow-up diagnoses in patients with messenger RNA (mRNA) high-risk HPV-positive, cytology-negative results. Methods We identified 1,157 patients with mRNA HPV-positive, cytology-negative cervical screening test results between June 2015 and June 2018. Reflex HPV 16/18/45 genotype results were documented in 1,018 women aged 30 years or older, 318 of whom had follow-up within 18 months. Results Histopathologic findings of cervical intraepithelial neoplasia 2 or worse (CIN2+) were diagnosed in 14 of 122 (11.5%) patients positive for HPV 16/18/45 vs in seven of 196 (3.6%) HPV 16/18/45–negative patients. Three patients with high-risk HPV–positive, cytology-negative cervical screening test results were diagnosed with stage I cervical adenocarcinomas following early colposcopic referral and biopsy after HPV 16/18/45–positive genotype results. Conclusions Immediate reflex HPV 16/18/45 genotyping of mRNA HPV-positive, cytology-negative patients led to early colposcopic referral and histopathologic diagnoses of three difficult-to-detect, low-stage, cervical adenocarcinomas and significantly increased overall early detection of CIN2+ lesions.

High-Risk Human Papillomavirus Testing, Genotyping, and Histopathologic Follow-up in Women With Abnormal Glandular Cells on Papanicolaou Tests

Abstract Objectives This study examined the association of high-risk human papillomavirus (hrHPV) status and HPV genotype with histopathologic follow-ups in women with an atypical glandular cell (AGC) interpretation. Methods Cases with AGC interpretation on a Papanicolaou (Pap) test were retrieved along with hrHPV testing, genotyping, and histologic follow-up results if available. Results A total of 561 AGC cases were identified, with histologic follow-up available for 471 cases (84%). The follow-up diagnoses included benign or reactive changes (60% of cases), low-grade cervical intraepithelial neoplasia (18%), high-grade cervical intraepithelial neoplasia (CIN2-3; 7%), cervical carcinoma (5%), and other malignancies (10%). Tests for hrHPV were positive in 128 of 426 (30%) cases, including HPV16 (30%), HPV18 (14%) and other HPV subtypes (56%). A positive hrHPV result significantly increased the risk of developing CIN2-3 or cervical carcinoma (odds ratio, 24.6; 95% CI, 9.9-58.9) and HPV16 or HPV18 further increased the risk (odds ratio, 49.5; 95% CI, 17.7-123.7). Conclusions Our data demonstrate that in women with an AGC Pap interpretation, a positive hrHPV result, especially type 16 or 18, is associated with an increased risk of developing cervical CIN2-3 or higher lesions, suggesting potential implications of hrHPV testing for the management of patients with an AGC result on a Pap test.

Impact of a Modified HistoGel Method for Processing Endocervical Curettage Specimens on Diagnostic Yield

Abstract Objectives Endocervical curettage (ECC) specimens may be limited by scant tissue. We evaluated whether a cellular concentration processing method could improve their diagnostic quality. Methods Between October 2018 and June 2019, ECC specimens were assigned chronologically to one of two groups: nonconcentrated ECC (NECC) or concentrated ECC (CECC). NECC specimens underwent routine histologic processing. CECC specimens were processed using a published HistoGel-based cell block method. We reviewed diagnoses for ECCs, concurrent cervical biopsies and/or loop electrosurgical excision procedures (LEEPs), and preceding Papanicolaou (Pap) smears. We performed multivariate logistic regression analyses to evaluate the impact of processing method on ECC adequacy and discordance between Pap smear and worst tissue diagnoses. Results NECC and CECC adequacy was 88.2% and 84.7% (P = .06). ECC adequacy was greater if concurrent biopsy/LEEP was performed (odds ratio [OR] = 1.76, P < .01). Discordance between Pap smear and worst tissue diagnoses was 9.5% and 13.3% (P = .04) for cases with NECC and CECC processing, although processing method was not significant in multivariate analysis (OR = 0.74, P = .11). Adequate ECC sampling and concurrent biopsy/LEEP were independently associated with concordance between Pap smear and worst tissue diagnosis (OR = 0.46, P < .01 and OR = 0.65, P = .02). Conclusions ECC processing method did not significantly affect either specimen adequacy (P = .06) or diagnostic discordance (P = .11) when controlled for other factors.

Role of High-Risk HPV Testing in Papanicolaou Tests With Atypical Glandular Cells With and Without Concurrent Squamous Cell Abnormalities

Abstract Objectives Data on Papanicolaou (Pap) tests with atypical glandular cells (AGCs) with concurrent squamous cell abnormalities (AGC + Sq) are limited. We evaluated histologic outcomes and the role of high-risk human papillomavirus (HR-HPV) testing in this setting compared with AGCs without concurrent squamous cell abnormalities (AGC-alone). Methods This study used a retrospective cohort of patients with Pap test diagnoses of AGC + Sq and AGC-alone between October 2013 and August 2021. Results We included 287 Pap tests from 278 patients. The HR-HPV test was positive in 55% of AGC + Sq cases and 14% of AGC-alone cases (P < .0001). Most AGC + Sq cases displayed squamous lesions (41.5%) or were benign (41.5%) on histology, whereas AGC-alone cases were predominantly benign (72%) or extracervical neoplasms (18%). AGC + Sq cases showed higher rates of significant histologic lesions (P = .0001), which were associated with positive HR-HPV status (P = .0012). In AGC-alone cases, HR-HPV status was associated with significant histology only in patients 50 years of age or younger. In both groups, 20% or more of HR-HPV–negative patients harbored significant lesions. Conclusions AGC + Sq represents a distinct group of patients. HR-HPV testing and patient age provide useful information in the evaluation of AGC, but triage based on HR-HPV testing is not recommended because of the potential for missing significant lesions.

The Clinical Utility of Extended High-Risk HPV Genotyping in Women With ASC-US Cytology

AbstractObjectivesExtended testing for high-risk human papillomavirus genotypes (hrHPVGTs) is increasingly investigated for risk stratification in cervical cancer screening.MethodsAge and hrHPVGT results from 16,993 women with atypical squamous cells of undetermined significance (ASC-US) cytology between November 2015 and August 2021 were studied and correlated with available histopathologic findings within 6 months.ResultsHigh-risk human papillomavirus (hrHPV)–positive rate was 66.9% in women with ASC-US cytology, and the most prevalent genotypes were HPV 52 (20.9%), 16 (15.7%), and 58 (12.8%). Single hrHPV genotypes and multiple HPV genotypes were detected in 77.2% and 22.8% of women with hrHPV-positive results. Cervical intraepithelial neoplasia grade 2 or more (CIN2+) severe lesions were identified in 19.5% of women with hrHPV-positive ASC-US. The greatest risk for CIN2+ was found in single genotype infections with HPV 16 (33.1%), followed by women with multiple genotype infections, including HPV 16 (32.7%), 82 (30.8%), and 31 (30.0%). hrHPVGT testing for genotypes 16, 31, 35, 45, 82, 58, 33, 52, and 18 was identified in 91.9% (965/1,050) of CIN2+ cases, with 88.9% sensitivity, 43.2% specificity, positive predictive value of 23.9%, and negative predictive value of 95.1%.ConclusionsExtended hrHPV genotyping for women with ASC-US cytology could identify those hrHPV genotypes (HPV 16, 31, 35, 45, 82, 58, 33, 52, 18) associated with higher risk of CIN2+ and allows for refined risk stratification of women being screened.

Adequacy in Endocervical Curettage

Abstract Objectives Specimen adequacy is an important quality assurance component of a cervical Papanicolaou test. Although consensus exists on minimal acceptable cellularity for cervical Papanicolaou tests, no such criteria exist for endocervical curettage (ECC) specimens. We sought to identify minimum acceptable cellularity for accurate diagnosis of high-grade dysplasia (HGD) on ECC. Methods All patients with HGD diagnosed in a loop electrosurgical excision procedure (LEEP) from May 8, 2018, to December 18, 2019, and an ECC in the preceding 6 months at our institution were included (n = 51). All ECCs performed before the LEEP were evaluated for cellularity of squamous cells using Aperio eSlide Manager (Leica Biosystems). Biopsy results concurrent with the ECC were noted. We compared the number of squamous cells in positive and negative ECC specimens using a t-test. The proportion of ECC specimens and concurrent biopsies undergoing immunohistochemical (IHC) staining for p16 were compared using the χ2 test. P < .05 was considered significant. Results Endocervical curettage specimens positive for HGD have increased cellularity compared with negative ECC specimens (mean cellularity, 10,165 vs 1,055; P < .05). Further, IHC staining for p16 was more likely to be performed on an ECC specimen positive for HGD than on a negative ECC specimen (50% vs 3%; P < .05). Biopsies performed concurrently with a negative ECC finding were more likely to undergo p16 IHC than biopsies performed concurrently with a positive ECC finding (51% vs 7%; P < .05). Finally, we observed no difference in the proportion of biopsies undergoing IHC staining for p16 when comparing biopsies positive for HGD with negative biopsies (37% vs 46%; P = .33). Conclusions We find cellularity of approximately 10,000 cells adequate to diagnose HGD in an ECC specimen and cellularity of approximately 1,000 cells to be inadequate. Further, we find p16 IHC commonly used as a “rule-in” test on ECC specimens at our institution. Biopsies accompanying an ECC specimen negative for HGD are more likely to undergo p16 IHC than those accompanying an ECC specimen positive for HGD, but there is no difference in the proportion of biopsies undergoing p16 IHC when comparing positive and negative results in the biopsies themselves. These findings further support the need for adequate cellularity for diagnosis in ECC, especially when a biopsy is technically difficult. Further areas for exploration include investigating laboratory procedures to maximize the cellularity of ECC specimens.

Comparison of Cervical Cancer Screen Results on Female-to-Male Transgender Patients With Female Patients

Abstract Objectives There are limited data on cervical screen results from female-to-male (FTM) transgender patients. Herein, we compiled demographic information and cervical screen testing on FTM transgender patients and compared with age-appropriate controls. Methods A search of our previous and current databases was performed for Papanicolaou (Pap) tests from patients taking testosterone and/or with a diagnosis of gender dysphoria, transsexualism, or transvestism. Patient data were reviewed. Relative risks of abnormal Pap smear and human papillomavirus (HPV) infection were calculated against age-matched controls. Results Eighty-nine Pap tests from FTM transgender individuals were identified, with a mean age of 31.3 years (range, 21-60 years). The Pap test diagnoses were distributed as follows: negative for intraepithelial lesion (n = 84, 94.4%), atypical squamous cells of undetermined significance (n = 0), low-grade intraepithelial lesion (n = 4, 4.5%), and high-grade squamous intraepithelial lesion (n = 1, 1.1%). Fifty (56.2%) patients had concurrent high-risk HPV testing with four (8%) positive results. Relative risk was 0.625 (95% confidence interval [CI], 0.25-1.59; P = .32) for an abnormal Pap test and 0.55 (95% CI, 0.19-1.52; P = .24) for HPV compared with 267 age-matched controls. Of note, 13.5% of patients older than 21 years had documentation of never having a prior Pap test in our medical record. Conclusions In our study, FTM transgender individuals were not at a higher or lower risk of HPV infection or abnormal Pap test result compared with women. However, larger studies are needed to support our findings.

Nationwide Prevalence and Genotype Distribution of High-Risk Human Papillomavirus Infection in China

Abstract Objectives Extended high-risk human papillomavirus (hrHPV) genotype testing has recently been introduced in routine cervical cancer screening. Changes in national and regional hrHPV genotype prevalence offer an objective baseline indicator of the future impact of mass HPV vaccination and HPV-based cervical screening. Methods This retrospective study reports nationwide hrHPV genotyping results from July 2018 to June 2019 in 29 KingMed Diagnostics laboratories throughout China. Results In total, 2,458,227 hrHPV genotyping results were documented from KingMed’s nationwide laboratory database during the study period. The overall prevalence of hrHPV-positive results was 19.1%, with twin peaks for highest hrHPV infection rates in women younger than 30 years of age (22.0%) and 50 years of age and older (21.8%). The most frequently detected hrHPV genotypes were HPV-52 (4.7%), HPV-16 (3.4%), HPV-53 (2.5%), HPV-58 (2.4%), HPV-51 (2.0%), and HPV-68 (1.6%). Overall, hrHPV-positive results varied regionally from 15.3% to 24.4%. Conclusions Nationwide hrHPV genotyping results from KingMed laboratories offer a baseline for measuring the future impact of large-scale HPV vaccination. High hrHPV infection rates in older (≥50 years) Chinese women likely reflect the limited extent of cervical screening in China. High rates of hrHPV infection and variable regional hrHPV genotype distribution may represent limiting factors for cost-effective implementation of hrHPV-based cervical screening in China.

The Potential Impact of High-Risk Human Papillomavirus–Negative Cervical Intraepithelial Neoplasia 2+ on Primary Human Papillomavirus Screening

Abstract Objectives To investigate the prevalence of high-risk human papillomavirus (HPV)–negative cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC) and to analyze the distribution of other genotypes in this subset. Methods In total, 431 women who underwent excisional surgical treatment for CIN or ICC at the European Institute of Oncology, Milan, Italy, from January 2016 to December 2017 were retrospectively analyzed. The Linear Array HPV genotyping test (Roche Diagnostics) was performed on a postaliquot from high-risk-HPV–negative liquid-based cervical specimens, when available. Patient characteristics and the prevalence of high-risk-HPV–negative CIN grade 2 or worse (CIN2+) were tabulated. We used t tests to compare age between high-risk-HPV–positive and high-risk-HPV–negative patients. Results Overall, 8.9% of CIN2+ and 7.5% of ICC cases were high-risk HPV negative. There was no age difference between high-risk-HPV–negative CIN2+ women (mean [SD], 41.3 [8.7] years) and high-risk-HPV–positive women (mean [SD], 39.5 [9.0] years) (P = .28). The Linear Array result was available in 22 cases. Most high-risk-HPV–negative patients were positive for a single other genotype infection (32.6%). HPV 73 was the most prevalent genotype, followed by HPV 53 and HPV 84. HPV 26 was detected in 1 case of ICC. Conclusions Our results showed a not-negligible proportion of high-risk-HPV–negative CIN2+, suggesting that cotesting would not miss these cases.

A comprehensive analysis of SOX17 expression by immunohistochemistry in human epithelial tumors, with an emphasis on gynecologic tumors

Abstract Objectives The objective of this study was to evaluate SOX17, a transcription factor from the Sry high-mobility group–related box superfamily, as a diagnostic marker to determine site of origin using both whole-tissue sections and tissue microarrays (TMAs). Methods SOX17 immunohistochemistry was performed on gynecologic and nongynecologic tissues (N = 1004) using whole-tissue sections and both internally constructed and commercially available TMAs. SOX17 nuclear reactivity was scored as positive or negative on the whole-tissue sections and using the semiquantitative H score method on TMAs. Results Using both whole-tissue sections and TMAs, SOX17 was positive in 94% (n = 155) of endometrial tumors and 96% (n = 242) of ovarian tumors. All breast cases (n = 241) and vulvar/cervical squamous cell carcinomas (n = 150) were negative. Among 1004 tumors from 20 sites, the only organs with positive tumors were ovary, uterus, and testis. Conclusions SOX17 is a sensitive and specific marker for gynecologic origin in the tissues tested and may be a valuable adjunct to PAX8 and other commonly used markers to confirm endometrial or ovarian origin. SOX17 expression is lower in mucinous tumors, endocervical adenocarcinoma, high-grade neuroendocrine tumors, and undifferentiated/dedifferentiated endometrial carcinoma.

Implementation of endometrial cancer molecular subtyping into a hybrid community-academic practice

Abstract Objectives We sought to confirm utility of our institution’s modified Proactive Molecular Risk Classifier for Endometrial Cancer protocol in our daily practice, which includes mismatch repair (MMR), p53, and L1 cell adhesion molecule (L1CAM) immunohistochemistry with in-house next-generation sequencing for POLE, TP53, and CTNNB1. Methods We conducted a retrospective review of all patients in our institution who underwent primary endometrial carcinoma resection from the year prior to protocol implementation (PRE; October 1, 2020, to September 30, 2021) through first year of implementation (POST; October 1, 2021, to September 30, 2022) to compare the distribution of molecular and traditional staging factors using GOG-249 criteria to assign clinical risk. Results In total, 136 of 260 PRE patients were classified as clinically low risk (LR), of whom 31 were MMR deficient. Of the 157 LR POST patients with endometrioid-type carcinoma, 45 were MMR deficient, 5 were POLE mutant, 5 were TP53 mutant, 56 were of no specific molecular profile (NSMP), and 46 did not receive full protocol testing. Of all 79 POST NSMP endometrioid-type cases, 18 were CTNNB1 mutated and 8 showed L1CAM expression. Conclusions Our protocol identified 22 (14%) of 157 LR tumors that harbored incipient intermediate- to high-risk molecular aberrations in TP53, CTNNB1, or L1CAM. Moving forward, results of ongoing trials assessing adjuvant therapy decisions based on molecular classification are necessary to confirm protocol utility and identify appropriate modifications.

HER2 in uterine serous carcinoma: Current state and clinical perspectives

Abstract Objectives Uterine cancer has the highest incidence and the second-highest mortality rate among gynecologic malignancies in the United States. Although uterine serous carcinoma (USC) represents less than 10% of endometrial carcinomas, it accounts for a disproportionate 50% of tumor relapses and 40% of endometrial cancer deaths. Over the past decade, clinical trials have focused on finding better treatments for this aggressive subtype of endometrial cancer, especially HER2-targeted therapy. Methods We conducted a literature search in PubMed to expand the understanding of HER2 in USC. Results HER2 has been established as an important biomarker with prognostic and therapeutic implications in USC. Intratumoral heterogeneity and lateral/basolateral membranous staining of HER2 as well as high discordance between HER2 immunohistochemistry and in situ hybridization are more common in USC than in breast carcinoma. Therefore, a universal HER2 testing and scoring system more suitable to endometrial cancer is needed and currently under investigation. Conclusions This review discusses the clinical perspective of HER2 overexpression/gene amplification in USC, the distinct HER2 staining pattern and the evaluation of HER2 in USC, the resistance mechanisms of HER2-targeted therapy in HER2-positive cancers, and likely areas of future investigation.

HER2 Gene Protein Assay: A Robust Tool for Evaluating HER2 Status and Intratumoral Heterogeneity in Endometrial Cancers

Abstract Objectives Human epidermal growth factor receptor 2 (HER2) status in endometrial cancer is usually determined by immunohistochemistry and/or in situ hybridization. We employed a novel HER2 gene protein assay (GPA) to simultaneously assesses HER2 gene amplification and protein expression in high-grade endometrial cancers. Methods We performed GPA in 180 endometrial cancers, including 106 serous carcinomas, 34 carcinosarcomas, and 40 mixed epithelial carcinomas. HER2 status was determined using the 2018 HER2 guidelines for breast carcinoma, and HER2 intratumoral heterogeneity (ITH) was examined. Clinicopathologic characteristics were collected and correlated with HER2 status. Results HER2 positivity was noted in 32% of serous carcinomas, significantly higher than in carcinosarcomas (5.9%) and mixed carcinomas (12.5%). HER2 ITH was detected in 32% of serous carcinomas, significantly greater than in carcinosarcomas (8.8%) and mixed carcinomas (10%). Patients with carcinosarcoma had a significantly lower overall survival than patients with serous or mixed epithelial carcinoma, but HER2 status caused no difference in survival in patients with serous carcinoma. Conclusions HER2 GPA can be used to accurately determine HER2 status in endometrial cancers and is a highly valuable tool for identifying HER2 heterogeneity.

Change of Practice Patterns Following an Educational Comment on Reports of Benign-Appearing Endometrial Cells in Papanicolaou Tests

Abstract Objectives Since the publication of our study demonstrating high negative predictive values (>99% for women in their 40s) of benign-appearing endometrial cells (nEMCs), we have begun to include an educational comment in Papanicolaou (Pap) test reports with nEMCs that recommends routine periodic screening for asymptomatic premenopausal women (APW). The current study evaluated how the inclusion of this comment has affected clinical practice patterns at our institution. Methods The 2017 to 2019 database identified 175 reports containing the educational comment in women aged 45 to 54 years with a follow-up time of 11 to 37 months. Data, including age, menopause status, symptoms, imaging, and outcome, were collected. The procedure rate and the impact of clinical modifiers were assessed. Results Thirty-seven (20.6%) patients had biopsies within 6 months, which decreased from 48.1% as we previously reported. All nine (5%) APW with biopsies triggered only by nEMCs had benign histopathology. The remaining 28 biopsied patients had abnormal bleeding or a thickened endometrium, or they were postmenopausal, including a 53-year-old patient with complex atypical hyperplasia. None of the 138 patients with conservative follow-up developed atypical/malignant lesions. Conclusions A qualifying educational note included in Pap reports significantly reduced follow-up biopsies in APW. Optimal follow-up of nEMCs should be based on relevant clinical modifiers.

Universal Mismatch Repair Protein Screening in Upper Tract Urothelial Carcinoma

AbstractObjectivesUniversal screening of upper tract urothelial carcinoma (UTUC) for Lynch syndrome by mismatch repair (MMR) protein immunohistochemistry (IHC) has been recommended by some investigators. Herein, we assess this recommendation retrospectively by simulating its performance on a retrospective, unselected cohort of UTUCs, with comparison to the established setting of colorectal and endometrial adenocarcinoma.MethodsWe assessed for complete loss of MMR protein (MLH1, MSH2, MSH6, and PMS2) IHC in 74 consecutive cases of UTUC and then tabulated clinical and pathologic factors. MMR findings from same-institution colorectal and endometrial adenocarcinomas were tabulated for comparison.ResultsWe observed loss of at least one MMR protein in 12% in our UTUC cohort (three MSH2/MSH6, three MSH6 only, one MLH1/PMS2, and two PMS2 only). Of these nine cases (seven males, two females, median age 67 years, five associated with colorectal adenocarcinoma), at least three (4% of the overall cohort) proved to be Lynch syndrome. Overall, MMR loss in UTUC was comparable to colorectal (11%; 50 of 471 cases) and endometrial (12%; 12 of 101 cases) adenocarcinomas.ConclusionsThe rate of MMR loss observed in UTUC was comparable to that in the established setting of colorectal and endometrial adenocarcinomas, supporting universal UTUC screening at our institution and others.

Congruence Between 1994 WHO Classification of Endometrial Hyperplasia and Endometrial Intraepithelial Neoplasia System

AbstractObjectivesTo assess congruence between World Health Organization (WHO) 1994 and endometrial intraepithelial neoplasia (EIN) classification systems of endometrial hyperplasia.MethodsSystematic review and meta-analysis were performed by searching electronic databases for studies that classified endometrial hyperplasia according to both WHO 1994 and EIN systems. Congruence was based on the rate of specimens classified as EIN in WHO categories, which should be virtually 0.000 in nonatypical hyperplasia (NAH) and 1.000 in atypical hyperplasia (AH). Subgroup analyses were performed based on architecture complexity.ResultsEight studies with 1,352 hyperplasias were included. Congruence with EIN criteria was fair in NAH (0.241) and moderate in AH (0.815). Subgroup analyses of NAH showed high congruence in simple NAH (0.065), null in complex NAH (0.517), null in simple AH (0.148), and high in complex AH (0.901).ConclusionsWHO 1994 system is not congruent with the EIN system and cannot be directly translated into a dual classification.

Assessment of microorganism detection in ThinPrep Papanicolaou tests utilizing the Hologic Genius Digital Diagnostics System

Abstract Objective The Hologic Genius Digital Diagnostics System (HGDDS) analyzes ThinPrep Papanicolaou (Pap) tests (TPPTs) to assist in detecting cervical lesions. The aim of this study was to determine the sensitivity of the HGDDS in identifying commonly diagnosed microorganisms in Pap tests. Methods A total of 305 TPPT cases were selected from Magee Women’s Hospital, University of Pittsburgh, consisting of 244 cases with microorganism diagnoses (a total of 262 cases of Actinomyces, Candida spp, herpes simplex virus [HSV], and Trichomonas) and 61 cases without microorganisms. Slides were scanned and then subjected to artificial intelligence (AI) analysis using the HGDDS and subsequently reviewed on a digital workstation by a cytologist, followed by a resident and a cytopathologist who made the final diagnoses. Results Diagnosis using the HGDDS demonstrated high sensitivity across all microorganisms (95.4%). Herpes simplex virus detection was comparatively lower (82.5%). Of the microorganisms, 85.2% were displayed in the first gallery of 30 images within row 5, 7.2% presented in the first gallery outside of row 5, and 7.6% presented in the hidden gallery of images. Among the 12 cases with missed diagnoses, 3 of 5 Candida spp and 3 of 7 HSV organisms were not presented within the 60 images selected by HGDDS. In another 6 cases, microorganisms were found within the 60 fields, but none were present in row 5. Conclusions Very high sensitivity was observed for TPPTs across 3 of 4 common microorganisms on the HGDDS, although sensitivity was relatively lower for detecting HSV. Understanding morphologic patterns of various microorganisms in detection misses by the HGDDS may help guide the implementation of AI-assisted cervical cancer screening systems.

Reduced cervical sampling of hysterectomy specimens with negative margins on conization: An opportunity to improve resource utilization

Abstract Objectives The current recommendation for hysterectomy specimens performed for cervical cancer following conization is that the entire cervix be submitted for histologic examination. Given the high cost of medical procedures and concerns regarding difficulties with laboratory staffing, we sought to evaluate the potential for selective histologic examination in this setting. Methods Post-conization hysterectomy cases were reviewed for the presence of residual disease in relation to the findings of the prior conization, with consideration of margin status. Residual disease was then assessed for clinical significance. The number of submitted blocks was recorded and the associated costs were estimated. Results Among 32 cases with invasive carcinoma, only cases with margins positive for invasive carcinoma on the conization specimen had residual invasion in the hysterectomy (n = 7), and there were no upgrades due to subtle microscopic disease; 1 case had a change in pathologic stage from pT1b1 to pT2b due to parametrial involvement in the setting of a grossly apparent lesion. Among 20 cases performed following a diagnosis of dysplasia, none were upgraded to invasive carcinoma. Based on protocol-based submission of the entire cervix, 16 blocks of cervix were submitted on average (range, 4-41). Conclusions We estimate that representative sections from each cervical quadrant would save approximately 2 work hours for laboratory staff per case and up to 6 hours for larger cases, reducing costs for the laboratory accordingly. Selective cervical sampling in the setting of negative margins on conization provides an opportunity for improved resource utilization without compromising patient care; as this is a small study, confirmation of these findings in a larger number of cases may be warranted. Additional studies are necessary to determine what other contexts in surgical pathology could benefit from a similar reductive approach.

The significance of extensive HPV genotyping for cervical high-grade neoplasia among women with atypical glandular cells

Abstract Objectives To examine the associated risk of cervical intraepithelial neoplasm grade 3+ (CIN3+) lesions in patients with AGC and extensive human papillomavirus (HPV) genotyping. Methods Cases with atypical glandular cell (AGC) interpretation on a Papanicolaou (Pap) test were identified along with associated extensive HPV genotyping and histologic follow-up results. Results Within this cohort of 469,694 Pap tests, 0.4% were diagnosed as AGCs. In total, 1267 cases had concurrent high-risk HPV (hrHPV) genotyping, and 40.3% were hrHPV positive. The percentage of AGC cases with cervical CIN3+ on histologic follow-up was 52.2% when hrHPV was positive, whereas it was 4.9% with a negative hrHPV result. The top 5 hrHPV genotypes associated with cervical CIN3+ in this cohort were HPV16, HPV18, HPV58, HPV52, and HPV33. Indeed, 92.8% of the hrHPV-associated CIN3+ lesions identified in this cohort were positive for at least one of these HPV genotypes. The sensitivity of detecting cervical CIN3+ lesions was 85.6% with the top 5 hrHPV genotypes (HPV16/18/58/52/33) and only increased to 89.0% when the additional 12 genotypes were included. Conclusions In patients with an AGC Pap, the risk of having a cervical CIN3+ lesion is greatly increased by positivity for hrHPV types 16, 18, 58, 52, and/or 33. Incorporating comprehensive HPV genotyping into AGC cytology allows for refined risk stratification and more tailored management strategies.

Characteristics and utility of high-resolution optical coherence microscopy images of endocervical canal lesions

Abstract Objectives To investigate optical coherence microscopy (OCM) imaging features and the application value of these high-resolution images for identifying endocervical canal lesions (ECLs), which is a clinical dilemma in cervical cancer screening programs. Methods In total, 520 OCM images were obtained by scanning the cervical canal lesions with an ultra-high-resolution OCM system (204 specimens from 73 patients). The OCM morphologic characteristics of ECLs were observed and summarized, and then 3 researchers performed a diagnostic test of OCM images of cervical canal lesions. The accuracy, sensitivity, specificity, positive predictive value, negative predictive value, 95% confidence interval of each parameter, and interinvestigator agreement (κ) were calculated. Results Normal endocervix, cysts, squamous metaplasia, high-grade squamous intraepithelial lesions involving glands, and invasive carcinoma had distinct OCM characteristics, which correlated well with corresponding H&E histologic sections. The accuracy, sensitivity, and specificity of the 3 researchers were 90.6%, 89.3% (95% CI, 86.5%-91.7%) and 91.6% (95% CI, 89.2%-93.5%), respectively. The positive predictive value was 90.1% (95% CI, 87.3%-92.4%), and the negative predictive value was 90.9% (95% CI, 88.5%-92.9%), with almost perfect agreement (κ = 0.874). Conclusions The application of the OCM system in cervical canal lesions is feasible and could help improve detection of occult ECLs in cervical cancer screening programs. This study lays the foundation for further research on OCM in cervical canal lesions in vivo, which also has a potential impact on projecting pathologic evaluation beyond what is currently possible, perhaps globally.

Impact of the Genius Digital Diagnostics System on workflow and accuracy compared with the ThinPrep Imaging System for review of ThinPrep Papanicolaou tests

Abstract Objective In this study, we compared the workflow of the Genius Digital Diagnostics System (Hologic, Inc) with our current workflow based on the ThinPrep Imaging System (Hologic, Inc) to assess potential efficiencies associated with digitalization of Papanicolaou screening. Methods Each step of the current workflow (glass slide movement and slide review) and the experimental workflow were documented. Substantial workflow efficiencies were associated with the reduction of glass slide movement observed with the experimental workflow of the Genius system compared with the ThinPrep system. Results The ThinPrep-based workflow required more than 5 hours of hands-on time at specific synchronized times throughout the day, whereas the hands-on time of the experimental Genius Digital Diagnostics System was just over an hour and allowed glass movement at flexible times. In addition to these workflow efficiencies, the Genius Digital Diagnostics System resulted in much shorter review times (70.1 seconds) than the ThinPrep Imaging system (138.0 seconds) while maintaining similar agreement to the sign-out diagnosis. Conclusions This study demonstrated that implementing a Genius Dx-based workflow may result in substantial efficiency gains, which can mitigate workforce shortages and improve turnaround time without compromising screening accuracy.

Amplification of MDM2 and Loss of p16 Expression: Do They Have a Role in Malignant Transformation of Ovarian Brenner Tumor?

Abstract Objectives To review the significance of MDM2 and cyclin D1 expression and loss of p16 expression in malignant and borderline Brenner tumors (BTs) of the ovary. Methods We describe 2 new cases of ovarian BT, 1 malignant and 1 borderline. We studied MDM2, p16, and cyclin D1 expression by immunohistochemistry in the benign, borderline, and malignant components of these 2 cases and in 5 additional cases of benign BT. We also reviewed and summarized the literature on the clinical, immunohistochemical and molecular characteristics of borderline and malignant BTs (BdBTs and MBTs). Results Nuclear expression of MDM2 was seen only in the MBT. Loss of p16 expression was seen in both BdBT and MBT. Cyclin D1 expression was in proportion to the degree of malignancy. Amplification of MDM2, loss of CDKN2A (p16-encoding gene), and amplification of CCND1 (cyclin D1–encoding gene) were confirmed by commercial next-generation sequencing in the case of MBT. Conclusions We are the first to report immunohistochemical expression of MDM2 in an MBT. Amplification of MDM2 and loss of p16 expression may have a role in malignant transformation of BT.

Interobserver reproducibility of cervical histology interpretation with and without p16 immunohistochemistry

Abstract Objectives Histopathological diagnosis of colposcopically identified cervical lesions is a critical step for the recognition of cervical cancer precursors requiring treatment. Although there have been efforts to standardize the histologic diagnosis of cervical biopsy specimens, in terms of terminology and use of biomarkers, there is no uniform approach in the pathology community. Adjunctive p16 immunohistochemistry (IHC) can highlight precancer diagnoses, with use recommendations outlined by the Lower Anogenital Squamous Terminology project. Methods We assessed the diagnostic reproducibility of cervical histopathological biopsy specimens with and without p16 staining among 2 expert pathologists. Results Interpretation of p16 IHC as positive vs negative was highly reproducible (92.5% agreement, κ = 0.85); greater variation was seen in the choice of which biopsy specimens required adjunctive p16 staining (78.0% agreement, κ = 0.43). Adjunctive p16 IHC did not significantly increase diagnostic agreement under multitiered grading systems (benign vs cervical intraepithelial neoplasia [CIN] 1/low-grade squamous intraepithelial lesion vs atypical squamous metaplasia vs CIN2/high-grade squamous intraepithelial lesion [HSIL] vs CIN3/HSIL-CIN3 vs cancer) (65.5% agreement, κ = 0.56 without p16; 70.0% agreement, κ = 0.58 with p16). However, when dichotomizing diagnoses based on clinical management (less than HSIL vs HSIL+), diagnostic agreement increased with p16 IHC (90.5% agreement, κ = 0.79 without p16; 92.0% agreement, κ = 0.84 with p16). For biopsy specimens taken from women positive for human papillomavirus (HPV) type 16, agreement was similar with or without adjunctive p16 (κ = 0.80 without p16; κ = 0.78-0.80 with p16). In contrast, p16 IHC substantially improved diagnostic agreement for cervical biopsy specimens taken from women positive for other high-risk HPV strains, producing improvements in κ from 0.03 to 0.24. Conclusions Adjunctive p16 immunostaining provides useful information in the evaluation of cervical biopsies for precancer. In our study, we have demonstrated that it is highly reproducible between 2 pathologists, although the decision of which biopsies warrant its use is less so. Furthermore, although p16 IHC showed a limited increase in diagnostic reproducibility for all biopsies included in our study, it did demonstrate a more sizable gain in biopsies negative for HPV 16 but positive for other high-risk genotypes. Further studies are needed to clarify the role of p16 IHC and how it can be optimized for the detection of cervical precancer, particularly in HPV-vaccinated populations where types other than HPV 16 are relatively more important.

Risk Assessment of Human Papillomavirus–Positive Cytology-Negative Cervical Cancer Screening in Black and White Women

Abstract Objectives As we move toward human papillomavirus (HPV) only as the preferred cervical cancer screening method, we performed a retrospective analysis of Black and White women with negative cytology (Papanicolaou negative [PAPneg]) and positive high-risk HPV (hrHPV) (HPVpos) results and determined follow-up. Methods We searched our pathology data system for patients with PAPneg/HPVpos results (2017-2019). Follow-up data were reviewed (39 months), and a comparison among race was performed. Results In total, 1,728 patients were identified (Black, 53%; White, 47%). Twenty-nine percent of the patients had no follow-up with no difference among the races. HPV 16 was more common among Whites (P < .01), while non-16/18 hrHPV was more common among Black patients (P = .01). A total of 30 (3.3%) Black and 26 (3.2%) White patients were diagnosed with cervical intraepithelial neoplasia grade 2/3 (CIN 2/3). More White women were diagnosed on biopsy alone (negative endocervical curettage) compared with Black women (20 vs 9, P < .01). Meanwhile, there were 21 Black and 6 White women with CIN 2/3 on endocervical curettage (P = .01). Conclusions Follow-up of women with PAPneg/HPVpos remains a challenge. There was no disparity in follow-up when cohorts were compared. However, Black women had higher numbers of high-grade intraepithelial lesions on endocervical curettage.

Juvenile Granulosa Cell Tumors of the Ovary

Abstract Objective To explore the clinical and pathologic features of ovarian juvenile granulosa cell tumors (JGCTs). Methods Clinical data, histopathologic observations, immunohistochemical results, FOXL2 mutation status, and follow-up information of 7 JGCT cases were studied. Results The patients most commonly presented with abdominal distension and pain (5 cases), followed by precocious puberty (1 case) and a pelvic mass (1 case). Six patients had stage I disease, and 1 had stage IV disease. The microscopic examinations typically showed lobular growth punctuated by variably sized and shaped follicles. Rare features included a reticular-cystic appearance mimicking a yolk sac tumor (2 cases), a lobular appearance similar to a sclerosing stromal tumor (1 case), strands and cords (1 case), pseudopapillary appearance (2 cases), spindle cell appearance (1 case), microcystic appearance (1 case), hobnail cells (1 case), and rhabdomyoid cells (1 case). No FOXL2 mutation was encountered. After a median follow-up of 53 months, only 1 patient with a strongly diffuse TP53-positive tumor died of the disease, and 2 successfully had babies. Conclusions JGCT is a rare neoplasm with a wide morphologic spectrum and is easily confused with other tumors. Familiarity with the characteristics, rare atypical appearances, and immunohistochemical results may aid in obtaining a correct diagnosis.

Morphologic and Immunocytochemical Features of High-Grade Serous Carcinoma of Ovary in Ascitic Fluid Effusion and Fine-Needle Aspiration Cytology

Abstract Objectives High-grade serous carcinoma (HGSC) is the most common ovarian malignancy. The role of cytopathology in obtaining tissue diagnosis before institution of neoadjuvant chemotherapy (NACT) was evaluated. Methods All histopathology-proven HGSC specimens between 2015 and 2018 with prior cytopathologic diagnosis by ascitic fluid evaluation or fine-needle aspiration (FNA) of ovarian mass were reviewed with cell block immunocytochemistry for CK7, CK20, PAX8, WT1, and p53. Results Of 288 cases of HGSC, pre-NACT cytology diagnosis was established in 32% (93/288), with specific HGSC diagnoses made on ascitic fluid in 88% (82/93) and by ovarian mass FNA in 12% (11/93). The ascitic fluid showed moderate/high cellularity with papillary clusters in 76% (71/93) cases. Cell block immunocytochemistry showed tumor cells positive for CK7, PAX8, and WT1. p53 showed mutant or null-type positivity in 65% (33/51) and 33% (17/51) of cases, respectively, with 100% concordance with subsequent histopathology specimens. Poor/intermediate response to chemotherapy was shown in 75% of cases. Conclusions Combined assessment of cytomorphology, cell block histomorphology, and ancillary immunohistochemical testing, including PAX8, WT1, and p53, allows for specific pre-NACT diagnoses of HGSC in ascitic fluid and ovarian FNA cytology. This practice allows for initiation of chemotherapy and diminution of disease burden prior to definitive surgical therapy.

Contributions of Liquid-Based (Papanicolaou) Cytology and Human Papillomavirus Testing in Cotesting for Detection of Cervical Cancer and Precancer in the United States

AbstractObjectivesGiven the recent debate challenging the contribution of cytology in cervical screening, we evaluated results of liquid-based cytology (LBC) and human papillomavirus (HPV) testing in cotesting preceding cervical cancer (CxCa) and precancer diagnoses in a national, heterogeneous population.MethodsWe assessed the results of cotesting, performed by Quest Diagnostics, in 13,633,071 women 30 years and older, tested 2010 to 2018. Cotest results preceding CxCa or precancer diagnoses were analyzed and stratified by histopathology.ResultsAmong all screening results, 1,615 cotests preceded 1,259 CxCa diagnoses, and 11,164 cotests preceded 8,048 cervical precancer diagnoses. More women who were subsequently diagnosed with CxCa within 1 year were identified by the LBC result than by the HPV result (85.1%, 1,015/1,193 vs 77.5%, 925/1,193). Among all women with CxCa, the overall rate of nondetection was 13.1% (212/1,615) for cotesting results (LBC negative/HPV negative) and this rate increased substantially when testing exceeded 12 months compared to within 1 year prediagnosis of either CxCa or precancer.ConclusionsAnalysis of 9-year cotest results from a national reference laboratory confirms the value of LBC element in cotesting. This supports that LBC/HPV cotesting enhances screening for the identification of CxCa in women 30 years and older, more so than LBC or HPV alone within cotesting.

Effects of Implementing the Dual Papanicolaou Test Interpretation of ASC-H and LSIL Following Bethesda 2014

Abstract Objectives To evaluate the impact of implementing the dual interpretation of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and low-grade squamous intraepithelial lesion (LSIL) after the Bethesda System 2014 and to compare it with other indeterminate interpretations. Methods Rates of high-risk human papillomavirus (HPV) positivity and histologic follow-up and the proportion of women with high-grade squamous intraepithelial lesion on histologic follow-up were compared for the combined interpretation of ASC-H and LSIL (ASCHL) and the categories of LSIL, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) and ASC-H. Results The percentage of ASCHL HPV-positive cases (86.0%) was similar to that of LSIL-H but significantly higher in comparison to that of ASC-H. The rates of cervical intraepithelial neoplasia grade 2 or higher (CIN 2+) and CIN 3+ for ASCHL (29.6% and 3.6%, respectively) were similar to those of LSIL-H and ASC-H. When stratified by HPV test results, the proportions of patients with CIN 2+ and CIN 3+ remained statistically similar to those with ASCHL and with LSIL-H and ASC-H. Conclusions Considering the similar risks of CIN 2+ and CIN 3+ for ASCHL and ASC-H, having a separate category of ASCHL for reporting cervical cytology appears to be redundant.

Impact of the Current US Preventive Services Task Force Recommendations for Cervical Cancer Screening in Young Women 21 to 29 Years Old

Abstract Objectives In 2012, the US Preventive Services Task Force decreased the recommended frequency of cervical cytology screening to once every 3 years and recommended against testing women younger than 21 years regardless of sexual history. We evaluated the impact of this in 21 to 29-year-old women at a tertiary care academic medical center in 2011 and 2017. Methods We retrospectively analyzed Papanicolaou test results at two time points in 21- to 29-year-old women. Results There was a decrease in the number of high-grade lesions in 21- to 25-year-old women (odds ratio [OR], 0.36) from 2011 to 2017. Within the 26- to 29-year-old patient group, there was a trend toward a higher percentage of high-grade squamous intraepithelial lesion (HSIL) in 2017 compared to 2011 on cytology, which did not reach statistical significance (OR, 1.46). However, follow-up histologic specimens showed a higher percentage of HSIL in 2017 compared to 2011 in this age group (OR, 2.16). Conclusions Our findings suggest that the cervical cancer screening guidelines introduced in 2012 have not had a detrimental impact on the outcomes of cervical cancer screening for 21- to 25-year-old women. However, we need to continue monitoring the effects of decreased screening in 26- to 29-year-old women.

Morules and β-catenin predict POLE mutation status in endometrial cancer: A pathway to more cost-effective diagnostic procedures

Abstract Objectives The characterization of DNA polymerase epsilon (POLE) mutations has transformed the classification of endometrial endometrioid carcinomas (EECs), highlighting the need for efficient identification methods. This study aims to examine the relationship between distinct morphologic features—namely, squamous morules and squamous differentiation (SD), as well as β-catenin expression—and the POLE mutation status in endometrial cancer (EC). Methods Our study included 35 POLE-mutated (POLEmut) EC cases and 395 non-POLEmut EEC cases. Results Notably, we observed no presence of morules in POLEmut cases, while SD was identified in 20% of instances. Conversely, morules and SD were identified in 12.7% and 26.1% of non-POLEmut EC cases, respectively, with morules consistently linked to a POLE wild-type status. The nuclear β-catenin expression is typically absent in tumors with wild-type POLE (wt-POLE) status. Conclusions Our findings suggest that the presence of either morules or nuclear β-catenin expression in EEC could practically rule out the presence of POLE mutations. These morphologic and immunohistochemical features can be used as preliminary screening tools for POLE mutations, offering significant savings in time and resources and potentially enhancing clinical decision-making and patient management strategies. However, further validation in larger, multi-institutional studies is required to fully understand the implications of these findings on clinical practice.

Cervical Screening Performance

Revision of interpretation criteria to define microsatellite instability in mismatch repair–deficient neoplasms with subtle electropherogram changes

Abstract Objectives To improve analytic performance characteristics of a microsatellite instability (MSI-V1.2) assay in endometrial cancers (ECs). Methods Nonneoplastic and neoplastic DNA from colorectal cancers (CRCs) and ECs were compared to define MSI by calculating base shifting of the highest peak and the 5% peak (the leftmost peak with a peak height >5% of the highest peak). Results We first demonstrated highly precise sizing by capillary electrophoresis. However, relative intensity of multiple peaks, characteristic for microsatellite amplicons, might show a 1-base, but not a 2-base or more, shift of the highest or 5% peak among duplicate runs of nonneoplastic DNA. This inherent bias of the polymerase chain reaction–based MSI assay may lead to false-positive interpretation if MSI was defined by a 1-base shift or more. Subsequently, MSI was evaluated by a 2-base shift or more of the highest peak (original criteria) or a 2-base shift or more of either the highest or 5% peak (revised criteria) without subjective interpretation of a subtle change of electropherogram configuration (the so-called shoulder pattern). While both criteria were highly sensitive in CRCs, the revised criteria improved sensitivity (83% vs 67%) and accuracy (89% vs 79%) and maintained 100% specificity in ECs. Conclusions The revised criteria provided sensitive, specific, and objective interpretation to examine subtle changes of MSI.

p53abn high-risk endometrial cancer with PPP2R1A mutation might not benefit from adjuvant chemotherapy

Abstract Objectives Recent studies have found that high-risk endometrial cancer frequently harbors mutations in tumor suppressor genes PPP2R1A and FBXW7. This study aimed to explore the prognostic utility of these genes and their potential to predict benefit from adjuvant treatment. Methods Tissue samples of 121 patients with high-risk endometrial cancer were collected. PPP2R1A, FBXW7, and POLE exonuclease domain mutations were detected using Sanger sequencing, while mismatch repair proteins and p53 expression were tested by immunohistochemistry. Results PPP2R1A and FBXW7 mutations were detected in 11.6% and 21.5% of tumors, respectively. PPP2R1A mutations occurred more frequently in nonendometrioid, high-grade, and advanced-stage tumors and were strongly correlated with poor prognosis. Importantly, PPP2R1A mutations were more frequent in the p53 abnormal (p53abn) subgroup than in the other 3 molecular subgroups (P = .011). In addition, patients with p53abn, PPP2R1A-mutated tumors showed poor prognosis regardless of whether they received adjuvant chemotherapy. In contrast, patients with p53abn, PPP2R1A wild-type tumors exhibited statistically significantly longer survival after adjuvant chemotherapy (P = .022). Similar associations were observed in the patients with p53abn, FBWX7 wild-type tumors. Conclusions PPP2R1A and FBXW7 status may be related to the current adjuvant chemotherapy outcome, particularly in endometrial cancer with the p53abn subtype. Thus, incorporating PPP2R1A and FBXW7 detection in the stratification and treatment decision-making process may be helpful.

Immunohistochemical Assessment of β-Catenin in Endometrial Carcinoma

Endometrial Cancer Characteristics and Risk of Recurrence

Abstract Objectives To describe clinicopathologic characteristics and survival outcomes of endometrial adenocarcinomas stratified by mismatch repair (MMR) status. Methods Single-institution, retrospective study of all women with endometrioid adenocarcinomas treated from January 2012 through December 2017. Patients were categorized into one of three groups based on MMR testing: intact MMR expression (MMR+), probable MMR mutation (MMR–), or MLH1 hypermethylation (hMLH1+). Demographics, pathologic characteristics, recurrence rates, and survival differences were analyzed. Results In total, 316 women were included in the analysis: 235 (74.4%) patients in the MMR+ group, 10 (3.1%) in the MMR– group, and 71 (22.5%) in the hMLH1+ group. Patients with hMLH1+ were significantly older, exhibited higher-grade histology and presence of lymphovascular space invasion, and were more likely to have received adjuvant treatment. The early stage hMLH1+ patients were more likely to recur (15.3% hMLH1+ vs 2.3% MMR+ vs 12.5% MMR–, P < .001). Hypermethylation remained a significant predictor of recurrence in multivariable analysis (odds ratio, 5.09; 95% confidence interval [CI], 1.54-16.86; P = .008). Recurrence-free survival was significantly reduced in early stage hMLH1+ (hazard ratio, 7.40; 95% CI, 2.80-21.62; P < .001). Conclusions Women with hMLH1+ endometrial cancer have worse prognostic features and recur more frequently, even in patients traditionally considered low risk for recurrence.

Sentinel Nodal Metastasis Detection in Endometrial Carcinoma With Microcystic, Elongated, and Fragmented (MELF) Pattern by Cytokeratin Immunostaining

Abstract Objectives The microcystic, elongated, and fragmented (MELF) pattern of myoinvasion in endometrial carcinoma (EC) is associated with an increased risk of lymph node metastasis. Our aim is to assess the role of cytokeratin immunohistochemical (IHC) stains in detecting sentinel nodal metastasis in MELF pattern tumors. Methods We recovered 19 MELF pattern EC hysterectomies with lymphadenectomy from our files. Negative nodes were subjected to cytokeratin AE1/AE3 IHC. Ten additional cases with sentinel lymph node (SLN) biopsies primarily assessed by IHC were also analyzed. Results Of the 19 cases of EC, 6 had positive lymph nodes based on H&E-stained sections at the time of their initial diagnosis. With the addition of IHC stains, 8 previously negative cases were found to have node metastases, and 3 of these were SLNs. Among the 10 cases primarily assessed by IHC, 5 had malignant cells in their SLNs. Conclusions Cytokeratin IHC staining detected malignant cells in 9 of 16 cases with SLNs in our sample of women with MELF pattern of myoinvasion. Immunohistochemical stains should be routinely performed on SLNs from all MELF-positive cases to detect occult lymph node metastases and isolated tumor cells.