Interobserver reproducibility of cervical histology interpretation with and without p16 immunohistochemistry.

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.

Racial and Ethnic Disparities in Cervical Cancer Incidence, Survival, and Mortality by Histologic Subtype.

PURPOSE: We conducted an integrated population-based analysis of histologic subtype-specific cervical cancer incidence, survival, and incidence-based mortality by race and ethnicity, with correction for hysterectomy prevalence. METHODS: Using the SEER 21 and 18 registries, we selected primary cases of malignant cervical cancer diagnosed among women ≥ 15 years. We evaluated age-adjusted incidence rates among cases diagnosed between 2000 and 2018 (SEER21) and incidence-based mortality rates among deaths from 2005 to 2018 (SEER18), per 100,000 person-years. Rates were stratified by histologic subtype and race/ethnicity (incidence and mortality), and stage, age at diagnosis, and county-level measures of social determinants of health (incidence only). Incidence and mortality rates were corrected for hysterectomy using data from the Behavioral Risk Factor Surveillance System. We estimated 5-year relative survival by histologic subtype and stratified by stage at diagnosis. RESULTS: Incidence rates of cervical squamous cell carcinoma were highest in Black and Hispanic women, while incidence rates of cervical adenocarcinoma (ADC) were highest among Hispanic and White women, particularly for localized ADC. County-level income and education variables were inversely associated with squamous cell carcinoma incidence rates in all racial and ethnic groups but had less influence on ADC incidence rates. Black women had the highest overall mortality rates and lowest 5-year relative survival, irrespective of subtype and stage. Disparities in survival were particularly pronounced for Black women with regional and distant ADC, compared with other racial/ethnic groups. CONCLUSION: Although Black women are less likely to be diagnosed with ADC compared with all other racial/ethnic groups, they experience the highest mortality rates for this subtype, likely attributed to the poor survival observed for Black women with regional and distant ADC.

Distinct mechanism of cervical cancer cell death caused by the investigational new drug SHetA2.

Drug-targetable vulnerabilities of cancer cells include their dependence on heat shock proteins (HSPs) to support elevated mitochondrial metabolism and counteract cell death factors. The investigational new drug SHetA2 targets these vulnerabilities in ovarian and endometrial cancer cells by disrupting complexes of the mortalin HSP with its client proteins (mitochondrial support proteins, metabolic enzymes, p53) leading to mitochondrial leakage of cytochrome c and apoptosis-inducing factor (AIF), and caspase-dependent apoptosis. Our objective was to evaluate the roles of mitochondrial damage and another SHetA2-target HSP protein, cytoplasmic heat shock cognate 70 (hsc70), in the mechanism of SHetA2 killing of cervical cancer cells. Cervical cancer cells responded to SHetA2 with excessive mitophagy that did not deter AIF leakage into the cytoplasm. Then, hsc70 was unable to prevent cytoplasmic AIF nuclear translocation and promotion of DNA damage and cell death, because SHetA2 disrupted hsc70/AIF complexes. The Cancer Genome Atlas analysis found that overexpression of hsc70, but not mortalin, was associated with worse cervical cancer patient survival. Use of specific inhibitors documented that AIF and mitophagy, but not caspases, contributed to the mechanism of SHetA2-induced cell death in cervical cancer cells. As validation, excessive mitophagy and lack of caspase activation were observed in SHetA2-inhibited xenograft tumors.

Diagnostic Cytopathology of Peritoneal Washings.

Peritoneal washings used for cytologic evaluation are collected at the outset of surgical exploration of women with gynecologic cancers to assist in determining extent of disease and follow-up therapy. While there are similarities to ascites, these samples have differences that must be recognized in order to avoid false positive interpretations. Non-neoplastic mesothelial alterations including heterogeneous reactive changes, endosalpingiosis , endometriosis and tumor rupture are typically not seen in ascites samples but can be seen in peritoneal washings from women with malignancies that have not extended to the peritoneal cavity. Awareness of these potential pitfalls and knowledge of the associated tumor type will facilitate accurate interpretation. When these caveats are recognized, peritoneal washing cytology results are a useful adjunct in helping to determine patient follow-up in women with gynecologic malignancies.

Cervical Precancers and Cancers Attributed to HPV Types by Race and Ethnicity: Implications for Vaccination, Screening, and Management.

BACKGROUND: Racial and ethnic variations in attribution of cervical precancer and cancer to human papillomavirus (HPV) types may result in different HPV vaccine protection, screening test coverage, and clinical management. METHODS: Pooling data from 7 US studies, we calculated the proportional attribution of precancers and cancers to HPV types using HPV DNA typing from diagnosis. All statistical tests were 2-sided. RESULTS: For all racial and ethnic groups, most cases of cervical intraepithelial neoplasia grade 3 (CIN3) (84.2%-90.8% of 5526) and squamous cell carcinoma (SCC) (90.4%-93.8% of 1138) were attributed to types targeted by the 9-valent vaccine. A higher proportion of CIN3s were attributed to nonvaccine HPV types among non-Hispanic Black women (15.8%) compared with non-Hispanic Asian or Pacific Islander (9.7%; P = .002), non-Hispanic White (9.2%; P < .001), and Hispanic (11.3%; P = .004) women. The proportion of SCCs attributed to 9-valent types was similar by race and ethnicity (P = .80). A higher proportion of CIN3s were attributed to nonvaccine HPV35 among non-Hispanic Black (9.0%) compared with non-Hispanic Asian or Pacific Islander (2.2%), non-Hispanic White (2.5%), and Hispanic (3.0%; all P < .001) women. Compared with CIN3, the proportion of SCCs attributed to HPV35 among non-Hispanic Black women (3.2%) was lower and closer to other groups (0.3%-2.1%; P = .70). CONCLUSION: The 9-valent HPV vaccine will prevent nearly all cervical precancers and invasive cancers among major racial and ethnic groups in the United States. Adding HPV35 to vaccines could prevent a small percentage of CIN3s and SCCs, with greater potential impact for CIN3s among Black women. HPV screening tests target high-risk HPV types, including HPV35. Future genotyping triage strategies could consider the importance of HPV35- and other HPV16-related types.

HPV51-associated Leiomyosarcoma: A Novel Class of TP53/RB1-Wildtype Tumor With Predilection for the Female Lower Reproductive Tract.

Inactivating mutations in tumor suppressor genes TP53 and RB1 are considered central drivers in leiomyosarcomas (LMSs). In high-risk human papillomavirus (HPV)-related tumors, a similar functional outcome is achieved through oncoproteins E6 and E7, which inactivate the p53 and RB1 proteins, respectively. Here, we hypothesized that HPV infection could provide an alternative mechanism for tumorigenesis in a subset of TP53/RB1-wildtype LMS. We evaluated tumor samples from 2585 consecutive unique patients carrying a diagnosis of gynecologic or soft tissue LMS. Tumor DNA and available RNA were analyzed by hybrid-capture-based next-generation sequencing/comprehensive genomic profiling of 406 genes and transcripts (FoundationOneHeme). Of the initial 2585 cases, we excluded 16 based on the presence of molecular alterations that are considered defining for sarcomas other than LMS. In the remaining 2569 cases, we searched for LMS that were TP53/RB1-wildtype (n=486 of 2569; 18.9%). We also searched LMS tumors for HPV sequences that we then classified into genotypes by de novo assembly of nonhuman sequencing reads followed by alignment to the RefSeq database. Among TP53/RB1-wildtype LMS, we identified 18 unique cases harboring HPV sequences. Surprisingly, most (n=11) were HPV51-positive, and these 11 represented all HPV51-positive tumors in our entire LMS database (n=11 of 2569; 0.4%). The absence of genomic alterations in TP53 or RB1 in HPV51-positive LMS represented a marked difference from HPV51-negative LMS (n=2558; 0% vs. 72% [P<0.00001], 0% vs. 53% [P=0.0002]). In addition, compared with HPV51-negative LMS, HPV51-positive LMS were significantly enriched for genomic alterations in ATRX (55% vs. 24%, P=0.027) and TSC1 (18% vs. 0.6%, P=0.0047). All HPV51-positive LMS were in women; median age was 54 years at surgery (range: 23 to 74 y). All known primary sites were from the gynecologic tract or adjacent anogenital area, including 5 cases of vaginal primary site. Histology was heterogeneous, with evaluable cases showing predominant epithelioid (n=5) and spindle (n=5) morphology. In situ hybridization confirmed the presence of high-risk HPV E6/E7 mRNA in tumor cells in three of three evaluable cases harboring HPV51 genomic sequences. Overall, in our pan-LMS analysis, HPV reads were identified in a subset of TP53/RB1-wildtype LMS. For all HPV51-associated LMS, the striking absence of any detectable TP53 or RB1 mutations and predilection for the female lower reproductive tract supports our hypothesis that high-risk HPV can be an alternative tumorigenic mechanism in this distinct class of LMS.

Automated Cervical Digitized Histology Whole-Slide Image Analysis Toolbox.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) is regarded as a potential precancerous state of the uterine cervix. Timely and appropriate early treatment of CIN can help reduce cervical cancer mortality. Accurate estimation of CIN grade correlated with human papillomavirus type, which is the primary cause of the disease, helps determine the patient's risk for developing the disease. Colposcopy is used to select women for biopsy. Expert pathologists examine the biopsied cervical epithelial tissue under a microscope. The examination can take a long time and is prone to error and often results in high inter-and intra-observer variability in outcomes. METHODOLOGY: We propose a novel image analysis toolbox that can automate CIN diagnosis using whole slide image (digitized biopsies) of cervical tissue samples. The toolbox is built as a four-step deep learning model that detects the epithelium regions, segments the detected epithelial portions, analyzes local vertical segment regions, and finally classifies each epithelium block with localized attention. We propose an epithelium detection network in this study and make use of our earlier research on epithelium segmentation and CIN classification to complete the design of the end-to-end CIN diagnosis toolbox. RESULTS: The results show that automated epithelium detection and segmentation for CIN classification yields comparable results to manually segmented epithelium CIN classification. CONCLUSION: This highlights the potential as a tool for automated digitized histology slide image analysis to assist expert pathologists.

Accuracy and Efficiency of Deep-Learning-Based Automation of Dual Stain Cytology in Cervical Cancer Screening.

BACKGROUND: With the advent of primary human papillomavirus testing followed by cytology for cervical cancer screening, visual interpretation of cytology slides remains the last subjective analysis step and suffers from low sensitivity and reproducibility. METHODS: We developed a cloud-based whole-slide imaging platform with a deep-learning classifier for p16/Ki-67 dual-stained (DS) slides trained on biopsy-based gold standards. We compared it with conventional Pap and manual DS in 3 epidemiological studies of cervical and anal precancers from Kaiser Permanente Northern California and the University of Oklahoma comprising 4253 patients. All statistical tests were 2-sided. RESULTS: In independent validation at Kaiser Permanente Northern California, artificial intelligence (AI)-based DS had lower positivity than cytology (P < .001) and manual DS (P < .001) with equal sensitivity and substantially higher specificity compared with both Pap (P < .001) and manual DS (P < .001), respectively. Compared with Pap, AI-based DS reduced referral to colposcopy by one-third (41.9% vs 60.1%, P < .001). At a higher cutoff, AI-based DS had similar performance to high-grade squamous intraepithelial lesions cytology, indicating a risk high enough to allow for immediate treatment. The classifier was robust, showing comparable performance in 2 cytology systems and in anal cytology. CONCLUSIONS: Automated DS evaluation removes the remaining subjective component from cervical cancer screening and delivers consistent quality for providers and patients. Moving from Pap to automated DS substantially reduces the number of colposcopies and also achieves excellent performance in a simulated fully vaccinated population. Through cloud-based implementation, this approach is globally accessible. Our results demonstrate that AI not only provides automation and objectivity but also delivers a substantial benefit for women by reduction of unnecessary colposcopies.

EpithNet: Deep Regression for Epithelium Segmentation in Cervical Histology Images.

BACKGROUND: Automated pathology techniques for detecting cervical cancer at the premalignant stage have advantages for women in areas with limited medical resources. METHODS: This article presents EpithNet, a deep learning approach for the critical step of automated epithelium segmentation in digitized cervical histology images. EpithNet employs three regression networks of varying dimensions of image input blocks (patches) surrounding a given pixel, with all blocks at a fixed resolution, using varying network depth. RESULTS: The proposed model was evaluated on 311 digitized histology epithelial images and the results indicate that the technique maximizes region-based information to improve pixel-wise probability estimates. EpithNet-mc model, formed by intermediate concatenation of the convolutional layers of the three models, was observed to achieve 94% Jaccard index (intersection over union) which is 26.4% higher than the benchmark model. CONCLUSIONS: EpithNet yields better epithelial segmentation results than state-of-the-art benchmark methods.

Association of HPV35 with cervical carcinogenesis among women of African ancestry: Evidence of viral-host interaction with implications for disease intervention.

HPV35 has been found in only ∼2% of invasive cervical cancers (ICC) worldwide but up to 10% in Sub-Saharan Africa, warranting further investigation and consideration of impact on preventive strategies. We studied HPV35 and ethnicity, in relation to the known steps in cervical carcinogenesis, using multiple large epidemiologic studies in the U.S. and internationally. Combining five U.S. studies, we measured HPV35 positivity and, in Northern California, observed HPV35 type-specific population prevalence and estimated 5-year risk of developing precancer when HPV35-positive. HPV35 genetic variation was examined for differences in carcinogenicity in 1053 HPV35+ cervical specimens from a U.S. cohort and an international collection. African-American women had more HPV35 (12.1% vs 5.1%, P < .001) and more HPV35-associated precancers (7.4% vs 2.1%, P < .001) compared to other ethnicities. Precancer risks after HPV35 infection did not vary by ethnicity (global P = .52). The HPV35 A2 sublineage showed an increased association with precancer/cancer in African-Americans (OR = 5.6 vs A1, 95% CI = 1.3-24.8) and A2 was more prevalent among ICC in Africa than other world regions (41.9% vs 10.4%, P < .01). Our analyses support a strong link between HPV35 and cervical carcinogenesis in women of African ancestry. Current HPV vaccines cover the majority of cervical precancer/cancer across all ethnic groups; additional analyses are required to determine whether the addition of HPV35 to the already highly effective nine-valent HPV vaccine would provide better protection for women in Africa or of African ancestry.

A Systematic Review of Tests for Postcolposcopy and Posttreatment Surveillance.

OBJECTIVE: For the 2019 ASCCP Risk-Based Management Consensus Guidelines, we conducted a systematic review of diagnostic assays for postcolposcopy and posttreatment management. MATERIALS AND METHODS: A literature search was conducted to identify articles reporting on tests/assays for cervical cancer screening, triage, postcolposcopy surveillance, and posttreatment surveillance published between 2012 and 2019 in PubMed and Embase. Titles and abstracts were evaluated by co-authors for inclusion. Included articles underwent full-text review, data abstraction, and quality assessment. Pooled absolute pretest and posttest risk estimates were calculated for studies evaluating management of patients after treatment. RESULTS: A total of 2,862 articles were identified through the search. Of 50 articles on postcolposcopy, 5 were included for data abstraction. Of 66 articles on posttreatment, 23 were included for data abstraction and were summarized in the meta-analysis. The pooled posttreatment risk of cervical intraepithelial neoplasia (CIN) 2+ in all studies was 4.8% (95% CI = 3.4%-6.8%), ranging from 0.4%-19.5% (τ = 0.57) in individual studies. Among individuals testing negative for human papillomavirus (HPV) posttreatment, the risk of CIN 2+ was 0.69% (95% CI = 0.3%-1.5%); among individuals testing positive for HPV posttreatment, the risk of CIN 2+ was 18.3% (95% CI = 12.1%-26.6%) in all studies. All risk estimates were substantially higher for liquid-based cytology. The HPV-cytology co-testing provided slightly better reassurance compared with HPV alone at the cost of much higher positivity. CONCLUSIONS: Despite a large number of published studies on postcolposcopy and posttreatment surveillance, only few met criteria for abstraction and were included in the meta-analysis. More high-quality studies are needed to evaluate assays and approaches that can improve management of patients with abnormal screening.

Reporting and Assessing the Quality of Diagnostic Accuracy Studies for Cervical Cancer Screening and Management.

OBJECTIVE: We adapted the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool for studies of cervical cancer screening and management and used the adapted tool to evaluate the quality of studies included in a systematic review supporting the 2019 Risk-Based Management Consensus Guidelines. METHODS: We evaluated the quality of all studies included in our systematic review for postcolposcopy (n = 5) and posttreatment (n = 23) surveillance using QUADAS-2 criteria. Subsequently, we adapted signaling questions to indications of cervical cancer screening and management. An iterative process was carried out to evaluate interrater agreement between 2 study authors (M.A.C. and N.W.). Discrepant ratings were discussed, and criteria were adapted accordingly. We also evaluated the influence of study quality on risk estimates and between study variation using stratified subgroup meta-analyses. RESULTS: Twelve signaling questions for bias assessment that were adapted to or newly developed for cervical cancer screening and management are described here. Interrater agreement on bias assessment increased from 70% to 83% during the adaptation process. Detailed assessment of bias and applicability showed that all studies on postcolposcopy management and 90% of studies on posttreatment management had high risk of bias in at least 1 domain. Most commonly, high risk of bias was observed for the patient selection domain, indicating the heterogeneity of study designs and clinical practice in reported studies. CONCLUSIONS: The adapted QUADAS-2 will have broad application for researchers, evidence evaluators, and journals who are interested in designing, conducting, evaluating, and publishing studies for cervical cancer screening and management.

Identification of HPV genotypes causing cervical precancer using tissue-based genotyping.

Identification of high-risk human papillomavirus genotypes causing cervical precancer is crucial for informing HPV vaccine development and efficacy studies, and for determining which types to include in next-generation genotyping assays. Co-occurrence of hrHPV infections is common and complicates carcinogenicity assessment; accurate attribution requires tissue-based genotyping of precancers. We included all women with cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) from the Biopsy Study, an observational study of 690 women enrolled between 2009 and 2012 at the University of Oklahoma. Tissue-based genotyping, including whole tissue sections (WTS) and laser-capture microdissection (LCM), was performed on all precancers with multiple hrHPV infections detected in cytology, totaling over 1,800 HPV genotyping assays. Genotype attribution was compared to hierarchical and proportional hrHPV-type attribution models. Of 276 women with CIN2+, 122 (44.2%) had multiple hrHPV genotypes in cytology. Of 114 women with genotyping data, 94 had one or more hrHPV detected in tissue. Seventy-one women (75.5%) had a single causal hrHPV genotype, while 23 women had multiple hrHPV genotypes causing CIN2+. Ten women had multiple causal infections in a single biopsy, contrary to the previous notion that each lesion is caused by a single type only. While HPV16 was the predominant causal hrHPV genotype using all approaches, the hierarchical model overattributed HPV16, whereas other causal hrHPV genotypes, particularly HPV18 and HPV35, were underattributed. Understanding true causal genotypes is important for the evaluation of vaccine efficacy, to estimate the extent of unmasking, and for type-specific risk assessment in screening and management.

Mutations in the HPV16 genome induced by APOBEC3 are associated with viral clearance.

HPV16 causes half of cervical cancers worldwide; for unknown reasons, most infections resolve within two years. Here, we analyze the viral genomes of 5,328 HPV16-positive case-control samples to investigate mutational signatures and the role of human APOBEC3-induced mutations in viral clearance and cervical carcinogenesis. We identify four de novo mutational signatures, one of which matches the COSMIC APOBEC-associated signature 2. The viral genomes of the precancer/cancer cases are less likely to contain within-host somatic HPV16 APOBEC3-induced mutations (Fisher's exact test, P = 6.2 x 10-14), and have a 30% lower nonsynonymous APOBEC3 mutation burden compared to controls. We replicate the low prevalence of HPV16 APOBEC3-induced mutations in 1,749 additional cases. APOBEC3 mutations also historically contribute to the evolution of HPV16 lineages. We demonstrate that cervical infections with a greater burden of somatic HPV16 APOBEC3-induced mutations are more likely to be benign or subsequently clear, suggesting they may reduce persistence, and thus progression, within the host.

DeepCIN: Attention-Based Cervical histology Image Classification with Sequential Feature Modeling for Pathologist-Level Accuracy.

BACKGROUND: Cervical cancer is one of the deadliest cancers affecting women globally. Cervical intraepithelial neoplasia (CIN) assessment using histopathological examination of cervical biopsy slides is subject to interobserver variability. Automated processing of digitized histopathology slides has the potential for more accurate classification for CIN grades from normal to increasing grades of pre-malignancy: CIN1, CIN2, and CIN3. METHODOLOGY: Cervix disease is generally understood to progress from the bottom (basement membrane) to the top of the epithelium. To model this relationship of disease severity to spatial distribution of abnormalities, we propose a network pipeline, DeepCIN, to analyze high-resolution epithelium images (manually extracted from whole-slide images) hierarchically by focusing on localized vertical regions and fusing this local information for determining Normal/CIN classification. The pipeline contains two classifier networks: (1) a cross-sectional, vertical segment-level sequence generator is trained using weak supervision to generate feature sequences from the vertical segments to preserve the bottom-to-top feature relationships in the epithelium image data and (2) an attention-based fusion network image-level classifier predicting the final CIN grade by merging vertical segment sequences. RESULTS: The model produces the CIN classification results and also determines the vertical segment contributions to CIN grade prediction. CONCLUSION: Experiments show that DeepCIN achieves pathologist-level CIN classification accuracy.

Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes.

BACKGROUND: Human papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies. METHODS: TypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA. RESULTS: We observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT. CONCLUSIONS: The agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy.

Development of the TypeSeq Assay for Detection of 51 Human Papillomavirus Genotypes by Next-Generation Sequencing.

We have developed a new human papillomavirus (HPV) genotyping assay for detection of 51 HPV genotypes by next-generation sequencing (NGS). The TypeSeq assay consists of 3 PCR steps that equalize viral load and each type's amplicon copies prior to genotyping by NGS, thereby maximizing multiple-type sensitivity with minimal sequencing reads. The analytical sensitivity of the TypeSeq assay is 10 copies per reaction for 49 of the 51 types, including 13 high-risk (HR) types. We tested 863 clinical cervical specimens previously evaluated with the Roche Linear Array HPV genotyping test (LA). TypeSeq achieved 94.4% positive agreement with LA for detection of any HR type. Positive agreement was 91.4% and 85.5% for HPV16 and HPV18, respectively. Low-risk (LR) types ranged from 40.0% positive agreement (HPV83) to 90.9% (HPV69). Our unique approach to HPV amplification achieved a multiple-type sensitivity comparable to that of LA, with 83.9% and 84.2% of specimens positive for multiple HPV types by TypeSeq or LA, respectively. A total of 48.2% of specimens showed perfect agreement for all 37 types common to both assays. The simplicity of our open-source TypeSeq assay allows for high-throughput yet scalable processing, with a single technician able to process up to 768 specimens within 3 days. By leveraging NGS sample multiplexing capabilities, the per-sample labor requirements are greatly reduced compared to those of traditional genotyping methods. These features and the broad spectrum of detectable types make TypeSeq highly suitable for a wide range of applications.

Human papillomavirus 16 sub-lineage dispersal and cervical cancer risk worldwide: Whole viral genome sequences from 7116 HPV16-positive women.

BACKGROUND: Human papillomavirus (HPV)16 can be separated into genetic sub-lineages (A1-4, B1-4, C1-4, D1-4) which may have differential cervical cancer risk. METHODS: A next-generation sequencing assay was used to whole-genome sequence 7116 HPV16-positive cervical samples from well-characterised international epidemiological studies, including 2076 controls, 1878 squamous cell carcinoma (SCC) and 186 adenocarcinoma/adenosquamous cell carcinoma (ADC), and to assign HPV16 sub-lineage. Logistic regression was used to estimate region-stratified country-adjusted odds ratios (OR) and 95%CI. RESULTS: A1 was the most globally widespread sub-lineage, with others showing stronger regional specificity (A3 and A4 for East Asia, B1-4 and C1-4 for Africa, D2 for the Americas, B4, C4 and D4 for North Africa). Increased cancer risks versus A1 were seen for A3, A4 and D (sub)lineages in regions where they were common: A3 in East Asia (OR=2.2, 95%CI:1.0-4.7); A4 in East Asia (6.6, 3.1-14.1) and North America (3.8, 1.7-8.3); and D in North (6.2, 4.1-9.3) and South/Central America (2.2, 0.8-5.7), where D lineages were also more frequent in ADC than SCC (3.2, 1.5-6.5; 12.1, 5.7-25.6, respectively). CONCLUSIONS: HPV16 genetic variation can strongly influence cervical cancer risk. However, burden of cervical cancer attributable to different sub-lineages worldwide is largely driven by historical HPV16 sub-lineage dispersal.

Multiple Luteinized Follicle Cysts of the Ovary in a Patient With a Pituitary Adenoma: Report of a Case and Review of the Literature.

The case of a 36-yr-old woman with a pituitary adenoma who was found to have bilateral ovarian masses is reported. The right ovary was removed, measured 15 cm in maximum dimension, and contained multiple cysts which on microscopic examination had the typical morphology of follicle cysts. The left ovary was grossly similar intraoperatively. Subsequent excision of the pituitary adenoma was followed ∼3 mo later by a return to normal size of the left ovary. The case represents an example of multiple luteinized follicle cysts, analogous to the phenomenon seen occasionally in pregnancy, but with a different clinical background. Periodic documentation of this phenomenon is present in the literature, predominantly the clinical literature with limited pathologic documentation of the nature of the process in many reports. As pertains to the evaluation of follicle cysts encountered during pregnancy the differential diagnosis is with a cystic granulosa cell tumor of either adult or juvenile types, more likely the latter. The cyst lining is identical to that of standard follicle cysts and contrasts with the immature mitotically active nuclei seen in a juvenile granulosa cell tumor. That neoplasm also usually shows follicular differentiation typically absent in follicle cysts. Pathologists should be aware of the rare occurrence of luteinized follicle cysts in patients with a pituitary adenoma to enable correct intraoperative and standard pathologic evaluation.

Cytologic patterns of cervical adenocarcinomas with emphasis on factors associated with underdiagnosis.

BACKGROUND: New cervical cancers continue to be diagnosed despite the success of Papanicolaou (Pap) tests. In an effort to identify pitfalls that limit the diagnosis of adenocarcinoma, the authors reviewed the cytologic characteristics of endocervical adenocarcinomas in their patient population. METHODS: Liquid-based cytology slides from 45 women who had concurrent, histologically confirmed cervical adenocarcinomas were reviewed retrospectively and semiquantitatively for 25 key cytologic traits. The original sign-out diagnosis, available clinical findings, and high-risk human papillomavirus (HR HPV) results also were noted. RESULTS: Abundant tumor cellularity, nuclear size from 3 to 6 times normal, abundant 3-dimensional tumor cell groups, round cell shape, and cytoplasmic neutrophils characterized the 23 cases that were identified correctly as adenocarcinomas. Key reasons for undercalls included low tumor cellularity and low-grade columnar morphology; these also tended to correlate with low-grade or unusual adenocarcinoma variants on histology. Overall, 73% of adenocarcinomas had a concurrent positive HR HPV test. CONCLUSIONS: Most endocervical adenocarcinomas can be diagnosed accurately in cases with classical features, but some cases continue to be problematic when evaluated based on cytologic features alone. Reflex HPV testing may help increase Pap test sensitivity for challenging cases that have atypical glandular cells of undetermined significance. Occasional cases with negative HR HPV test results remain of concern.