Evaluation of transport media for laboratory detection of SARS-CoV-2 in upper respiratory tract swab specimens.

The reduced availability of commercial swabs and transport media for testing and administrative demands for increased testing capacity during the coronavirus disease 2019 (COVID-19) public health emergency has seriously challenged national laboratory testing programs, forcing many to use nontraditional collection devices, often without typical analytical assessment of their suitability in testing. Five common transport media (four commercial and one in-house) were evaluated for their suitability in the collection of nasopharyngeal swab specimens for subsequent molecular detection of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). Results suggest that these transport media provide dependable temporal stability of the SARS-CoV-2 virus without significant analytical interference of molecular assays. These findings are not only important for addressing critical laboratory supply chain shortages of transport media in the current COVID-19 health crisis but also for future pandemic planning, when again supplies of commercially available transport media might be depleted.

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.

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.

A prospective study of risk-based colposcopy demonstrates improved detection of cervical precancers.

BACKGROUND: Sensitivity for detection of precancers at colposcopy and reassurance provided by a negative colposcopy are in need of systematic study and improvement. OBJECTIVE: We sought to evaluate whether selecting the appropriate women for multiple targeted cervical biopsies based on screening cytology, human papillomavirus testing, and colposcopic impression could improve accuracy and efficiency of cervical precancer detection. STUDY DESIGN: In all, 690 women aged 18-67 years referred to colposcopy subsequent to abnormal cervical cancer screening results were included in the study (ClinicalTrials.gov: NCT00339989). Up to 4 cervical biopsies were taken during colposcopy to evaluate the incremental benefit of multiple biopsies. Cervical cytology, human papillomavirus genotyping, and colposcopy impression were used to establish up to 24 different risk strata. Outcomes for the primary analysis were cervical precancers, which included p16+ cervical intraepithelial neoplasia 2 and all cervical intraepithelial neoplasia 3 that were detected by colposcopy-guided biopsy during the colposcopy visit. Later outcomes in women without cervical intraepithelial neoplasia 2+ at baseline were abstracted from electronic medical records. RESULTS: The risk of detecting precancer ranged from 2-82% across 24 strata based on colposcopy impression, cytology, and human papillomavirus genotyping. The risk of precancer in the lowest stratum increased only marginally with multiple biopsies. Women in the highest-risk strata had risks of precancer consistent with immediate treatment. In other risk strata, multiple biopsies substantially improved detection of cervical precancer. Among 361 women with cervical intraepithelial neoplasia <2 at baseline, 195 (54%) had follow-up cytology or histology data with a median follow-up time of 508 days. Lack of detection of precancer at initial colposcopy that included multiple biopsies predicted low risk of precancer during follow-up. CONCLUSION: Risk assessment at the colposcopy visit makes identification of cervical precancers more effective and efficient. Not finding precancer after a multiple-biopsy protocol provides high reassurance and allows releasing women back to regular screening.

Diagnosis of Cervical Precancers by Endocervical Curettage at Colposcopy of Women With Abnormal Cervical Cytology.

OBJECTIVE: To evaluate the performance of routine endocervical curettage (ECC) for diagnosing high-grade cervical intraepithelial neoplasia (CIN) 2 or worse and additional precancers not otherwise detected by ectocervical biopsies. METHODS: In a secondary analysis of the Biopsy Study, a cross-sectional study conducted between 2009 and 2012 at the University of Oklahoma Health and Sciences Center that found an incremental increase in detection of cervical precancers by multiple biopsies at colposcopy, ECC was performed in most women aged 30 years or older. Cervical intraepithelial neoplasia 2 or worse yield by ECC alone was evaluated in analyses stratified by cervical cytology (atypical squamous cells of undetermined significance [ASC-US] or low-grade squamous intraepithelial lesions [LSIL] compared with atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesions [ASC-H] or high-grade squamous intraepithelial lesions [HSIL] or worse), colposcopic impression (less than high-grade compared with high-grade), human papillomavirus (HPV)-16 infection status, whether the examination was satisfactory, and by ECC indications per the current guidelines for cervical cancer screening. The diagnostic value of ECC for detecting additional disease was evaluated by the number of lesion-directed ectocervical biopsies. RESULTS: Of the 204 women aged 30 years or older, 181 (88.7%) underwent ECC. Overall ECC detected 14.4% CIN 2 or worse (95% CI 10.0-20.2%). Endocervical curettage was more likely to find disease in the endocervix among women with high-grade cytology, positive HPV-16 infection, or high-grade colposcopic impressions (respective P values <.05). Among women with ASC-US or LSIL cytology, those with an unsatisfactory examination had a 13.0% CIN 2 or worse yield on ECC (95% CI 6.1-25.7); when colposcopic examination was normal or satisfactory with visible abnormal lesions, ECC detected less than 5% CIN 2 or worse in the endocervix. An ASC-H or HSIL or worse cytology was associated with a CIN 2 or worse yield of 25.8% by ECC (95% CI 16.6-37.9%). However, ECC found only 3.9% (95% CI 1.9-7.8%) additional CIN 2 or worse beyond the cumulative disease detected by up to four biopsies of visible acetowhite ectocervical lesions. Additional CIN 2 or worse yield by ECC increased when fewer lesion-directed biopsies were taken (P<.05). CONCLUSION: The additional yield of CIN 2 or worse by ECC in a colposcopy with up to four ectocervical biopsies was low. Based on our findings, we recommend routine ECC be performed in women aged 45 years old or older with HPV-16 infection and in any woman aged 30 years or older with HSIL or worse or ASC-H cytology, high-grade colposcopic impression, or ASC-US or LSIL cytology and an unsatisfactory examination. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT00339989.

Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer.

Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin-fixed, paraffin-embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neoplasia grade 3, and cancer using a bead-based microarray covering 1,500 CpG sites in over 800 genes. Methylation levels in individual CpG sites were compared using a t-test, and results were summarized by computing p-values. A total of 12 candidate genes (ADCYAP1, ASCL1, ATP10, CADM1, DCC, DBC1, HS3ST2, MOS, MYOD1, SOX1, SOX17 and TMEFF2) identified by DNA methylation profiling, plus an additional three genes identified from the literature (EPB41L3, MAL and miR-124) were chosen for validation in an independent set of 167 liquid-based cytology specimens using pyrosequencing and targeted, next-generation bisulfite sequencing. Of the 15 candidate gene markers, 10 had an area under the curve (AUC) of ≥ 0.75 for discrimination of high grade squamous intraepithelial lesions or worse (HSIL+) from

Comparison of Colposcopic Impression Based on Live Colposcopy and Evaluation of Static Digital Images.

OBJECTIVE: The aim of the study was to evaluate the agreement and compare diagnostic accuracy of colposcopic impressions from live colposcopy versus evaluation of static digital images. MATERIALS AND METHODS: Live impressions and corresponding static images obtained during colposcopy of 690 women were independently compared. Diagnostic accuracy was calculated for colposcopic impressions from both methods, varying hypothetical thresholds for colposcopically directed cervical biopsies (acetowhitening or worse, low grade or worse, high grade or worse). Stratified analyses investigated the impact of referral cytology, human papillomavirus 16 infection, and age on colposcopic impression. RESULTS: Overall agreement between live and static colposcopic visualization was 43.0% (κ = 0.20; 95% CI = 0.14-0.26) over normal, acetowhitening, low-grade, and high-grade impressions. Classification of acetowhitening or worse impressions showed the highest agreement (92.2%; κ = 0.39; 95% CI = 0.21-0.57); both methods achieved more than 95% sensitivity for CIN 2+. Agreement between live and static colposcopic visualization was 69.3% for rating low-grade or worse impressions (κ = 0.23; 95% CI = 0.14-0.33) and 71% when rating high-grade impressions (κ = 0.33; 95% CI = 0.24-0.42). Live colposcopic impressions were more likely to be rated low grade or worse (p < .01; odds ratio = 3.5; 95% CI = 2.4-5.0), yielding higher sensitivity for CIN 2+ at this threshold than static image assessment (95.4% vs 79.8%, p < .01). Overall, colposcopic impressions were more likely rated high grade on live assessment among women referred with high-grade cytology (odds ratio = 3.3; 95% CI = 1.8-6.4), significantly improving the sensitivity for CIN 2+ (66.3% vs 48.5%, p < .01). CONCLUSIONS: Colposcopic impressions of acetowhitening or worse are highly sensitive for identifying cervical precancers and reproducible on static image-based pattern recognition.

Detection of HPV DNA in paraffin-embedded cervical samples: a comparison of four genotyping methods.

BACKGROUND: Identification of human papillomavirus (HPV) DNA in cervical tissue is important for understanding cervical carcinogenesis and for evaluating cervical cancer prevention approaches. However, HPV genotyping using formalin-fixed, paraffin-embedded (FFPE) tissues is technically challenging. We evaluated the performance of four commonly used genotyping methods on FFPE cervical specimens conducted in different laboratories and compared to genotyping results from cytological samples. METHODS: We included 60 pairs of exfoliated-cell and FFPE specimens from women with histologically confirmed cervical intraepithelial lesions grade 2 or 3. Cytology specimens were genotyped using the Linear Array assay. Four expert laboratories processed tissue specimens using different preparation methods and then genotyped the resultant sample preparations using four different HPV genotyping methods: SPF10-PCR DEIA LiPA25 (version 1), Inno-LiPA, Linear Array and the Onclarity assay. Percentage agreement, kappa statistics and McNemar's chi-square were calculated for each comparison of different methods and specimen types. RESULTS: Overall agreement with respect to carcinogenic HPV status for FFPE samples between different methods was: 81.7, 86.7 and 91.7% for Onclarity versus Inno-LiPA, Linear Array and SPF-LiPA25, respectively; 81.7 and 85.0% for Linear Array versus Inno-LiPA and SPF-LiPA25, respectively; and 86.7% for SPF-LiPA25 versus Inno-LiPA. Type-specific agreement was >88.3% for all pair-wise comparisons. Comparisons with cytology specimens resulted in overall agreements from 80 to 95% depending on the method and type-specific agreement was >90% for most comparisons. CONCLUSIONS: Our data demonstrate that the four genotyping methods run by expert laboratories reliably detect HPV DNA in FFPE specimens with some variation in genotype-specific detection.

Multiple biopsies and detection of cervical cancer precursors at colposcopy.

PURPOSE: Women with abnormal cervical cancer screening results are referred to colposcopy and biopsy for diagnosis of cervical cancer precursors (high-grade squamous intraepithelial lesions [HSILs]). Colposcopy with a single biopsy can miss identification of HSILs. No systematic study has quantified the improved detection of HSIL by taking multiple lesion-directed biopsies. METHODS: The Biopsy Study was an observational study of 690 women referred to colposcopy after abnormal cervical cancer screening results. Up to four directed biopsies were taken from distinct acetowhite lesions and ranked by colposcopic impression. A nondirected biopsy of a normal-appearing area was added if fewer than four directed biopsies were taken. HSIL identified by any biopsy was the reference standard of disease used to evaluate the incremental yield and sensitivity of multiple biopsies. RESULTS: In the overall population, sensitivities for detecting HSIL increased from 60.6% (95% CI, 54.8% to 66.6%) from a single biopsy to 85.6% (95% CI, 80.3% to 90.2%) after two biopsies and to 95.6% (95% CI, 91.3% to 99.2%) after three biopsies. A significant increase in sensitivity of multiple biopsies was observed in all subgroups. The highest increase in yield of HSIL was observed for women with a high-grade colposcopic impression, HSIL cytology, and human papillomavirus (HPV) type 16 positivity. Only 2% of all HSILs diagnosed in the participants were detected by biopsies of normal-appearing transformation zone. CONCLUSION: Collection of additional lesion-directed biopsies during colposcopy increased detection of histologic HSIL, regardless of patient characteristics. Taking additional biopsies when multiple lesions are present should become the standard practice of colposcopic biopsy.

Evaluation of clinical performance of a novel urine-based HPV detection assay among women attending a colposcopy clinic.

BACKGROUND: Human papillomavirus (HPV) testing in urine offers a convenient approach for cervical cancer screening but has previously suffered from limited clinical sensitivity. OBJECTIVES: We evaluated clinical performance of the prototype Trovagene HPV test, a novel polymerase chain reaction assay that targets the E1 region of the HPV genome and detects and amplifies short fragments of cell-free HPV DNA in urine. STUDY DESIGN: We conducted a pilot study among 72 women referred to colposcopy following abnormal screening. Participants provided a urine sample prior to clinician-collected cervical sampling and colposcopically-directed punch biopsy. Trovagene HPV test results on urine samples were compared with cervical and urine testing by Linear Array HPV Genotyping Test (LA-HPV) for detection of histologically-confirmed cervical precancerous lesions. RESULTS: There was high concordance between urine samples tested by the Trovagene HPV test and corresponding cervical (87.5%) and urine (81.9%) samples tested by LA-HPV. The Trovagene HPV test had high sensitivity (92.3% for detecting CIN2/3, and 100% for CIN3), comparable to LA-HPV testing on cervical samples (96.0% and 100%, respectively), and higher than LA-HPV testing on urine samples (80.8% and 90.0%, respectively). In this referral population, the specificity of the Trovagene urine HPV test was non-significantly lower (29% for CIN2/3 and 25% for CIN3) than corresponding estimates of LA-HPV testing on cervical (36% and 28%, respectively) and urine (42% and 38%, respectively) samples. CONCLUSIONS: This pilot study suggests that the Trovagene HPV test has high sensitivity for urine-based detection of cervical precancer and merits evaluation in larger studies.

Factors associated with reduced accuracy in Papanicolaou tests for patients with invasive cervical cancer.

BACKGROUND: Recent proposals to lengthen the interval in cervical cancer screening highlight the importance of the accurate interpretation of screening tests. Tumor debris present in Papanicolaou (Pap) tests from women with invasive cancer is known to hamper interpretation. The current study evaluated limiting factors in Pap tests from women with invasive cervical cancer. METHODS: A total of 3003 women with the spectrum of cervical lesions who had ThinPrep (Hologic Inc, Marlborough, Mass) Pap and human papillomavirus (HPV) genotyping tests performed were grouped by their most severe histologic diagnosis. Cytologic and HPV results were analyzed by cross-sectional analysis. RESULTS: The unsatisfactory rate of cytology specimens from patients with cancer (3.1%) was significantly higher than those from patients with cervical intraepithelial neoplasia of type 3 or less (0.8%) (P < .001). The percentage of samples with qualified adequacy was 34.8% in specimens from patients with cancer compared with only 3.6% from specimens from those without cancer (P < .001). The unsatisfactory and qualified adequacy rates were higher in squamous cancers compared with adenocarcinomas. However, adenocarcinomas were identified less frequently than squamous cancers (37.0% vs 61.7%) in the Pap tests. HPV tests were positive in 84.4% of unsatisfactory cases including 8 of 9 cancer cases, although 8.5% of cancers tested negative for HPV. CONCLUSIONS: Unsatisfactory and suboptimal ThinPrep Pap tests were increased in cancer cases compared with lesser histologic diagnoses. This was found to be particularly true for squamous cancers. Specimens from adenocarcinomas had fewer adequacy problems but were less frequently recognized as malignant. HPV tests were positive in the majority of unsatisfactory Pap tests in women with carcinoma, suggesting that HPV testing in women aged > 30 years can help to identify high-risk women with unsatisfactory Pap tests.

Comparison of human papillomavirus detections in urine, vulvar, and cervical samples from women attending a colposcopy clinic.

While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.

Evaluation of a multiplex panel of immune-related markers in cervical secretions: a methodologic study.

Although persistent carcinogenic human papillomavirus (HPV) infection is necessary for cervical carcinogenesis, the cofactors involved in HPV persistence and disease progression are poorly understood. Chronic cervical inflammation may increase risk, but few studies have measured immune markers (cytokines, chemokines and soluble receptors) in cervical secretions. We evaluated the performance of 74 multiplexed, bead-based immune markers in cervical secretions from three groups of women with biopsy evaluation of cervical intraepithelial neoplasia (CIN), (i) 25% detectability and >80% interclass correlation coefficients (ICCs) acceptable for epidemiologic studies. Within-batch coefficients of variation (CVs) of ≥25% indicated room for assay improvement. Secondarily, we explored associations between marker levels and CIN/HPV status adjusted for matching variables, assay batch, age and number of sexual partners. Sixty-two markers (84%) had >25% detectability and ICCs > 80%. Of those, 53 (85%) had CVs < 25%. Using these preliminary data, we found that HPV positivity was associated with increased eotaxin-1 [odds ratio (OR): 15.63, 95% confidence interval (CI): 1.26-200.00] and G-CSF (OR: 12.99, 95% CI: 1.10-142.86) among CIN-negative women. There was suggestive evidence that higher chemoattractant marker levels were associated with CIN2/3 (e.g., MIP-1delta, OR: 4.48, 95% CI: 0.87-23.04 versus

The differential diagnosis of pilocytic astrocytoma with atypical features and malignant glioma: an analysis of 16 cases with emphasis on distinguishing molecular features.

Rare pilocytic astrocytomas (PA) have atypical histologic and clinicoradiologic features that raise the differential diagnosis of glioblastoma. Whether ancillary studies can supplement histopathologic examination in placing these cases accurately on the spectrum of WHO Grade I PA to higher-grade glioma is not always clear, partly because these cases are not common. Here, ten PAs with atypical clinicoradiologic and histologic features and six pediatric glioblastoma multiforme (pGBMs) were analyzed for BRAF V600E, IDH1, IDH2, and TP53 mutations. Ki-67, p53, and p16 protein expression were also examined by immunohistochemistry. BRAF-KIAA1549 fusion status was assessed in the PA subgroup. The rate of BRAF-KIAA1549 fusion was high in these PAs (5/7 tumors) including four extracerebellar examples. A single BRAF V600E mutation was identified in the fusion-negative extracerebellar PA of a very young child who succumbed to the disease. TP53 mutations were present only in malignant gliomas, including three pGBMs and one case designated as PA with anaplastic features (with consultation opinion of pGBM). IDH1 and IDH2 were wild type in all cases, consistent with earlier findings that IDH mutations are not typical in high-grade gliomas of patients ≤14 years of age. Immunohistochemical studies showed substantial overlap in Ki-67 labeling indices, an imperfect correlation between p53 labeling and TP53 mutation status, and complete p16 loss in only two pGBMs but in no PAs. These results suggest that (a) BRAF-KIAA1549 fusion may be common in PAs with atypical clinicoradiologic and histologic features, including those at extracerebellar sites, (b) BRAF V600E mutation is uncommon in extracerebellar PAs, and (c) TP53 mutation analysis remains a valuable tool in identifying childhood gliomas that will likely behave in a malignant fashion.

Chromosomal gains measured in cytology samples from women with abnormal cervical cancer screening results.

OBJECTIVE: Chromosomal gains at 3q26, 5p15 and 20q13 have been described in cervical precancer and cancer. We evaluated a novel fluorescence in situ hybridization (FISH) assay that detects gains at these three loci simultaneously as a possible biomarker for detecting cervical precancer. METHODS: Chromosomal copy numbers at 3q26, 5p15, 20q13 and the centromere of chromosome7 (cen7) in liquid-based cytology specimens from 168 women enrolled in the Biopsy Study were determined by FISH. The number of cells with ≥ 3 or ≥ 4 signals for a genomic locus was enumerated and diagnostic test performance measures were calculated using receiver operating characteristic (ROC) analyses. Sensitivity and specificity values were determined for the detection of CIN2+ and/or HSIL. RESULTS: The median number of cells with ≥ 3 signals increased with the severity of cervical lesion for each genomic locus (p-trend<0.02 for each locus). ROC analysis for the number of cells with ≥ 3 signals resulted in area under the curve values of 0.70 (95% CI: 0.54-0.86), 0.67 (0.52-0.83), 0.67 (0.51-0.83) and 0.78 (0.64-0.92) for 3q26, 5p15, 20q13 and cen7, respectively, for the detection of CIN2+ and/or HSIL. Positivity for gains at multiple loci resulted in only slightly better test performance measures than those for the individual probes for four distinct combinations of probes. CONCLUSIONS: Chromosomal gains at 3q26, 5p15, 20q13 and cen7 are associated with severity of cervical lesions. Further studies are required to quantify risk stratification of FISH assays for cervical cancer screening.

The role of co-factors in the progression from human papillomavirus infection to cervical cancer.

OBJECTIVE: Co-factors for cervical cancer, including oral contraceptive (OC) use, smoking and multiparity have been identified; however, the stage at which they act in cervical carcinogenesis is not clear. We compared established risk factors among women with CIN2 and CIN3 to evaluate the heterogeneity of these factors in precancer and also assessed their role during cervical carcinogenesis. METHODS: The current analysis included 2783 women with various stages of cervical disease who were enrolled in the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED) and the Biopsy Study. Associations of co-factors within cervical precancer and at different stages of cervical carcinogenesis were estimated using logistic regression. RESULTS: Long-term OC use (10+years vs. never: OR=2.42, 95% CI: [1.13-5.15]), multiparity (3+ births vs. nulliparous: OR=1.54 [1.04-2.28]), smoking (ever vs. never: OR=1.95 [1.48-2.58]), and no Pap test in the previous five years (2.05 [1.32-3.17]) were positively associated with CIN3 compared to CIN2. We observed that long-term OC use, parity and smoking were associated with an increased risk of CIN3 compared to

Heterogeneity of high-grade cervical intraepithelial neoplasia related to HPV16: implications for natural history and management.

Factors associated with progression from cervical intraepithelial neoplasia (CIN) grades 2 and 3 to invasive cancer are not well understood; most CIN2 and CIN3 do not progress to cancer. Among carcinogenic human papillomavirus (HPV) types, infections with HPV16 have the highest risk of progressing to cancer. We evaluated the heterogeneity of risk factors, lesion size, colposcopic impression and colposcopic biopsy results in relation to HPV16 status among 627 women with CIN2 or CIN3 in women referred to colposcopy at the University of Oklahoma. Loop excision specimens were evaluated in 12 radial segments to estimate lesion size. The mean age at CIN3 was 27.7 years for HPV16-positive women (n = 225) and 33.6 years for HPV16-negative women (n = 104). The average lesion size did not differ by HPV16 status (p = 0.83). Among HPV16-positive women with CIN3, lesions were significantly larger in women 30 years and older (p = 0.03). Colposcopic impression was worse in women with HPV16 infections (p = 0.009), but the detection of CIN3 at the preceding biopsy was not improved in HPV16-positive women. CIN3 is detected at the same lesion size, but at much younger age in women with HPV16 infections, suggesting faster growth. CIN2 lesion size in women without HPV16 peaks below 30 years and then decreases, suggesting frequent regression, whereas HPV16-related CIN2 is more likely to persist. Lesion size seems to be an important determinant of colposcopy and biopsy performance. Genotyping for HPV16 in cervical cancer screening can improve risk stratification but may pose challenges to finding small lesions in colposcopy.

Reproducibility of linear array for human papillomavirus genotyping.

We conducted a Linear Array test/retest analysis using cytologic specimens from 198 women. A total of 67.2% of samples had the same human papillomavirus (HPV) types detected in both tests (type-specific positive agreement was 83.3% overall [Kappa = 0.9] and 86.8% for carcinogenic types [Kappa = 0.92]). Discordance was highest with a low hybridization signal strength. Overall, Linear Array was highly reproducible.