Roy Zhang

Multiple human papillomavirus genotype infections in cervical cancer progression in the study to understand cervical cancer early endpoints and determinants

Determining the causal attribution of human papillomavirus (HPV) genotypes to cervical disease is important to estimate the effect of HPV vaccination and to establish a type spectrum for HPV‐based screening. We analyzed the prevalence of HPV infections and their attribution to cervical disease in a population of 1,670 women referred to colposcopy for abnormal cytology at the University of Oklahoma. HPV genotyping was performed from cytology specimens using the Linear Array assay that detects 37 HPV genotypes. We used different methods of type attribution to revised cervical disease categories. We found very high prevalence of multiple HPV infections with up to 14 genotypes detected in single specimens. In all disease categories except for cancers, there was a significant trend of having more infections at a younger age. We did not see type interactions in multiple genotype infections. HPV16 was the most frequent genotype at all disease categories. Based on different attribution strategies, the attribution of vaccine genotypes (6, 11, 16, 18) ranged from 50.5 to 67.3% in cancers (n = 107), from 25.6 to 74.8% in CIN3 (n = 305), from 15.2 to 52.2% in CIN2 (n = 427), and from 6.6 to 26.0% in

Performance of p16/Ki-67 immunostaining to detect cervical cancer precursors in a colposcopy referral population

Purpose: Cytology-based screening has limited sensitivity to detect prevalent cervical precancers. Human papilloma virus (HPV) DNA testing is highly sensitive and provides a high, long-term reassurance of low risk of cervical cancer. However, the specificity of HPV DNA testing is limited, requiring additional, more disease-specific markers for efficient screening approaches. Experimental Design: Liquid-based cytology samples were collected from 625 women referred to colposcopy. A slide was stained using the CINtec plus cytology assay. Pap cytology and HPV genotyping were conducted from the same vial. Clinical performance characteristics were calculated for all women, stratified by age, and for women referred with a low-grade squamous intraepithelial lesion (LSIL) Pap. Results: p16/Ki-67 positivity increased with histologic severity, from 26.8% in normal histology, 46.5% in CIN1, 82.8% in CIN2 to 92.8% in CIN3. Among women with CIN3, p16/Ki-67 positivity increased from 77.8% for women younger than 30 years without HPV16 to 100% for women 30 years and older with HPV16. The sensitivity and specificity to detect CIN3+ were 93.2% and 46.1%, respectively, and increased to 97.2% and 60.0% among women 30 years and older. In women with high-risk (HR)-HPV–positive atypical squamous cells of undetermined significance (ASC-US) and LSIL, sensitivity and specificity for detection of CIN3 were 90.6% and 48.6%, respectively. Conclusions: p16/Ki-67 testing could reduce referral to colposcopy by almost half while detecting the most severe cases of CIN3. The high sensitivity of p16/Ki-67 with significantly improved specificity compared with HPV testing makes p16/Ki-67 a viable option for LSIL triage. Further studies are required to evaluate p16/Ki-67 as triage marker in HPV-based screening strategies. Clin Cancer Res; 18(15); 4154–62. ©2012 AACR.

Grading the severity of cervical neoplasia based on combined histopathology, cytopathology, and HPV genotype distribution among 1,700 women referred to colposcopy in Oklahoma.

Diagnosis and treatment of cervical cancer precursors rely on colposcopic biopsy, which is sometimes hampered by incorrect biopsy placement and the unclear prognostic value of poorly reproducible diagnoses such as cervical intraepithelial neoplasia (CIN) Grade 1 and 2. Searching for discrete disease categories that incorporate the value of cytology and that reflect the causal role of particular HPV types, we analyzed histology, cytology and HPV genotype distributions in the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED). This cross‐sectional study comprises ∼1,700 women referred to colposcopy or treatment for the spectrum of cervical disease, including 439 women with

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.

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 <CIN2 (1.97 [1.12-3.46]; 2.23 [1.59-3.11]; 2.60 [2.04-3.30], respectively), whereas associations were not significantly different (OC use, parity) or showed decreased risk (smoking) when comparing cancer to CIN3. CONCLUSIONS Differences in established risk factors suggest that CIN3 is a more specific definition of precancer than CIN2. Hormonally-related factors and smoking play a role in the transition from human papillomavirus infection to precancer.

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.

Accuracy of cervical specimens obtained for biomarker studies in women with CIN3

OBJECTIVE We developed a protocol to collect representative cervical specimens based on colposcopic evaluation from women treated with loop electrosurgical excision procedure (LEEP). METHODS We analyzed the histology of biopsies targeting the worst and a normal area on the cervical surface in 74 women referred for LEEP because of cervical intraepithelial neoplasia grade 3 (CIN3) detected in a previous biopsy. Lesions and normal tissue were identified in colposcopy, marked, and removed after LEEP. Cervical cytology specimens collected at the same time were analyzed using Pap cytology and human papillomavirus (HPV) genotyping. RESULTS All but two women had an abnormal colposcopic impression with 59 of 68 (87%) showing an impression of CIN2 or greater. In 19 of 58 (33%) women, the histology result of the frozen specimen targeting the worst lesion was < or =CIN1. In 18 of 46 (40%) women, the histology of the frozen specimen targeting normal tissue was CIN2+. A concordant histology result in specimens targeting the worst lesion was associated with a greater extension of the CIN3 in the LEEP (p trend=0.002) and a HSIL cytology result (p trend=0.02). CONCLUSION It is challenging to sample representative cervical tissue. Even in women with confirmed CIN3, colposcopy performance to identify the worst lesion on the cervix was limited. Correctly identified CIN3s were more likely to be larger lesions that may have a higher risk of progression to cancer.

Automated Analysis of Fluorescent in situ Hybridization (FISH) Labeled Genetic Biomarkers in Assisting Cervical Cancer Diagnosis

The numerical and/or structural deviation of some chromosomes (i.e., monosomy and polysomy of chromosomes 3 and X) are routinely used as positive genetic biomarkers to diagnose cervical cancer and predict the disease progression. Among the available diagnostic methods to analyze the aneusomy of chromosomes 3 and X, fluorescence in situ hybridization (FISH) technology has demonstrated significant advantages in assisting clinicians to more accurately detect and diagnose cervical carcinoma at an early stage, in particular for the women at a high risk for progression of low-grade and high-grade squamous intraepithelium lesions (LSIL and HSIL). In order to increase the diagnostic accuracy, consistency, and efficiency from that of manual FISH analysis, this study aims to develop and test an automated FISH analysis method that includes a two-stage scheme. In the first stage, an interactive multiple-threshold algorithm is utilized to segment potential interphase nuclei candidates distributed in different intensity levels and a rule-based classifier is implemented to identify analyzable interphase cells. In the second stage, FISH labeled biomarker spots of chromosomes 3 and X are segmented by a top-hat transform. The independent FISH spots are then detected by a knowledge-based classifier, which enables recognition of the splitting and stringy FISH signals. Finally, the ratio of abnormal interphase cells with numerical changes of chromosomes 3 and X is calculated to detect positive cases. The experimental results of four test cases showed high agreement of FISH analysis results between the automated scheme and the cytogeneticists analysis including 92.7% to 98.7% agreement in cell segmentation and 4.4% to 11.0% difference in cell classification. This preliminary study demonstrates the feasibility of potentially applying the automatic FISH analysis method to expedite the screening and detecting cervical cancer at an early stage.

Comparison of Human Papillomavirus Detections in Urine, Vulvar, and Cervical Samples from Women Attending a Colposcopy Clinic

ABSTRACT 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.

Clinical and Pathological Heterogeneity of Cervical Intraepithelial Neoplasia Grade 3

Objective Cervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US. Methods We examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance. Results CIN3 cases varied substantially by size (1–10 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results. Conclusions We demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion.