Daisy R. Lee

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women

While infection with high‐risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV‐16 DNA‐positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT‐PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV‐16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20–7.76), smoking (OR = 3.0, 95% CI 1.19–7.56), high viral load (OR = 5.27, 95% CI 2.05–13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1–47.58) and high transcript copies (OR = 5.56, 95% CI 2.05–13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV‐16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV‐16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening.

Detection of Human Papillomavirus in Archival Tissues: Comparison of In Situ Hybridization and Polymerase Chain Reaction:

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues.

Gene expression profile of cervical tissue compared to exfoliated cells: Impact on biomarker discovery

BackgroundExfoliated cervical cells are used in cytology-based cancer screening and may also be a source for molecular biomarkers indicative of neoplastic changes in the underlying tissue. However, because of keratinization and terminal differentiation it is not clear that these cells have an mRNA profile representative of cervical tissue, and that the profile can distinguish the lesions targeted for early detection.ResultsWe used whole genome microarrays (25,353 unique genes) to compare the transcription profiles from seven samples of normal exfoliated cells and one cervical tissue. We detected 10,158 genes in exfoliated cells, 14,544 in the tissue and 7320 genes in both samples. For both sample types the genes grouped into the same major gene ontology (GO) categories in the same order, with exfoliated cells, having on average 20% fewer genes in each category. We also compared microarray results of samples from women with cervical intraepithelial neoplasia grade 3 (CIN3, n = 15) to those from age and race matched women without significant abnormalities (CIN1, CIN0; n = 15). We used three microarray-adapted statistical packages to identify differential gene expression. The six genes identified in common were two to four fold upregulated in CIN3 samples. One of these genes, the ubiquitin-conjugating enzyme E2 variant 1, participates in the degradation of p53 through interaction with the oncogenic HPV E6 protein.ConclusionThe findings encourage further exploration of gene expression using exfoliated cells to identify and validate applicable biomarkers. We conclude that the gene expression profile of exfoliated cervical cells partially represents that of tissue and is complex enough to provide potential differentiation between disease and non-disease.

Optimization of SELDI-TOF protein profiling for analysis of cervical mucous

Cervical mucous, produced in the region where cervical neoplasia occurs, is thought to be a good choice for discovery of biomarkers to improve cervical cancer screening. In this study, SELDI-TOF MS analysis was used to evaluate parameters for protein profiling of mucous. Proteins were extracted from mucous collected with Weck-Cel sponges. Several parameters like extraction reagent, loading protein concentration, matrix type, bind/wash conditions and sample fractionation, on different protein chip surfaces were evaluated. SELDI peak number and consistency in the resulting spectra were used to evaluate each condition. Analysis of spectra generated by different protein chips revealed an average of 30 peaks in the 2.5-30 kDa mass range using sinnapinic acid in the unfractionated sample. Sample concentration and buffer conditions evaluated did not lead to large alterations in the profiles. Quality control spectra were reproducible with intra- and inter-assay intensity CV for CM10, H50 and Q10 arrays being less than 20% and 30% respectively. IMAC30-Cu chips had higher intra- and inter-assay CVs at 25% and 35%. Current data showed that optimizing pre-analytical parameters can help in standardization and reproducibility of protein profiles produced by cervical mucous, and thus can be used for protein biomarker discovery with the SELDI platform.

Molecular markers for early detection of cervical neoplasia

Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, National Center forInfectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Healthand Human Services, Atlanta, GA, USA1. IntroductionIn the United States, cervical cancer screening pro-grams based on exfoliated cervical cytology (Papsmears) have significantly reduced the incidence of in-vasive cervical cancer. As a result of this success,current cervical cancer screening programs in the USare not directed at invasive disease, but at detection ofthe precursors of carcinoma, referred to as dysplasias,squamous intraepithelial lesions (SIL) or cervical in-traepithelial neoplasias (CIN). The progressive histo-logicandcytologicchangesthatoccurduringthemulti-step process of cervical carcinogenesis can be dividedinto multiple stages, early lesions known as CIN 1 orLSIL and high grade lesions known as CIN 2, 3 orHSIL. The natural history of these cervical cancer pre-cursor lesions is difficult to study because they are usu-ally biopsied or otherwise treated as soon as detected.However, it is clear that CIN 1 and 2 lesions are morelikelytoregressthantoprogresstoinvasivedisease[37,69]. While the risk of progression is greatest for CIN 3lesions, not all of these lesions progress and regressionis recognizedto occurin a significantbut variablenum-ber of cases [69]. Because of the slow rate of diseaseprogression, targeting early detection at CIN 3 lesionsis an effective strategy to avoid invasive cancer and at

Age-Group Differences in Human Papillomavirus Types and Cofactors for Cervical Intraepithelial Neoplasia 3 among Women Referred to Colposcopy

Background: Recommendations for high-risk human papillomavirus (HR-HPV) testing as an adjunct to cytology for cervical cancer screening differ by age group, because HR-HPV tests lack adequate specificity in women aged <30. Here, we assess age-group differences in HPV types and other risk factors for cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) versus CIN0–2 in women from four colposcopy clinics. Methods: Women ages 18 to 69 (n = 1,658) were enrolled and completed structured interviews to elicit data on behavioral risk factors prior to their examinations. HPV genotyping was done on exfoliated cervical cell samples. We estimated relative risks (RR) for HPV types and cofactors for CIN3+, overall and stratified by age group. Results: After 2 years of follow-up, we identified 178 CIN3+, 1,305 CIN0–2, and 175 indeterminate outcomes. Nonvaccine HR-HPV types were only associated with CIN3+ among women ≥30 (RR = 2.3, 95% CI: 1.5–3.4; <30: RR = 0.9). Among all HR-HPV–positive women, adjusting for age, significant cofactors for CIN3+ included current smoking (RR = 1.5), former smoking (RR = 1.8), regular Pap screening (RR = 0.7), current regular condom use (RR = 0.5), and parity ≥5 (RR = 1.6, Ptrend for increasing parity = 0.07). However, the parity association differed by age group (≥30: RR = 1.8, Ptrend = 0.008; <30: RR = 0.9; Ptrend =.55). Conclusion: Subgroup variation by age in the risk of CIN3+ points to the importance of the timing of exposures in relation to CIN3+ detection. Impact: Future screening strategies need to consider natural history and secular trends in cofactor prevalence in the pursuit of appropriately sensitive and specific screening tools applied to appropriate age groups. Cancer Epidemiol Biomarkers Prev; 21(1); 111–21. ©2011 AACR.

Interphase cytogenetic analysis of 1q12 satellite III DNA in melanocytic lesions: increased aneuploidy with malignant histology.

To examine the relationship of chromosome 1 copy number to melanocytic tumorigenesis, interphase cytogenetic analysis of 1q12 satellite III DNA was performed on the spectrum of melanocytic lesions comprising Clarks tumor progression model. Results showed increased copy number in a “step off” pattern between melanoma in-situ and the intraepidermal component of invasive melanoma rather than a progression between each lesional group. These findings support Clarks concept of independent clonal expansion of a cell population giving rise to the vertical growth phase and further demonstrates increased chromosome 1 copy number as a late event in melanoma tumor progression.

An unusual cervical carcinoma showing exception to epitheliotropism of human papillomavirus.

Human papillomaviruses (HPV) infect epithelial tissues but have not been previously detected within mesenchymal cells. During a systematic investigation of FIGO stage Ib cervical cancers with colorimetric in situ hybridization, we detected HPV 16 DNA within the stromal compartment of an unusual undifferentiated carcinoma. The mesenchymal nature of the HPV-containing cells was confirmed by immunohistochemistry and electron microscopy. No viral particles were identified. Sequencing the majority of the HPV 16 genome identified few changes from the revised reference clone; all previously reported in other HPV 16 variants. These viral changes are unlikely to explain the exceptional mesenchymal localization of the HPV 16 DNA in this case.

Human papillomavirus in amniotic fluid

BackgroundThere is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes.MethodsResidual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV.ResultsThere were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples.ConclusionGiven the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.

Evaluation of RNA markers for early detection of cervical neoplasia in exfoliated cervical cells

Numerous molecular biomarkers have been suggested for early detection of cervical cancer, but their usefulness in routinely collected exfoliated cells remains uncertain. We used quantitative reverse transcription-PCR to evaluate expression of 40 candidate genes as markers for high-grade cervical intraepithelial neoplasia (CIN) in exfoliated cervical cells collected at the time of colposcopy. Samples from the 93 women with CIN3 or cancer were compared with those from 186 women without disease matched (1:2) for age, race, and high-risk human papillomavirus status. Normalized threshold cycles (Ct) for each gene were analyzed by receiver operating characteristics to determine their diagnostic performance in a split sample validation approach. Six markers were confirmed by an area under the curve >0.6 in both sample sets: claudin 1 (0.75), minichromosome maintenance deficient 5 (0.71) and 7 (0.64), cell division cycle 6 homologue (0.71), antigen identified by monoclonal antibody Ki-67 (0.66), and SHC SH2-domain binding protein 1 (0.61). The sensitivity for individual markers was relatively low and a combination of five genes to a panel resulted in 60% sensitivity with 76% specificity, not positively increasing this performance. Although the results did not indicate superiority of RNA markers for cervical cancer screening, their performance in detecting disease in women referred for colposcopy suggests that the genes and pathways they highlight could be useful in alternative detection formats or in combination with other screening indicators. (Cancer Epidemiol Biomarkers Prev 2007;16(2):295–301)