Rosemary E. Zuna

Human Tumor Antigens Tn and Sialyl Tn Arise from Mutations in Cosmc

Neoplastic lesions typically express specific carbohydrate antigens on glycolipids, mucins, and other glycoproteins. Such antigens are often under epigenetic control and are subject to reversion and loss upon therapeutic selective pressure. We report here that two of the most common tumor-associated carbohydrate antigens, Tn and sialyl Tn (STn), result from somatic mutations in the gene Cosmc that encodes a molecular chaperone required for formation of the active T-synthase. Diverse neoplastic lesions, including colon cancer and melanoma-derived cells lines, expressed both Tn and STn antigen due to loss-of-function mutations in Cosmc. In addition, two human cervical cancer specimens that showed expression of the Tn/STn antigens were also found to have mutations in Cosmc and loss of heterozygosity for the cross-linked Cosmc locus. This is the first example of somatic mutations in multiple types of cancers that cause global alterations in cell surface carbohydrate antigen expression.

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.

Worldwide genomic diversity of the high-risk human papillomavirus types 31, 35, 52, and 58, four close relatives of human papillomavirus type 16

ABSTRACT Among the more than one hundred formally described human papillomavirus (HPV) types, 18 are referred to as high-risk HPV types due to their association with anogenital cancer. Despite pathogenic similarities, these types form three remotely related taxonomic groups. One of these groups is called HPV species 9 and is formed by HPV-16, the most common and best-studied type, together with HPV-31, -33, -35, -52, -58, and -67. Previous worldwide comparisons of HPV-16 samples showed about 2% nucleotide diversity between isolates, which were subsequently termed variants. The distribution of divergent variants has been found to correlate frequently with the geographic origin and the ethnicity of the infected patients and led to the concept of unique African, European, Asian, and Native American HPV-16 variants. In the current study, we address the question of whether geography and ethnicity also correlate with sequence variations found for HPV-31, -35, -52, and -58. This was done by sequencing the long control region in samples derived from Europe, Asia, and Africa, and from immigrant populations in North and South America. We observed maximal divergence between any two variants within each of these four HPV types ranging from 1.8 to 3.6% based on nucleotide exchanges and, occasionally, on insertions and deletions. Similar to the case with HPV-16, these mutations are not random but indicate a relationship between the variants in form of phylogenetic trees. An interesting example is presented by a 16-bp insert in select variants of HPV-35, which appears to have given rise to additional variants by nucleotide exchanges within the insert. All trees showed distinct phylogenetic topologies, ranging from dichotomic branching in the case of HPV-31 to star phylogenies of the other three types. No clear similarities between these types or between these types and HPV-16 exist. While variant branches in some types were specific for Europe, Africa, or East Asia, none of the four trees reflected human evolution and spread to the extent illustrated by HPV-16. One possible explanation is that the rare HPV types that we studied spread and thereby diversified more slowly than the more abundant HPV-16 and may have established much of todays variant diversity already before the worldwide spread of humans 100,000 years ago. Most variants had prototypic amino acid sequences within the E6 oncoprotein and a segment of the L1 capsid protein. Some had one, two, or three amino acid substitutions in these regions, which might indicate biological and pathogenic diversity between the variants of each HPV type.

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

Human Papillomavirus Cofactors by Disease Progression and Human Papillomavirus Types in the Study to Understand Cervical Cancer Early Endpoints and Determinants

Human papillomavirus (HPV) cofactors for cervical cancer include smoking, multiparity, and oral contraceptive use, but their mechanisms of action are not fully understood. It is also unknown whether cofactors vary by HPV genotypes. The Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED) is a cross-sectional study comprising women referred to the University of Oklahoma from November 2003 to September 2007 for abnormal cervical screening results. Detailed questionnaire data and liquid cytology specimens were collected and the latter was genotyped for HPV using the LINEAR ARRAY HPV Genotyping Test. The present analysis includes women with both questionnaire and HPV data and diagnosed with <CIN1 (n = 535), CIN1 (n = 497), CIN2 (n = 336), CIN3 (n = 292), and cancer (n = 80). We evaluated HPV types and cofactors among HPV-infected women by calculating odds ratios (OR) and 95% confidence intervals (95% CI) for CIN3 and CIN2 separately compared with <CIN2 using a polytomous logistic regression model; cancers were excluded from further analysis due to the substantially higher ages of these women. We found that HPV-infected women with minor histologic or cytologic abnormalities (e.g., CIN1, ASCUS, and LSIL) were indistinguishable from those with normal histology/cytology and were thus combined to form the referent group (<CIN2). Among women positive for oncogenic HPV, current smokers had a 2.5-fold increased risk for CIN3 (95% CI, 1.8-3.6) compared with nonsmokers. Among HPV16-infected women, current smokers had elevated risk for both CIN2 (OR, 1.9; 95% CI, 1.1-3.2) and CIN3 (OR, 2.7; 95% CI, 1.6-4.6). Our data suggest that non-HPV16-related CIN2 likely reflects a combination of CIN1 and CIN3 diagnosis, whereas HPV16-related CIN2 may indicate a precancerous state. Investigations on the molecular distinctions along the disease continuum of cervical pathogenesis by HPV type are needed. (Cancer Epidemiol Biomarkers Prev 2009;18(1):113–20)

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo.

Aberrant activation of Wingless-type (Wnt)/β-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2′-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G2/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of β-catenin and a significant inhibition of the Wnt/β-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/β-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

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.

Association of HPV16 E6 variants with diagnostic severity in cervical cytology samples of 354 women in a US population

It has been suggested that DNA sequence variants of HPV16 contribute to differences in the behavior of individual cervical lesions. To address this question, we have analyzed the association of HPV16 variants with diagnostic severity in 354 HPV16‐positive Oklahoman women. HPV16 variant status was determined by PCR amplification and DNA sequencing of the E6 open reading frame. European sequences were identified in 86% of samples and 14% were non‐European. Of the 51 non‐European cases, 61% were Asian‐American, 23% African and 16% were Native American variants. European prototype and related variants were present in comparable numbers (43% each) but the relative proportion of each differed with diagnostic category. In general, the proportion of European variants and non‐European variants increased with diagnostic severity while the European prototype decreased. When adjusted for age and race (white, black or Hispanic), the increased risk for carcinoma/severe dysplasia for non‐European variants was statistically significant with an odds ratio of 3.8 (1.3–10.7). However, the analogous comparison for the European variants, although also showing increased association with carcinoma/severe dysplasia, did not reach statistical significance (OR = 1.6 (95% CI 0.7–3.6). Overall, HPV16 European sequences (both prototype and related variants), were predominant in Oklahoman women including those with cancers. This suggests that while there appear to be differences among the HPV16‐variant categories in risk for progression to invasive cancer, all variant categories are associated with the development of invasive cancer.

Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling

Significance Cervical cancer (CxCa) is the second most frequent cancer in women and the third leading cause of cancer death in women worldwide. Our global analysis of gene expression in normal, precancerous, and cancerous cervical tissue shows increased DNA replication/repair and cell proliferation followed by substantial metabolic shifts. We observed a dramatic, progressive decrease in estrogen receptor alpha (ERα) in tumor progression, and ranking specimens by estrogen-responsive gene expression correlated remarkably with histopathology. Whereas ERα expression shuts off in tumor epithelium, stromal fibroblasts in the microenvironment retain ERα, and the data indicate estrogen-related alteration of several candidate stroma–tumor signaling pathways. Our findings strongly support a role of stromal estrogen signaling in CxCa development with implications for CxCa management and control. To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1– CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA− precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1−, CD34−, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma–tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.