Racial/ethnic variation in the prevalence of vaccine-related human papillomavirus genotypes.

Objective: There are currently three licensed human papillomavirus (HPV) vaccines that protect against cervical cancer. Here we compare the prevalence of bi-, quadri-, and nonavalent vaccine-related HPV genotypes in a multi-ethnic sample of non-Hispanic white, non-Hispanic black, Hispanic, and Asian women. Design: Patients in this analysis (n = 419) represent a subset of women with a previous abnormal Pap test participating in a clinical trial. HPV genotyping was conducted using the Roche Linear Array. Prevalent HPV genotypes were grouped according to their inclusion in each of the vaccines: bivalent (16, 18), quadrivalent (16, 18, 6, 11), and nonavalent (16, 18, 31, 33, 45, 52, 58, 6, 11). Results: The prevalence of HPV genotypes covered by the bi-/quadrivalent vaccines was lowest among non-Hispanic black (15%) and Hispanic women (20%), compared to non-Hispanic white (38%) and Asian women (38%). Across all racial/ethnic groups, a large proportion of infections (38%-49%) were with genotypes included in the nonavalent vaccine. However, the prevalence of HPV genotypes not covered by any vaccine was significantly higher among non-Hispanic black (36%) and Hispanic women (42%), compared to non-Hispanic white (24%) and Asian women (16%) (p < 0.001). Racial/ethnic differences in HPV genotype prevalence were observed when controlling for demographic and sexual behavior characteristics, as well as when restricting the analysis to women with CIN 2+. Conclusion: Our data suggest racial/ethnic differences in the prevalence of vaccine-related HPV genotypes. In particular, non-Hispanic black and Hispanic women had the lowest prevalence of HPV genotypes covered by the bi-/quadrivalent vaccines. While a large proportion of their infections were covered by the nonavalent vaccine, non-Hispanic black and Hispanic women also had the highest prevalence of HPV genotypes not covered by any vaccine.

Comparing the Performance of Hybrid Capture II and Polymerase Chain Reaction (PCR) for the Identification of Cervical Dysplasia in the Screening and Diagnostic Settings.

OBJECTIVE: Both PCR and Hybrid Capture II (HCII) have been used for identifying cervical dysplasia; however, comparisons on the performance between these two tests show inconsistent results. We evaluated the performance of HCII and PCR MY09/11 in both screening and diagnostic populations in sub-sample of 1,675 non-pregnant women from a cohort in three clinical centers in the United States and Canada. METHODS: Sensitivity, specificity, positive predictive value, negative predictive value, and concordance between the two tests were calculated. RESULTS: Specificity of HCII in detecting low-grade squamous intraepithelial lesion (LSIL) was higher in the screening group (88.7%; 95% CI: 86.2%-90.8%) compared to the diagnostic group (46.3%; 95% CI: 42.1%-50.6%); however, specificity of PCR was low in both the screening (32.8%; 95% CI: 29.6%-36.2%) and diagnostic (14.4%; 95% CI: 11.6%-17.6%) groups. There was comparable sensitivity by both tests in both groups to detect high-grade squamous intraepithelial lesion (HSIL); however, HCII was more specific (89.1%; 95% CI: 86.8%-91.0%; 66.2%; 95% CI: 62.0%-70.1%) than PCR (33.3%; 95% CI: 30.2%-36.5%; 17.9%; 95% CI: 14.8%-21.6%) in the screening and diagnostic groups, respectively. Overall agreement for HPV positivity was approximately 50% between HCII and PCR MY09/11; with more positive results coming from the PCR MY09/11. CONCLUSION: In the current study, PCR MY09/11 was more sensitive but less specific than HCII in detecting LSIL, and HCII was more sensitive and specific in detecting HSIL than PCR in both screening and diagnostic groups.

Associations between human herpesvirus-6, human papillomavirus and cervical cancer.

Cervical cancer (CxCa) is the second most common cancer among women globally. Human papillomavirus (HPV) infection is thought to be a necessary, but not sufficient, causal factor in CxCa development. Why some women are able to clear HPV infection with no adverse effects, whereas others develop cancer, remains unclear. HHV-6 has demonstrated transformative abilities and has been shown to be present in the genital tract. However, based on the current evidence, we cannot conclude that HHV-6 is a co-factor in HPV-associated carcinogenesis. Nonetheless, future research is warranted because of several crucial gaps in the literature.

Epidemiologic differentiation of diagnostic and screening populations for the assessment of cervical dysplasia using optical technologies.

BACKGROUND: We report here the logistic modeling of the epidemiologic differences between a diagnostic population and a screening population recruited for the study of optical technologies for cervical cancer detection. OBJECTIVES: The goal of this analysis was to determine if there were differences in the sociodemographic or clinical factors between subjects recruited to our diagnostic and screening trials. METHODS: Epidemiologic data were obtained from a risk factor interview as a component of a multicenter Phase II clinical trial that used fluorescence and reflectance point spectroscopy to diagnose cervical disease. Participants with recent or past abnormal findings on a Papanicolaou smear were grouped into the diagnostic (high-risk) population, whereas those with a history of normal findings on Papanicolaou smears and no cervical treatments were grouped into the screening (low-risk) population. RESULTS: Our model revealed that nonwhite race, higher than a high school education, and peri- and postmenopausal status were associated with the screening population. A history of genital infections, current oral contraceptive use, human papillomavirus positivity (by Hybrid Capture II and consensus polymerase chain reaction), and worst histological diagnosis at clinic visit were important predictors of being in the diagnostic group. CONCLUSIONS: We were successful in recruiting 2 distinctive populations and anticipate being able to use these results to more correctly classify women at higher risk for cervical lesions in our future studies of optical spectroscopy.

Optical technologies and molecular imaging for cervical neoplasia: a program project update.

There is an urgent global need for effective and affordable approaches to cervical cancer screening and diagnosis. In developing nations, cervical malignancies remain the leading cause of cancer-related deaths in women. This reality may be difficult to accept given that these deaths are largely preventable; where cervical screening programs have been implemented, cervical cancer-related deaths have decreased dramatically. In developed countries, the challenges of cervical disease stem from high costs and overtreatment. The National Cancer Institute-funded Program Project is evaluating the applicability of optical technologies in cervical cancer. The mandate of the project is to create tools for disease detection and diagnosis that are inexpensive, require minimal expertise, are more accurate than existing modalities, and can be feasibly implemented in a variety of clinical settings. This article presents the status and long-term goals of the project.

Accuracy of optical spectroscopy for the detection of cervical intraepithelial neoplasia: Testing a device as an adjunct to colposcopy.

Testing emerging technologies involves the evaluation of biologic plausibility, technical efficacy, clinical effectiveness, patient satisfaction, and cost-effectiveness. The objective of this study was to select an effective classification algorithm for optical spectroscopy as an adjunct to colposcopy and obtain preliminary estimates of its accuracy for the detection of CIN 2 or worse. We recruited 1,000 patients from screening and prevention clinics and 850 patients from colposcopy clinics at two comprehensive cancer centers and a community hospital. Optical spectroscopy was performed, and 4,864 biopsies were obtained from the sites measured, including abnormal and normal colposcopic areas. The gold standard was the histologic report of biopsies, read 2 to 3 times by histopathologists blinded to the cytologic, histopathologic, and spectroscopic results. We calculated sensitivities, specificities, receiver operating characteristic (ROC) curves, and areas under the ROC curves. We identified a cutpoint for an algorithm based on optical spectroscopy that yielded an estimated sensitivity of 1.00 [95% confidence interval (CI) = 0.92-1.00] and an estimated specificity of 0.71 [95% CI = 0.62-0.79] in a combined screening and diagnostic population. The positive and negative predictive values were 0.58 and 1.00, respectively. The area under the ROC curve was 0.85 (95% CI = 0.81-0.89). The per-patient and per-site performance were similar in the diagnostic and poorer in the screening settings. Like colposcopy, the device performs best in a diagnostic population. Alternative statistical approaches demonstrate that the analysis is robust and that spectroscopy works as well as or slightly better than colposcopy for the detection of CIN 2 to cancer.

Prospective evaluation of a portable depth-sensitive optical spectroscopy device to identify oral neoplasia.

A portable, depth-sensitive clinical spectroscopy device for noninvasive early diagnosis of oral cancer is described. We carried out a pilot study to evaluate the ability of the device to identify oral neoplasia using a previously developed diagnostic algorithm. A total of 79 oral sites in 33 subjects, including 28 patients with oral lesions and 5 healthy volunteers, were measured and analyzed. Measurements of 54 nonkeratinized oral sites yielded an area under the receiver operating characteristic curve of 0.90. Measurements of 25 keratinized oral sites yielded an area under the receiver operating characteristic curve of 0.83.

Delivery of optical contrast agents using Triton-X100, part 1: reversible permeabilization of live cells for intracellular labeling.

Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility of Triton-X100-mediated permeabilization in live mammalian cells. We report a series of studies to characterize macromolecule delivery in cells following Triton-X100 treatment. Using this approach, we demonstrate that molecules ranging from 1 to 150 kDa in molecular weight can be reproducibly delivered into live cells by controlling the moles of Triton-X100 relative to the number of cells to be treated. When Triton-X100 is administered at or near the minimum effective concentration, cell permeabilization is generally reversed within 24 h, and treated cells continue to proliferate and show metabolic activity during the restoration of membrane integrity. We conclude that Triton-X100 is a promising permeabilization agent for efficient and reproducible delivery of optical contrast agents into live mammalian cells.

Delivery of optical contrast agents using Triton-X100, part 2: enhanced mucosal permeation for the detection of cancer biomarkers.

Uniform delivery of optical contrast agents through mucosal tissue has proven a significant challenge. Topical permeation enhancers that have proven useful for skin demonstrate limited success in mucosal tissue. We sought to develop a topical permeation strategy capable of delivering tissue-impermeant molecular-specific contrast agents through mucosal epithelium in a uniform, controlled manner. We demonstrate that Triton-X100 can be utilized to deliver targeted and untargeted optical contrast agents through freshly excised normal mucosal epithelium and epithelial cancer. Macromolecules up to 150 kDa in size were successfully delivered via transcellular and paracellular routes. The depth of Triton-mediated permeation was modulated by varying the treatment time and concentration. Uniform epithelial penetration to a depth of 500 mum was achieved in approximately 1.5 h for molecules of 40 kDa or less. Larger optical probes required longer treatment times. Coadministration of molecular-specific contrast agents with Triton-X100 treatment facilitated simultaneous labeling of biomarkers on the cell membrane, in the cytoplasm, and in the nucleus with high specificity. Together, these data suggest that Triton-X100 is a promising topical permeation enhancer for mucosal delivery of tissue-impermeant molecular-specific optical contrast agents.

The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.

p53 codon 72 polymorphism associated with risk of human papillomavirus-associated squamous cell carcinoma of the oropharynx in never-smokers.

The tumor suppressor p53 protein can be bound, degraded and inactivated by the human papillomavirus (HPV) E6 oncoprotein. The p53 protein's susceptibility to this oncoprotein may be influenced by the p53 codon 72 polymorphism, but the role of such a polymorphism in the development of HPV16-associated squamous cell carcinoma of the oropharynx (SCCOP) has not been established. To investigate the role of the p53 codon 72 polymorphism in the risk of HPV16-associated SCCOP, we conducted a hospital-based case-control study of 188 non-Hispanic white patients with newly diagnosed SCCOP and 342 cancer-free control subjects frequency matched by age (+/-5 years), sex, tobacco smoking status and alcohol drinking status. We found that HPV16 seropositivity was associated with an increased risk of SCCOP [adjusted odds ratio (OR), 5.7; 95% confidence interval (CI), 3.7-8.7], especially among never-smokers (adjusted OR, 14.1; 95% CI, 6.0-32.9) and among subjects with the p53 codon 72 variant genotypes [Arginine (Arg)/Proline (Pro) and Pro/Pro] (adjusted OR, 9.2; 95% CI, 4.7-17.7). A significant multiplicative interaction on the risk of SCCOP was also found between the p53 codon 72 polymorphism and HPV16 seropositivity (P = 0.05). Among never-smokers, the risk of SCCOP for those who had both HPV16 seropositivity and p53 codon 72 variant genotypes (Arg/Pro + Pro/Pro) was particularly high (adjusted OR, 22.5; 95% CI, 4.8-106.2). These findings suggest that p53 codon 72 variant genotypes modify the risk of HPV16-associated SCCOP and may be markers of genetic susceptibility to HPV16-associated SCCOP, especially among never-smokers.

Epidemiologic modeling of cervical dysplasia with molecular and cytopathological markers.

OBJECTIVES: Conventional cervical screening is insufficient at identifying patients who are likely to progress from cervical dysplasia to carcinoma. Traditional epidemiologic studies have identified potential factors to aid in the discrimination between those lesions likely to progress from those likely to regress; however, there is still much to be learned. To examine the role of traditional epidemiologic factors in conjunction with molecular markers of human papillomavirus activity, we studied a group of women attending colposcopy clinics in Houston, TX, and Vancouver, BC, between October 2000 and July 2003. METHODS: Quantitative real-time PCR was used to measure mRNA expression of the human papillomavirus E7 gene, and quantitative cytology was used to gather information about the DNA index and chromatin features of the cells from these women. Logistic regression was used to establish predictor variables for histologic grade based on the epidemiologic risk factors and the molecular markers. RESULTS: The most predictive factors were mRNA level, DNA index, parity, and age. The ROC curve for the individual logits indicated excellent discrimination. CONCLUSION: In accordance with other authors, these results suggest that molecular markers of the malignant process should be included in analyses looking to predict the progression potential of cervical lesions.

Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Human papillomavirus-related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture.

BACKGROUND: Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real-time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples. METHODS: Human papillomavirus (HPV) 16 and 18 E7 mRNA expression was measured using quantitative RT-PCR. DNA index and chromatin measures were taken from image cytology samples. The chromatin features, through discriminant analysis, were aggregated into a score, and both measurements were related to mRNA expression. RESULTS: mRNA level and DNA index show an increasing trend over increasing histological grades. However, DNA index and chromatin score were not correlated to mRNA levels in these samples. Chromatin score differed by mRNA type found with HPV 18 infected samples having a higher score than those with HPV 16. Samples infected with HPV 16 and HPV 18 had even higher chromatin scores. CONCLUSIONS: DNA index and chromatin score were not directly correlated with mRNA levels. However both mRNA and DNA index were related to histological grade, and chromatin score was associated with HPV type. Therefore, DNA index and mRNA levels could be independent predictors of cervical dysplasia, and chromatin score could be related to the viral integration process in cells infected with HPV 18 or dual infections.

Kappa statistics to measure interrater and intrarater agreement for 1790 cervical biopsy specimens among twelve pathologists: qualitative histopathologic analysis and methodologic issues.

BACKGROUND: As part of a program project to evaluate emerging optical technologies for cervical neoplasia, we performed fluorescence and reflectance spectroscopic examinations of patients with abnormal Papanicolaou smears. Biopsy specimens were taken from each area and measured optically, and study pathologists performed qualitative histopathologic readings. Several methodologic issues arose in this analysis: (1) the interpathologist and intrapathologist agreement between institutions for the 1790 biopsy specimens; (2) the interinstitutional agreement among the two institutions conducting the trials on 117 randomly chosen biopsy specimens; (3) the interinstitutional agreement among the two institutions and a third expert gynecologic pathologist to ensure the expert readings were comparable to those outside both institutions on 117 randomly chosen biopsy specimens; and (4) an additional three reviews of the 106 difficult biopsy specimens by all three institutions. METHODS: All 1790 specimens from 850 patients were reviewed three times at each institution in blinded fashion; those for which the first and second reviews were identical were not reviewed a third time. A randomly selected sample of 117 specimens was randomly ordered and read by study pathologists at The University of Texas M. D. Anderson Cancer Center, British Columbia Cancer Agency (BCCA), and Brigham and Women's Hospital (BWH). The 106 difficult cases were treated in the same manner as the randomized and random-ordered cases. Generalized, unweighted, and weighted kappas and their 95% confidence intervals were used to assess agreement. Binary comparisons were used to compare diagnostic categories. FINDINGS: The kappas for the three readings of the overall data set using eight-category World Health Organization (WHO) criteria were as follows: 0.66 for the generalized, 0.72 for weighted, and ranged from 0.59 to 0.94 unweighted binary categories; those read using four-category Bethesda criteria: 0.70 for generalized, 0.69 for weighted, and 0.56-0.94 for unweighted binary categories. For the pool versus the study pathologist readings, the eight-category kappa was 0.51 for generalized, 0.72 for weighted, and 0.56-0.82 for unweighted binary categories; for those read using Bethesda criteria: 0.70 for generalized, 0.70 for weighted, and 0.59-0.82 for the unweighted binary categories. The interpathologist and intrapathologist readings were fair by Landis standards at the low end of the diagnostic scale (atypia, human papillomavirus, and CIN1) and substantial to almost perfect at the high end (CIN2, CIN3, and CIS). The randomly selected and randomly ordered sample of 117 specimens read with the WHO system yielded a generalized kappa of 0.45; among the three institutions (M. D. Anderson Cancer Center vs. BCCA, M. D. Anderson vs. BWH, and BCCA vs. BWH), the unweighted kappas were 0.46, 0.41, and 0.49 and the weighted were 0.65, 0.66, and 0.68, respectively; for the Bethesda, a generalized kappa of 0.65, unweighted kappas of 0.66, 0.65, and 0.47, and weighted of 0.74, 0.72, and 0.74. The difficult specimens read with the WHO system yielded a generalized kappa of 0.23; among the three institutions the unweighted kappas were 0.20, 0.30, and 0.37, and the weighted were 0.17, 0.34, and 0.31; for the Bethesda, a generalized kappa of 0.25; among the three institutions, the unweighted kappas were 0.21, 0.32, and 0.37, and the weighted were: 0.07, 0.21, and 0.37, respectively. INTERPRETATION: Kappas in this expert group of pathologists were in the moderate, substantial, and almost perfect ranges for the overall and randomized samples. The randomized sample was representative of the larger sample. The kappa of the specimens for which disagreements arose was, predictably, in the slight range. Our findings will aid both the correlations with optical measurements using fluorescence and reflectance spectroscopy and the quantitative histopathologic analysis of these study specimens.

Subvisual chromatin changes in cervical epithelium measured by texture image analysis and correlated with HPV.

OBJECTIVES: In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture II test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. METHODS: A group of 1544 patients entered the optical technology trials, generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture II test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. RESULTS: The sensitivity of the classification was 52-54% and the specificity was 77-78%. Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. CONCLUSIONS: Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV-associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.

Correlation of human papillomavirus type 16 and human papillomavirus type 18 e7 messenger RNA levels with degree of cervical dysplasia.

Infection with certain types of human papillomavirus (HPV) is a necessary event in the development of cervical carcinoma; however, not all women who become infected with HPV will progress to cancer. Much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation. Little is known about the additional factors needed to drive the process. Quantitative real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL). Prevalence of mRNA transcripts was lower among normal women (27%) than for women with LSIL (40%) and HSIL (37%). Mean levels ranged from 2.0 (ln scale per 20 ng cDNA) among normal women to 4.2 among those with HSIL, with a significant trend (P=0.008). This trend was only significant for HPV 18 transcripts if separately analyzed by HPV type. The transcriptional activity of HPV 18 is higher than that of HPV 16 and increases with increasing level of dysplasia. This is in concert with the findings of other studies, and reinforces the notion that HPV 18 is a more aggressive viral type. Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from a cervical swab, thus providing a way to better screen women who may progress to higher grade lesions or invasive carcinoma from those who will spontaneously regress.

Differential susceptibility of metastatic and primary oral cancer cells to TRAIL-induced apoptosis.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immune surveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by its inhibitors, known as cystatins. We examined the TRAIL-mediated cytotoxicity rates of clonally-related primary and metastatic oral cancer (OC) cells and correlated them with the expression levels of TRAIL receptors, cathepsin B and cystatins A, B, C and M. Two pairs of primary (686Tu and 101A) and metastatic (686Ln and 101B) OC cell lines were treated with various concentrations (5 to 1000 ng/ml) of recombinant human TRAIL protein for 14 h, and cell viability and apoptotic rate were determined. In both pairs of cell lines, primary OC cells revealed greater susceptibility to TRAIL than their metastatic counterparts. The protein synthesis inhibitor cycloheximide markedly increased the TRAIL sensitivity of these cell lines, whereas the CB-specific chemical inhibitor CA-074 markedly reduced the sensitivity of primary OC cells to TRAIL. DNA laddering and M30 CytoDEATH immunodetection assays confirmed that TRAIL-induced OC cell death is an apoptotic process. Expression levels of TRAIL death (DR4 and DR5) and decoy (DcR1 and DcR2) receptors were not different between primary and metastatic OC cells. However, expression levels of cystatins were higher in metastatic OC cells than in their respective primary cells, whereas CB levels remain unchanged. Cathepsin B is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells may cause their greater resistance to TRAIL-induced apoptosis. Our data suggest that high expression of cystatins in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.

Correlation of protease-activated receptor-1 with differentiation markers in squamous cell carcinoma of the head and neck and its implication in lymph node metastasis.

PURPOSE: Protease-activated receptor-1 (PAR-1) is a G-protein-coupled receptor that contributes to multiple signal transduction pathways. Although the functions of PAR-1 in many normal cells, such as platelets and astrocytes, have been well studied, its roles in cancer progression and metastasis have not been fully elucidated, and studies to date appear contradictory. EXPERIMENTAL DESIGN: To clarify the function of PAR-1 in metastasis of squamous cell carcinoma of the head and neck (SCCHN), we examined PAR-1 expression in clinical specimens by immunohistochemistry and in SCCHN cell lines by immunoblotting. Furthermore, par-1 cDNA-transfected SCCHN cell lines were also used to verify PAR-1-mediated pathway. RESULTS: The metastatic tumors showed a lower percentage of PAR-1-positive cells (46%) and lower levels of PAR-1 expression (median weight index = 10) than node negative primary tumors (80% and median weight index = 60, respectively). In addition, expression level of PAR-1 positively correlated with levels of keratinocyte differentiation markers keratin-1, -10, and -11. Additional studies using sense and antisense par-1 cDNA-transfected SCCHN cell lines illustrated that the presence of PAR-1 was required for the expression of involucrin, a keratinocyte differentiation marker. PAR-1 expression also contributes to activation of the mitogen-activated protein kinase (MAPK) pathway. Blocking MAPK activation by a mitogen-activated protein/extracellular signal-regulated kinase inhibitor, not by a phosphatidylinositol 3'-kinase inhibitor, reduced level of involucrin, suggesting that regulation of involucrin by PAR-1 is partially through the MAPK signaling pathway. CONCLUSIONS: Our study suggests that PAR-1 signaling induces differentiation markers in SCCHN cells, and its expression is conversely correlated with cervical lymph node metastasis.