Racial/ethnic differences in HPV 16/18 genotypes and integration status among women with a history of cytological abnormalities.

OBJECTIVE: HPV genotype distribution varies by race/ethnicity, but is unclear whether there are racial/ethnic variations in HPV 16/18 integration in the host genome. We describe HPV16/18 infection and integration status in a racially/ethnically diverse sample of women with a recent abnormal Pap test. METHODS: Patients (n=640) represent a subset of women participating in a clinical trial. Cervical swabs were tested for HPV16/18 DNA using type-specific polymerase chain reaction assays. Viral integration status was assessed using type-specific integration assays and categorized as fully integrated, fully non-integrated, or mixed. Unconditional logistic regression was used to generate unadjusted (OR) and adjusted odds ratios (aOR) to assess the association between self-reported race/ethnicity and risk of these outcomes. RESULTS: Hispanic and non-Hispanic black women had half the odds of prevalent HPV16 compared to non-Hispanic white women (aORs: 0.43 and 0.45, respectively). The prevalence odds of HPV18 was less than half among Hispanic women (aOR: 0.48), but not significantly different between black and white women (aOR: 0.72). Among women with prevalent HPV16, the odds of fully integrated viral DNA were significantly higher among black women (aORs: 2.78) and marginally higher among Hispanic women (aOR: 1.93). No racial/ethnic differences were observed for HPV18 DNA integration. CONCLUSIONS: While HPV16 and 18 infections were less prevalent among Hispanic and black women compared to whites, their HPV16 DNA was more likely to be present in a fully integrated state. This could potentially contribute to the higher rates of abnormal cytology and cervical dysplasia observed among Hispanic and black women.

Human papillomavirus infection: biology, epidemiology, and prevention.

Over the past several decades, knowledge of the biology and epidemiology of human papillomavirus (HPV) infection has increased tremendously. However, there are still many unanswered questions concerning the interaction of the virus with its host. The virus has been identified as a necessary causal agent for cervical squamous neoplasia and has been linked to the development of neoplasia in several other mucosal sites. The viral oncogenes E6 and E7 are the major players in the virus' scheme to evade the immune system and use the host cell replication machinery to survive. Many risk factors for infection with HPV have been identified; however, the focus now centers on identifying risk factors for persistence of the infection as it is likely that transient infections play a very small role in the overall development of clinical disease. Prevention measures to date have centered around screening programs, mostly for cervical cancer, including the perfection of screening techniques and inclusion of molecular testing for HPV into screening regimens. The development of prophylactic and therapeutic vaccines has also increased as primary prevention measures appear to have the best hope for long-term effects on cancer incidence.

A pilot study for a screening trial of cervical fluorescence spectroscopy.

Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. In preparation for a multicenter phase II screening trial, a pilot trial was conducted to test data collection and patient examination procedures, use data forms, time procedures, and identify problems with preliminary data analysis. Women 18 years of age and older underwent a questionnaire, a complete history, and a physical examination, including a pan-colposcopy of the lower genital tract. A fiber-optic probe measured fluorescence excitation-emission matrices at 1-3 cervical sites for 58 women. The data collection procedures, data forms, and procedure times worked well, although collection times for all the clinical data take an average of 28 min. The clinical team followed procedures well, and the data could be retrieved from the database at all sites. The multivariate analysis algorithm correctly identified squamous normal tissue 99% of the time and columnar normal tissue only 7%. The assessment of ploidy from monolayer samples was not accurate in this small sample. The study was successful as a pilot trial. We learned who participated, who withdrew, how often abnormalities were present, and that algorithms that have worked extremely well in previous studies do not work as well when a few study parameters are changed. The current algorithm for diagnosis identified squamous normal tissue very accurately and did less well for columnar normal tissue. Inflammation may be an explanation for this phenomenon. Fluorescence spectroscopy is a promising technology for the detection of epithelial precancers and cancers. The screening trial of fluorescence and reflectance spectroscopy was successful.

Molecular imaging of carcinogenesis with immuno-targeted nanoparticles.

Molecular characterization of cancer could have important clinical benefits such as earlier cancer detection based on molecular characterization, the ability to predict the risk of cancer progression, real time margin detection, the ability to rationally select molecular therapy and to monitor response to the therapy. We present a new class of molecular specific contrast agents for optical imaging of carcinogenesis in vivo - gold nanoparticles conjugated with monoclonal antibodies specific for cancer biomarkers.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Persistent productive Epstein-Barr virus replication in normal epithelial cells in vivo.

Productive Epstein-Barr virus (EBV) replication characterizes hairy leukoplakia, an oral epithelial lesion typically occurring in individuals infected with human immunodeficiency virus (HIV). Serial tongue biopsy specimens were obtained from HIV-infected subjects before, during, and after valacyclovir treatment. EBV replication was detected by Southern hybridization to linear terminal EBV genome fragments, reverse-transcriptase polymerase chain reaction amplification of EBV replicative gene transcripts, immunohistochemical detection of EBV replicative protein, and in situ hybridization to EBV DNA. EBV replication was detected in both hairy leukoplakia and normal tongue tissues. Valacyclovir treatment completely abrogated EBV replication in vivo, resulting in resolution of hairy leukoplakia when it was present. EBV replication returned in normal tongue epithelial cells after valacyclovir treatment. These data suggest that normal oral epithelium supports persistent EBV infection in individuals infected with HIV and that productive EBV replication is necessary but not sufficient for the pathogenesis of oral hairy leukoplakia.

Papillary squamous cell carcinomas of the upper aerodigestive tract: a clinicopathologic and molecular study.

BACKGROUND: The limited studies and the small number of published cases of papillary squamous cell carcinoma have precluded accurate assessment of the biologic characteristics of this lesion. METHODS: Thirty-eight of the carcinomas were studied. In-situ hybridization and polymerase chain reaction were performed to detect human papilloma virus (HPV) and p53 expression. RESULTS: HPV was found in 4 of 14 assessable carcinomas by in-situ hybridization and in 5 of 14 by polymerase chain reaction. The most frequently identified HPVs were HPVs in 6/11 and 16/18 patients. In general, a reciprocal relationship was found between p53 and HPV prevalence. The most lethal site for this tumor was the sinonasal tract, whereas patients with papillary squamous cell carcinomas of the larynx had the best outlook. Eleven of 25 (44%) assessable patients died of disease (mean time interval, 2 year). CONCLUSIONS: Papillary squamous cell carcinoma of the upper aerodigestive tract is a distinct variant of squamous cell carcinoma. As such and because of its putative association with HPV, papillary squamous cell carcinoma could be an informative model for defining how viral oncogenes cooperate with other factors in genomic instability, carcinogenesis, and tumor development.

Endogenous p53 gene status predicts the response of human squamous cell carcinomas to wild-type p53.

Prior reports suggest that p53 protein status may influence the response to gene transduction with wild-type (wt) p53. Adenoviral vectors containing the p53 gene were administered to normal keratinocytes, to squamous cell carcinoma (SCC) lines with varied p53 protein status (absent, mutant, wt, or degraded by papillomavirus), as well as to tumors formed in severe combined immunodeficient mice. The percentage of cells undergoing apoptosis, G1 growth arrest, WAF1/p21 induction, and in vivo tumor progression were studied after wt p53 gene transduction. Apoptosis developed first in normal keratinocytes, next in SCCs lacking p53 protein, and last in SCCs with mutant or degraded p53 protein. All of the cell lines studied demonstrated an increase in WAF1/p21 protein, but only those lacking p53 protein showed G1 arrest. Tumors lacking p53 protein were more susceptible to p53 overexpression than those containing mutant or degraded p53 protein. The endogenous p53 protein status of SCCs appears to influence the outcome of p53 gene transduction.

Correlation of cisplatin sensitivity with differential alteration of EGFR expression in head and neck cancer cells.

Overexpression of human epidermal growth factor (EGFR) has been associated with a variety of human malignancies. The exact role of EGFR in human malignancies and its correlation with chemotherapeutiveness response has not been determined. Using a quantitative RT-PCR method, we previously studied the effects of cisplatin treatment on levels of EGFR mRNA in human papillomavirus (HPV)-positive head and neck cancer cell lines. In this report we extended these studies to HPV-negative head and neck cancer cells. We also compared the growth inhibition and 50% inhibitory concentration (IC50) of cisplatin between these cells. We found that three of four HPV-negative cell lines had 3 to 5 times higher cisplatin IC50 values as compared to two HPV-positive cell lines. EGFR mRNA levels were increased after exposure to cisplatin in the cell lines with the higher IC50 values, while EGFR levels were reduced after cisplatin exposure in the cell lines with the lower IC50 values. These results suggest that the cisplatin sensitivity of head and neck cancer cells corresponds to subsequent alteration of EGFR levels following cisplatin treatment.

Ribozyme ablation demonstrates that the cardiac subtype of the voltage-sensitive calcium channel is the molecular transducer of 1, 25-dihydroxyvitamin D(3)-stimulated calcium influx in osteoblastic cells.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) stimulates transmembrane influx of Ca(2+) through L-type voltage-sensitive Ca(2+) channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca(2+) influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca(2+)-dependent signaling. 1, 25(OH)(2)D(3) also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore-forming alpha(1) subunit and four additional subunits. The alpha(1) subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the alpha(1C) cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)(2)D(3)-regulated VSCC remained unknown. We created a ribozyme specifically cleaving alpha(1C) mRNA. To increase target ablation efficiency, the ribozyme was inserted into U1 small nuclear RNA (snRNA) by engineering the U1 snRNA expression cassette, conferring the ribozyme transcript with stabilizing stem-loops at both sides and the Sm binding site that facilitates localization into nucleoplasm. After transfection of ROS 17/2.8 cells with U1 ribozyme-encoding vector, stable clonal cells were selected in which the expression of alpha(1C) transcript and protein were strikingly reduced. Ca(2+) influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1, 25(OH)(2)D(3)-regulated Ca(2+) responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca(2+) influx in ROS 17/2.8 osteoblastic cells.

Local inflammation may influence oral tumor cell differentiation.

The mRNA levels of keratin K13, involucrin, protein kinase C alpha and epsilon, and interferon-gamma and its receptors were examined in biopsies from human oral squamous cell carcinomas. Expression of all the genes was elevated in the histologically more differentiated tumors, but it was at or below normal (perilesional control) levels in the poorly differentiated ones. For the same set of biopsies, we had previously shown that the well differentiated tumors expressed higher levels of T cell markers. As interferon-gamma stimulates differentiation, its secretion by inflammatory cells at the tumor site may influence the differentiation status of the tumor.

Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors.

Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.

Differentiation-dependent expression of growth regulatory cytokines and their receptors in squamous cell carcinomas of the head and neck.

Cytokines, such as IFN-alpha and TNF-alpha are capable of affecting keratinocyte proliferation in the microenvironment of the tumor. Their elevated expression along with high levels of their receptor mRNAs was determined by a semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) method in biopsies of head and neck squamous cell carcinomas that were established as histologically well or moderately differentiated. In contrast, tumors with poor differentiation exhibited low levels of these growth suppressive factors, although levels of their receptors were elevated. In fact, expression of these growth suppressive cytokines highly correlated with the histological status of tumors suggesting a role of these agents in growth regulation of those tumors. Apparently, growth signaling in these tumors differs in the availability of either the ligand or the receptor.

Expression of human papillomavirus E7 mRNA in human oral and cervical neoplasia and cell lines.

Human papillomaviruses (HPVs) have been strongly linked to progression of human cancers, such as cervical and oral cancers. Two HPV oncoproteins, E6 and E7, can inhibit the tumor suppressor proteins, p53 and pRB, respectively, resulting in a deregulation of the cell cycle. In order to further test the significance of HPV expression in oral and cervical carcinogenesis, we analyzed HPV E7 mRNA in oral and cervical neoplasia and cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that HPV E7 mRNA was present in 90% of patients with oral neoplasia and 100% of patients with cervical neoplasia. Quantitative RT-PCR and western blot analysis on both transformed cervical and oral epithelial cell lines demonstrated that the mRNA level of HPV-16 E7 corresponded to E7 protein level, suggesting that HPV oncogene expression is primarily regulated at the transcriptional or post-transcription level. The potential clinical application of quantitative RT-PCR for HPV E7 mRNA expression in cancer screening and treatment evaluation requires further investigation.

Differentiation and cathepsin D expression in human oral tumors.

OBJECTIVES/HYPOTHESIS: This study aimed to ascertain whether cathepsin D expression could be related to the stage of differentiation of oral tumors. STUDY DESIGN: Human oral biopsies of 10 squamous cell carcinomas and of the corresponding perilesional normal tissues were used. The tumors had all been clinically graded as advanced stage but nonmetastatic; five were classified histopathologically as poorly differentiated. METHODS: The gene expression of cathepsin D and keratin K13 in the biopsies was measured by reverse transcription polymerase chain reaction. Ratios of tumor-to-control readings helped compensate for sample variability. RESULTS: Keratin K13, as a suprabasal cell marker, tended to confirm the histological grading of the tumors (but was not otherwise useful in distinguishing tumors from normal tissue). Substantial overexpression of cathepsin D was found in the poorly differentiated tumors. CONCLUSIONS: Cathepsin D overexpression is considered a prognostic indicator of metastasis. In this sample, it was also associated with dedifferentiation. Cathepsin D might serve as a valuable gauge in clinical exploration of the connection between dedifferentiation and metastasis.

Interleukin-1 regulates interleukin-6 secretion in human oral squamous cell carcinoma in vitro: possible influence of p53 but not human papillomavirus E6/E7.

We have previously shown that interleukin-1 (IL-1) and IL-6 are constitutively produced by human oral squamous cell carcinoma (SCC) and some derived cell lines but not by cultured normal oral keratinocytes. To elucidate possible cytokine regulatory pathways that may contribute to oral SCC growth and/or progression, we tested the hypotheses that exogenous and/or endogenous IL-1 regulates IL-6 production in vitro. We investigated the effects of exogenous IL-1 and IL-6 on secondary cytokine secretion. Our studies revealed that IL-1 strongly up-regulated IL-6 protein secretion in all three cell lines tested. This effect was completely abrogated by IL-1 receptor antagonist. IL-1 receptor antagonist also inhibited the secretion of IL-1alpha and IL-1beta in two of three cell lines. These data show for the first time that IL-1 strongly up-regulates IL-6 and support the notion of autocrine regulation of IL-1 in certain oral SCC cell lines. Additionally, because human papillomavirus (HPV) infection and p53 mutation have been implicated in the malignant transformation of SCC, we explored a second hypothesis, that HPV and/or p53 mutation contribute to cytokine dis-regulation. We investigated HPV DNA presence, transcriptional activation of HPV E6/E7 (in HPV DNA-positive cell lines), and p53 gene status in our cell lines. No association between HPV DNA and cytokine expression was found. However, the oral SCC cell lines secreting the most IL-6 had mutant rather than wild-type p53.

Differential expression of epidermal growth factor receptor in human head and neck cancers.

BACKGROUND: Over-expression of human epidermal growth factor receptor (EGFR) is associated with a variety of human malignancies, including head and neck cancer. It has also been studied for its effect on cancer cell responses to chemotherapy. To accurately measure changes in EGFR expression that might be of diagnostic or prognostic importance in head and neck cancers, a quantitative assay for the direct detection of EGFR messenger ribonucleic acid (mRNA) was developed. METHODS: Our method was based on competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that was able to measure EGFR mRNA levels undetectable by northern-blot analysis. We measured EGFR mRNA by RT-PCR in human head and neck cancers and their corresponding adjacent, histologically normal tissues and in cisplatin-treated and untreated oral epithelial cell lines. RESULTS: All the tumor samples had higher EGFR mRNA levels than their corresponding adjacent normal tissues. It is also shown that EGFR mRNA levels in normal oral epithelial cells were elevated after exposure to cisplatin. In contrast, EGFR mRNA levels in oral cancer cells were decreased after the exposure, suggesting that increased EGFR expression may have different functions in cancer cells and in normal cells under stress. CONCLUSIONS: Accurate monitoring of EGFR expression may be a useful marker for diagnosis, treatment, and prognosis of head and neck cancer.

Tumor differentiation-dependent local immunity in human head and neck cancers.

Distribution of markers of local cell-mediated immunity was examined in oral tumors exhibiting different histological stages of differentiation. Using a RT-PCR-based semiquantitative technique we determined levels of Langerhans cells, CD4- and CD8-positive T-cells, macrophages/NK cells, beta2-microglobulin and IFN-gamma mRNAs from tissue biopsies. A positive correlation was found between levels of these immunological markers and the tumor differentiation stage. Since tumor differentiation may correlate with the prognosis and response to various treatment modalities, our results may be useful clinically.

Head and neck squamous cell growth suppression using adenovirus-p53-FLAG: a potential marker for gene therapy trials.

The recombinant wild-type p53 adenovirus has been proven effective against the growth of human head and neck squamous cell cancer (SCCHN) cell lines iir vitro and in a nude mouse model. The addition of a FLAG peptide sequence was used in this study, along with the p53 adenovirus vector as a marker of the site of the gene therapy activity. It provides clear evidence of the exogenous gene product within the transduced carcinoma cells. No alterations in transcription or translation of the p53 gene product were noted with the addition of the FLAG sequence to the original p53 adenovirus vector. Immunohistochemical analysis displayed simultaneous expression of the p53 and FLAG proteins in the infected cells. The p53 protein remained localized to the nucleus, whereas the FLAG protein was additionally noted in the cytoplasm. In vitro growth suppression assays and in vivo microscopic residual tumor model experiments in nude mice showed a similar tumoricidal effect with the p53-FLAG adenovirus vector to that with the previously studied p53 adenovirus vector without the addition of the FLAG sequence. We conclude that the addition of the FLAG octapeptide sequence allows identification of those cells that have been affected by the molecular therapy independent of the endogenous gene expression of the cells. This novel molecular tracer may prove useful in characterizing infection efficiency and in gene therapy trials.

Differentiation markers in oral carcinoma cell lines and tumors.

Cell lines are useful as models if they retain the relevant characteristics of the tissue of origin. We compared two human squamous carcinoma cell lines derived from tumors of the tongue that vary in their extent of differentiation, with human biopsies of carcinomas of the tongue that were either poorly or well-differentiated. The mRNA levels of suprabasal cell proteins (keratin K13, involucrin, transglutaminase) and of protein kinase C (PKC) isozymes were measured by RT-PCR. Apart from PKC beta and PKC delta (mostly expressed by Langerhans cells and missing in culture), qualitatively similar patterns were found in vitro and in vivo. The more differentiated cells had expression levels moderately lower to higher than the normal controls. The poorly differentiated cells generally had substantially lower levels.