Gene expression profiles in HPV-infected head and neck cancer.

Epidemiological and laboratory evidence indicate that, in addition to tobacco and alcohol, human papillomaviruses (HPV) play an important aetiological role in a subset of head and neck squamous cell carcinoma (HNSCC). To evaluate the molecular pathogenesis of HPV-infected HNSCC, we compared gene expression patterns between HPV-positive and -negative HNSCC tumours using cDNA microarrays. Tumour tissue was collected from 42 histologically confirmed HNSCC patients from an inner-city area of New York. Total DNA and RNA were extracted and purified from frozen tumour samples and gene expression levels were compared to a universal human reference RNA standard using a 27 323 cDNA microarray chip. HPV detection and genotyping were performed using an MY09/11-PCR system and RT-PCR. HPV was detected in 29% of HNSCC tumours. Most harboured only HPV16 and expressed the HPV16-E6 oncogene. HPV prevalence was highest in pharyngeal tumours (45%). Gene expression patterns that differentiated HPV-positive from negative tumours were compared by supervised classification analysis, and a multiple-gene signature was found to predict HPV16 prevalence in primary HNSCC with a false discovery rate < 0.2. Focusing on never-smokers, we further identified a distinct subset of 123 genes that were specifically dysregulated in HPV16-positive HNSCC. Overexpressed genes in HPV-positive HNSCC tumours included the retinoblastoma-binding protein (p18), replication factor-C gene, and an E2F-dimerization partner transcription factor (TFDP2) that have also been found to be overexpressed in cervical cancer. An additional subset of genes involved in viral defence and immune response, including interleukins and interferon-induced proteins, was found to be down-regulated in HPV-positive tumours, supporting a characteristic and unique transcriptional profile in HPV-induced HNSCC.

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray.

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

Three-dimensional analysis of caloric nystagmus in the rhesus monkey.

The aim of this study was to investigate whether caloric nystagmus contains response components that can be attributed to a stimulation of the vertical semicircular canals. Three dimensional eye movement recordings with a dual search coil technique revealed important horizontal, vertical and torsional nystagmus components following irrigation of the external ear canal with cold water in various head positions relative to gravity. Horizontal nystagmus components, i.e. lateral semicircular canal vectors, followed a cosine function of both the pitch and yaw angle of the head relative to gravity, confirming a mainly thermovective mechanism for stimulation of the horizontal canals. Vertical and torsional nystagmus components behaved differently following left and right ear irrigations. Right-left symmetrical vectors emerged only when the vertical and torsional components were transposed into vectors of single semicircular canal directions. The intensity of these vertical semicircular canal vectors as a function of the position of the corresponding canal relative to gravity, however, excludes important thermovective mechanisms acting at the vertical canals. It remains an open question whether these vertical canal vectors represent a non-thermovective caloric stimulation of vertical canal afferents.

[The vertical component in a caloric nystagmus and the existence of a second phase of the nystagmus--the possibility of canal otolithic interaction in normal subjects].

To clarify the existence of the vertical component during a caloric nystagmus and the existence of a second phase of the nystagmus, 194 induced incidents of a caloric nystagmus in 29 normal subjects have been analyzed. Each nystagmus episode was recorded by using ENG and an infra-red video camera. The caloric stimuli were given by pouring 5 ml of water at 20 degrees C into the ear at an ear-up position. After irrigation, each subject then assumed a supine or a prone position, with the head bent 30 degrees forward in either position. All recordings contained vertical components that depended on the supine or prone head position and not on the side of the stimulated ear, i.e., an up-beating nystagmus resulted in the supine position and a down-beating nystagmus in the prone position. Further, the vertical component was far stronger in the prone position. In contrast, the horizontal component had larger velocities and was of longer duration in the supine position than in the prone position. When the first phase of the caloric nystagmus ended, the body position was changed 90 degrees, i.e., to a sitting position or a right-ear-down or left-ear-down position. All trials showed a horizontal component during the secondary phase when the head assumed the sitting position. As for the ear-down positions, only when the irrigated ear was moved upwards from the prone position during the secondary phase, an up-beating vertical nystagmus resulted in almost all the trials. These findings suggest that a caloric nystagmus may originate not only from the lateral semicircular canal but also from the vertical canals, and the second phase of a caloric nystagmus may be strongly influenced by the otolithic organs.