Telomerase reactivation is an early event in laryngeal carcinogenesis.

The exact role and timing of reactivation of telomerase, a key enzyme implicated in cellular immortalization and transformation in the multistep process of laryngeal carcinogenesis, is still unknown. We attempted to (1) determine that quantitative differences exist in the levels of telomerase catalytic subunit (hTERT) mRNA expression among different grades of laryngeal epithelial abnormalities classified according to the Ljubljana classification; (2) determine that telomerase reactivation is an important, most probably early event in laryngeal carcinogenesis; and (3) analyze whether the relative quantity of hTERT mRNA can be used as a molecular biomarker in the early detection of precancerous lesions. The relative quantity of hTERT mRNA, expressed as an hTERT index, was analyzed in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis by using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. The presence and relative quantity of hTERT mRNA in laryngeal epithelium increases progressively with the degree of epithelial abnormalities. hTERT mRNA was detectable in 1/15 normal laryngeal epithelia (7%, mean hTERT index 0.02), 3/15 simple hyperplasias (20%, mean hTERT index 0.09), 10/27 abnormal hyperplasias (37%, mean hTERT index 0.18), 9/12 atypical hyperplasias (75%, mean hTERT index 0.74), 8/9 intraepithelial carcinomas (89%, mean hTERT index 1.82), and 53/62 invasive laryngeal squamous cell carcinomas (85%, mean hTERT index 2.51). Statistical analysis revealed two groups of laryngeal epithelial changes with significant differences in the levels of hTERT mRNA expression (P <.0033): (1) normal and reactive hyperplastic laryngeal epithelium (simple and abnormal hyperplasia) and (2) atypical hyperplasia (precancerous lesion), intraepithelial and invasive laryngeal squamous cell carcinoma. The results of the present study suggest that telomerase reactivation is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for progression of these epithelial abnormalities toward invasive laryngeal squamous cell carcinoma.

Hybrid Capture II HPV Test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail.

BACKGROUND: Hybrid Capture II HPV Test (HCII) (Digene Corporation, Gaithersburg, MD) is a signal amplified hybridization microplate-based assay designated to detect 18 human papillomavirus (HPV) genotypes using two probe cocktails, for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 and low-risk HPV genotypes 6, 11, 42, 43 and 44. At present, HCII is the only commercially available HPV assay with sufficient scientific data to support its performance in the clinical setting. OBJECTIVES: To determine the specificity and accuracy of HCII high-risk probe cocktail for detection of 13 HPV genotypes included in the high-risk probe cocktail. STUDY DESIGN: Cervical samples obtained from 325 women recognized as HPV positive using the HCII high-risk probe cocktail were included in the study. HPV genotypes were determined by restriction fragment analysis of PGMY09/PGMY11 polymerase chain reaction (PCR) products using seven restriction endonucleases. RESULTS: A 450 bp fragment of HPV L1 gene was successfully amplified from 312 out of 325 samples. Of the 312 PCR-positive samples, 280 samples were associated with the expected high-risk HPV genotypes and 32 samples with the HPV genotypes not included in the HCII high-risk probe cocktail. Thus, HPV53 was detected in 8 samples, HPV66 in 4 samples, HPV54 in 3 samples, HPV6, HPV26, HPV70 each in 2 samples, and HPV11, HPV40, HPV42, HPV61, HPV73, HPV81, MM4, IS39, CP6108 each in 1 sample. In 2 samples, we were not able to determine the HPV genotype. CONCLUSIONS: Our study shows that HCII high-risk probe cocktail detects at least 15 HPV genotypes not included in the current HCII high-risk probe cocktail. The potential impact of HCII high-risk probe cocktail cross-reactivity with phylogenetically related and unrelated HPV genotypes, including genotypes currently considered to be low-risk HPVs, remains to be determined.

Comparative evaluation of three commercial assays for quantitative measurement of hepatitis B virus DNA in serum samples.

BACKGROUND/AIMS: Quantitative determination of HBV DNA in serum samples is indispensable for predicting disease progression and for monitoring the antiviral treatment in patients with chronic HBV infection. METHODOLOGY: Three commercial assays for quantification of HBV DNA: Digene Hybrid-Capture HBV DNA Assay, Bayer Quantiplex HBV DNA Assay and Roche Amplicor HBV Monitor Test were comparatively evaluated under the routine conditions of diagnostic virology laboratory, using 61 serum samples obtained from 55 Slovenian patients with chronic hepatitis B. RESULTS: HBV DNA was detected by Amplicor, Quantiplex and Hybrid-Capture in 38 (62.3%), 34 (55.7%) and 27 (44.3%) samples, respectively. The sensitivity of Amplicor and Quantiplex assays did not differ significantly (p = 0.13), while both Amplicor and Quantiplex assays were found to be significantly more sensitive than Hybrid-Capture (p = 0.003 and p = 0.02, respectively). For a given sample, the highest correlation was observed between HBV DNA loads determined by Quantiplex and Hybrid-Capture assays (r = 0.85, p < 0.0001). CONCLUSIONS: Amplicor HBV Monitor Test seems to be the most sensitive assay for the detection of HBV DNA in serum samples and can be clinically used for monitoring patients with chronic HBV infection.

Seroprevalence of HIV-1 subtypes A-E among HIV-1 infected individuals from Slovenia.

To investigate the prevalence of HIV-1 subtypes A-E in Slovenia, 82 HIV-1 infected individuals were tested for the presence of HIV-1 subtype specific antibodies using a research competitive peptide enzyme immuno assay supplied by Boehringer Mannheim. In 74 individuals unambiguous results were obtained. As in other European countries, the majority of Slovenian HIV-1 infected individuals (86.5%) were infected with subtype B. Infections with subtypes C, A, D and E were detected in 8.1%, 2.7%, 1.3% and 1.3% individuals, respectively.