RESUMEN
AIMS: Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. METHODS AND RESULTS: Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker gamma-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. CONCLUSIONS: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.
Asunto(s)
Biomarcadores de Tumor/análisis , ADN de Neoplasias/genética , Neoplasias de la Boca/química , Neoplasias de la Boca/genética , Ploidias , Lesiones Precancerosas/química , Lesiones Precancerosas/genética , Aneuploidia , Anticuerpos Antinucleares , Anticuerpos Monoclonales , Carcinoma in Situ/química , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , ADN de Neoplasias/análisis , Histonas/análisis , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Antígeno Ki-67/análisis , Neoplasias de la Boca/diagnóstico , Adhesión en Parafina , Lesiones Precancerosas/diagnóstico , Estudios RetrospectivosRESUMEN
The objective of the study was to determine maximum phonation time reliability as a function of the number of trials, days, and raters in dysphonic and control subjects. Two groups of adult subjects participated in this reliability study: a group of outpatients with functional or organic dysphonia versus a group of healthy control subjects matched by age and gender. Over a period of maximally 6 weeks, three video recordings were made of five subjects' maximum phonation time trials. A panel of five experts were responsible for all measurements, including a repeated measurement of the subjects' first recordings. Patients showed significantly shorter maximum phonation times compared with healthy controls (on average, 6.6 seconds shorter). The averaged interclass correlation coefficient (ICC) over all raters per trial for the first day was 0.998. The averaged reliability coefficient per rater and per trial for repeated measurements of the first day's data was 0.997, indicating high intrarater reliability. The mean reliability coefficient per day for one trial was 0.939. When using five trials, the reliability increased to 0.987. The reliability over five trials for a single day was 0.836; for 2 days, 0.911; and for 3 days, 0.935. To conclude, the maximum phonation time has proven to be a highly reliable measure in voice assessment. A single rater is sufficient to provide highly reliable measurements.