Different methods of cell quantification can lead to different results: a comparison of digital methods using a pilot study of dendritic cells in HIV-positive patients.

BACKGROUND: Although new digital pathology tools have improved the positive cell quantification, there is a heterogeneity of the quantification methods in the literature. The aim of this study was to evaluate and propose a novel dendritic cells quantification method in squamous cell carcinoma comparing it with a conventional quantification method. MATERIAL AND METHODS: Twenty-six squamous cell carcinomas HIV-positive cases affecting the oropharynx, lips and oral cavity were selected. Immunohistochemistry for CD1a, CD83, and CD207 was performed. The immunohistochemical stains were evaluated by automated examination using a positive pixel count algorithm. A conventional quantification method (unspecific area method; UA) and a novel method (specific area method; SA) were performed obtaining the corresponding density of positive dendritic cells for the intratumoral and peritumoral regions. The Mann-Whitney U test was used to verify the influence of the quantification methods on the positive cell counting according to the evaluated regions. Data were subjected to the ANOVA and Student's t-test to verify the influence of the tumour location, stage, histological grade, and amount of inflammation on the dendritic cells density counting. RESULTS: The cell quantification method affected the dendritic cells counting independently of the evaluated region (P-value <0.05). Significant differences between methods were also observed according to the tumour features evaluations. CONCLUSIONS: The positive cell quantification method influences the dendritic cells density results. Unlike the conventional method (UA method), the novel SA method avoids non-target areas included in the hotspots improving the reliability and reproducibility of the density cell quantification.

Immunohistochemical profile of odontogenic epithelium in developing dog teeth (Canis familiaris).

Tumors of the jaw bones and oral soft tissue are relatively common lesions in dogs. The aim of this study was to find cell markers to differentiate odontogenic epithelium from nonodontogenic epithelium for future research on the pathogenesis and pathology of odontogenic neoplasms in dogs. Keratin 14 and 19 staining was observed in odontogenic and nonodontogenic epithelium, whereas amelogenin and p75 neurotrophin receptor immunoreactivity was observed in certain odontogenic epithelial cells at various stages of development but not in other epithelial cells. Calretinin staining was observed in the alveolar epithelial cells directly overlying the developing tooth germ in 28 of 39 sections (71.8%), as well as the dental laminae in 30 of 35 sections (85.7%) and Serres rests in 24 of 28 sections (85.7%). Focal positivity was detected in the respiratory mucosa, some hair follicles, and fusion epithelium of the palate, but no calretinin staining was observed in other oral epithelial cells; therefore, calretinin has potential to be utilized as a marker to differentiate odontogenic form nonodontogenic epithelium.

The suitability of paraffin-embedded material to predict metastatic potential of oral squamous cell carcinoma.

BACKGROUND: The presence of regional metastasis in oral squamous cell carcinoma (OSCC) is an important prognostic factor. This study was undertaken to identify histological features and biological markers from paraffin-embedded primary OSCC that may predict the presence of regional metastases. MATERIALS AND METHODS: Fifty-three en-bloc primary OSCC resections were divided into two groups, 26 with lymph node metastases and 27 without metastases. The pattern of infiltration, presence of vascular or perineural infiltration and tumour necrosis were evaluated while expression of p53, p21 and Rb were assessed in the two groups. DNA ploidy status was also determined with a flow cytometer. RESULTS: The presence of DNA aneuploidy was found to be the only statistically significant predictor of regional metastases. Seventy-seven per cent of the primary OSCC with lymph node metastases showed DNA aneurploidy. CONCLUSION: DNA flow cytometry obtained from archival material could be used as a parameter to predict regional metastases.

Fhit protein expression in oral epithelium: immunohistochemical evaluation of three antisera.

BACKGROUND: A number of studies have shown that the Fhit tumour suppressor protein is abundantly expressed in normal epithelial cells of human organs and that this expression is lost or reduced in the majority of cancers arising in these epithelial tissues. A variety of antiFhit sera have been used but a systematic comparison of the different antisera has not yet been reported. MATERIALS AND METHODS: We compared the Fhit expression pattern in the epithelium of fibrous epuli, oral lichen planus, oral epithelial dysplasia and oral squamous cell carcinomas (OSCC) using three different Fhit antisera. RESULTS: The antigstFhit sera from two sources gave very similar results for all types of oral lesions except for lichen planus and showed that about 60% of OSCCs have lost Fhit expression. CONCLUSION: Although different staining patterns were found for the three antisera, all three could be used for evaluation of Fhit expression in OSCC.

FHIT RNA and protein expression in oral squamous cell carcinomas.

BACKGROUND: To investigate the possible role of FHIT, a possible tumour suppressor gene, in oral carcinogenesis, we examined 17 oral squamous cell carcinomas (OSCCs) for genetic alterations. MATERIALS AND METHODS: Fresh tissue was obtained during surgery, snap-frozen in liquid nitrogen and stored at -70 degrees C. Nested PCR amplification to examine the integrity of FHIT mRNA was performed on the reverse transcribed complementary DNA obtained from the frozen normal and tumour tissue. Immunohistochemistry was done on formal in-fixed paraffin-embedded tissue protein from the same cases using a polyclonal antiserum against the full length Fhit. RESULTS: Twelve out 17 (71%) OSCCs showed reduced or absent Fhit protein and half of the cases with reduced Fhit protein exhibited aberrant RT-PCR products. CONCLUSION: Immunohistochemical detection of Fhit protein expression in OSCCs is the more sensitive method to determine the status of Fhit in these tumours, in agreement with previous studies of other tumour types.

Immunohistochemical evaluation of Fhit protein expression in oral squamous cell carcinomas.

The expression of Fhit (fragile histidine triad) protein in oral squamous cell carcinoma (OSCC) and adjacent oral epithelium was evaluated by immunohistochemistry on formalin-fixed paraffin-embedded blocks of 32 cases of OSCC. Rabbit polyclonal anti-GST-Fhit antiserum at 1:600 was used, after antigen enhancement in a microwave pressure cooker, in a saturated lead thiocyanate solution. This antiserum has been shown specifically to detect human Fhit by immunohistochemistry at dilutions up to 1:10,000. The Fhit protein expression was evaluated using both the intensity and extent of staining. Normal stratified squamous epithelium showed strong positivity, especially in the stratum spinosum and areas of keratinisation. Basal and parabasal cells were negative or expressed low levels of Fhit relative to the squamous epithelium. Mild and moderate epithelial dysplasia showed Fhit expression in the superficial layers, while Fhit expression was absent from severely dysplastic lesions. A reduction or loss of Fhit expression was found in 21 (66%) of the OSCC. The alterations in Fhit protein expression in OSCC, and not in normal tissues, are consistent with the proposal that Fhit inactivation plays a role in oral carcinogenesis.

Warthin's tumour is not an Epstein-Barr virus related disease.

BACKGROUND: The histogenesis of Warthin's tumour (WT) is controversial. A possible role for Epstein-Barr virus (EBV) has been suggested. MATERIALS AND METHODS: Twenty formaldehyde-fixed, paraffin-embedded blocks of WT from the parotid gland were examined for the presence of EBV. In situ hybridisation was performed using EBV encoded small nuclear RNAs (EBER1/2) probes labelled with fluorescein isothiocyanate. An EBV-positive P3HR-1 cell line processed to paraffin wax was used as a positive control and a brain section as negative control. RESULTS: EBER1/2 could not be found in the neoplastic epithelial cells in any of the tumours nor in the adjacent normal parotid tissues. Individual positive lymphocytes were present in 7 tumours. CONCLUSIONS: These results indicated that EBV is not involved in the pathogenesis of WT.