A novel evaluation method for Ki-67 immunostaining in paraffin-embedded tissues.

The Ki-67 labeling index is traditionally used to investigate tumor aggressiveness. However, no diagnostic or prognostic value has been associated to the heterogeneous pattern of nuclear positivity. The aims of this study were to develop a classification for the patterns of Ki-67-positive nuclei; to search scientific evidence for the Ki-67 expression and location throughout the cell cycle; and to develop a protocol to apply the classification of patterns of Ki-67-positive nuclei in squamous epithelium with different proliferative activities. Based on empirical observation of paraffin sections submitted to immunohistochemistry for the determination of Ki-67 labeling index and literature review about Ki-67 expression, we created a classification of the patterns of nuclear positivity (NP1, NP2, NP3, NP4, and mitosis). A semi-automatic protocol was developed to identify and quantify the Ki-67 immunostaining patterns in target tissues. Two observers evaluated 7000 nuclei twice to test the intraobserver reliability, and six evaluated 1000 nuclei to the interobserver evaluation. The results showed that the immunohistochemical patterns of Ki-67 are similar in the tumoral and non-tumoral epithelium and were classified without difficulty. There was a high intraobserver reliability (Spearman correlation coefficient > 0.9) and moderate interobserver agreement (k = 0.523). Statistical analysis showed that non-malignant epithelial specimens presented a higher number of NP1 (geographic tongue = 83.8 ± 21.8; no lesion = 107.6 ± 52.7; and mild dysplasia = 86.6 ± 25.8) when compared to carcinoma in Situ (46.8 ± 34.8) and invasive carcinoma (72.6 ± 37.9). The statistical evaluation showed significant difference (p < 0.05). Thus, we propose a new way to evaluate Ki-67, where the pattern of its expression may be associated with the dynamics of the cell cycle. Future proof of this association will validate the use of the classification for its possible impact on cancer prognosis and guidance on personalized therapy.

Computerized Evaluation of the Immunoexpression of Ki-67 Protein in Odontogenic Keratocyst and Dentigerous Cyst.

Evaluation and comparison of odontogenic keratocysts and detigerous cysts immunoexpression and immunostaining intensities of Ki-67 antigen by assessing the whole extent of the epithelium (all epithelium layers in combination) and each layer individually. Ki-67 immunoexpression was evaluated in 15 odontogenic keratocysts and 6 dentigerous cysts using automated methods and the Aperio Technologies Inc. computer system. No statistically significant differences were observed in immunoexpression nor in immunostaining intensities between both lesions. Also, no statistically significant differences were found between odontogenic keratocysts from maxilla versus mandible nor primary versus recurrent. However, odontogenic keratocyst showed a significantly higher cellular proliferation index in the suprabasal layers compared to the basal layer. Assessment of the cellular proliferation index through a computerized system enabled the evaluation of all epithelial tissue without field selection. The increased Ki-67 immunoexpression in suprabasal layers of odontogenic keratocyst suggests a different biological behavior and more aggressive proliferation potential when compared to dentigerous cyst. The same result was found in recurrent odontogenic keratocysts when compared with primary ones. The odontogenic keratocysts of the maxilla and mandible have similar Ki-67 immunoexpression. The evaluation of cellular proliferation only by immunohistochemical analysis with Ki-67 antigen does not provide enough data to elucidate the biological behavior of odontogenic keratocyst.

Complete genome sequences of two new virus isolates associated with cotton blue disease resistance breaking in Brazil.

Since 2006, Brazilian cotton (Gossypium hirsutum) crops planted with cultivars that are resistant to cotton blue disease have developed a new disease termed "atypical" cotton blue disease or atypical vein mosaic disease. Here, we describe the complete genomes of two virus isolates associated with this disease. The new virus isolates, called CLRDV-Acr3 and CLRDV-IMA2, were found to have a high degree of nucleotide and amino acid sequence similarity to previously described isolates of cotton leafroll dwarf virus, the causal agent of cotton blue disease. However, their P0 proteins were 86.1 % identical. These results show that this new disease is caused by a new CLRDV genotype that seems to have acquired the ability to overcome cotton blue disease resistance.