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Summary p2l protein (p21) inhibitor of cyclin-dependent kinases is a critical downstream effector in the p53-specific pathway of growth control. p21 can also be induced by p53-independent pathways in relation to terminal differentiation. We investigated p21 immunoreactivity in normal breast and in 91 breast carcinomas [three in situ ductal carcinomas (DCIS) with microinfiltration and 88 infiltrating carcinomas, 17 of which with an associated DCIS; 57 node negative and 34 node positive] with long-term follow-up (median= 58 months). Seven additional breast carcinomas with known p53 gene mutations were investigated. In normal breast p21 expression was seen in the nuclei of rare luminal cells of acinar structures, and in occasional myoepithelial cells. Poorly differentiated DCIS showed high p21 expression, whereas well-differentiated DCIS tumours showed few p21-reactive cells. p21 was seen in 82 (90%) infiltrating tumours; staining was heterogeneous; the percentage of reactive nuclei ranged from 1% to 35%. High p21 expression (more than 10% of reactive cells) was seen in 24 (26%) cases, and was associated with high tumour grade (P=0.032); no associations were seen with tumour size, metastases, oestrogen receptor status, MIBI expression and p53 expression. p21 expression in cases with p53 gene mutations was low in six cases and high in one. High p21 expression was associated with short relapse-free survival (P=0.003).

BackgroundKi67 labeling index (Ki67 LI), the percentage Ki67 immunoreactive cells, is a measure of tumor proliferation, with important clinical relevance in breast cancer, and it is extremely important to standardize its evaluation.AimTo test the efficacy of computer assisted image analysis (CAIA) applied to completely digitized slides and to assess its feasibility in routine practice and compare the results obtained using two different Ki67 monoclonal antibodies.Materials and methods315 consecutive breast cancer routinely immunostained for Ki-67 (223 with SP6 and 92 with MM1 antibodies previously examined by an experienced pathologist, have been re-evaluated using Aperio Scanscope Xs.ResultsMean human Ki67 LI values were 36%± 14.% and 28% ± 18% respectively for SP6 and MM1 antibodies; mean CAM Ki67 LI values were 31%± 19% and 22% ± 18% respectively for SP6 and MM1. Human and CAIA evaluation are statistically highly correlated (Pearson: 0.859, p<0.0001), although human LI are systematically higher. An interobserver variation study on CAIA performed on 84 cases showed that the correlation between the two evaluations was linear to an excellent degree.DiscussionOur study shows that a) CAIA can be easily adopted in routine practice, b) human and CAIA Ki67 LI are highly correlated, although human LI are systematically higher, c) Ki67 LI using different evaluation methods and different antibodies shows important differences in cut-off values.

Receptor status, proliferative activity, loss of differentiation, inactivation of tumor suppressor genes, and overexpression of oncogenes are related events that may affect the prognosis of patients with breast cancer. Ninety-seven unselected breast carcinomas were immunostained for estrogen and progesterone receptors, Ki-67 proliferation-associated antigen, p53 tumor suppressor gene product (p53), and c-erbB-2 protein. Immunohistochemical results and clinical data were compared. Altered p53 expression (regarded as indirect indication of inactivating gene alterations) was found in 25.8% of cases and was associated with a high Ki-67 labeling index, high mitotic count, and high histologic grade, with c-erbB-2 overexpression, and with negative estrogen and progesterone receptor status. p53 immunostaining could be found also in cytologic samples and correlated with p53 immunoreactivity on frozen sections of the corresponding tumors. c-erbB-2 protein overexpression was seen in 24.7% of cases and was associated with p53 altered expression and negative receptor status. Double immunohistochemical staining showed p53 and c-erbB-2 immunoreactivity in the same cells. Median and mean +/- standard deviation Ki-67 labeling index values were 15 and 16.32 +/- 10.05, respectively. Ki-67 labeling index was correlated with high mitotic count and was positively associated with histologic grade, negative progesterone receptor status, and p53 expression. Estrogen receptor status was not associated with any histologic or clinical parameters, whereas progesterone receptor status was associated with grading. The direct relation of p53 protein alterations with c-erbB-2 overexpression may be interpreted in light of the multistep model of tumor progression. Cases with altered expression of both p53 and c-erbB-2 proteins could be interpreted as having lost one inhibitory control mechanism of cell proliferation and having gained one activator of the malignant potential. However, in comparing cases with the p53 + c-erbB-2 + phenotype with cases showing positivity for only one of these gene products, no association with higher stages was seen. Detection of p53 altered expression on cytologic samples of malignant tumors may have diagnostic relevance, and p53 immunostaining may prove to be an additional diagnostic criterion in cytologic diagnosis.

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