Comparative diagnostic and prognostic performances of the hematoxylin-eosin and phospho-histone H3 mitotic count and Ki-67 index in adrenocortical carcinoma.

Mitotic count on hematoxylin and eosin slides is a fundamental morphological criterion in the diagnosis and grading of adrenocortical carcinoma in any scoring system employed. Moreover, it is the unique term strongly associated with patient's prognosis. Phospho-histone H3 is a mitosis-specific antibody, which was already proven to facilitate mitotic count in melanoma and other tumors. Therefore, a study was designed to assess the diagnostic and prognostic role of phospho-histone H3 in 52 adrenocortical carcinomas, comparing manual and computerized count to standard manual hematoxylin- and eosin-based method and Ki-67 index. Manual hematoxylin and eosin and phospho-histone H3 mitotic counts were highly correlated (r=0.9077, P<0.0001), better than computer-assisted phospho-histone H3 evaluations, and had an excellent inter-observer reproducibility at Bland-Altman analysis. Three of 15 cases having <5 mitotic figures per 50 high-power fields by standard count on hematoxylin and eosin gained the mitotic figure point of Weiss Score after a manual count on phospho-histone H3 slides. Traditional mitotic count confirmed to be a strong predictor of overall survival (P=0.0043), better than phospho-histone H3-based evaluation (P=0.051), but not as strong as the Ki-67 index (P<0.0001). The latter further segregated adrenocortical carcinomas into three prognostic groups, stratifying cases by low (<20%), intermediate (20-50%), and high (>50%) Ki-67 values. We conclude that (a) phospho-histone H3 staining is a useful diagnostic complementary tool to standard hematoxylin and eosin mitotic count, enabling optimal mitotic figure evaluation (including atypical mitotic figures) even in adrenocortical carcinomas with a low mitotic index and with a very high reproducibility; (b) Ki-67 proved to be the best prognostic indicator of overall survival, being superior to the mitotic index, irrespective of the method (standard on hematoxylin and eosin or phospho-histone H3-based) used to count mitotic figures.

A collection of primary tissue cultures of tumors from vacuum packed and cooled surgical specimens: a feasibility study.

Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84-100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.