Overall, the performance of the 4M panel was superior to the performance of the same panel without CHC (the 3M panel). With staining by at least two markers, the accuracy was 97% and 84.3% in nonsmall and small HCCs, respectively, and this was superior to the accuracy of the panel without the addition of CHC (86% and 76.9%, respectively). For small HCCs, the addition Alisertib in vivo of CHC to the panel consistently increased the sensitivity from 46.8% to 63.8%. Interestingly enough, for nonsmall HCCs, even though the material was sampled with 20- to 21-gauge needles, the accuracy of the novel panel (97%) was better than the accuracy that we previously reported

(78.4%) with a 3M panel in an analogous HCC series sampled with 16- to 18-gauge needles.6 This means that the addition of CHC not only counterbalances the putative loss of sensitivity of thinner core materials but also increases the diagnostic accuracy. Although the use of a 4M panel is more elaborate and time-consuming for pathologists, the unitary cost of

an additional immunoreaction to the panel (approximately $15-20) is much less expensive than confirmatory additional imaging4 or repeat biopsy. When we dissected our HCC series into subpopulations CHIR-99021 solubility dmso according not only to size but also to grading (G1 versus G2/G3), the panel accuracy remained excellent and greater than 90% for G2/G3 HCCs, regardless of the size (Table 5). This confirmed for us that the performance of the 4M panel is optimal when tumor differentiation is compromised; in other words, the individual markers of the panel cooperatively stain HCCs that have progressed. Unfortunately, these are cases for which the pathological diagnosis can be rendered on morphological grounds without the use of staining beyond H&E. Interestingly, although the tumor size was not an issue in G2/G3 HCCs, it was a major issue in well-differentiated (G1) HCCs. Indeed, in this HCC group, which was the most difficult to evaluate, the accuracy of the panel was still excellent in nonsmall G1 HCCs (93.9%)

but dropped to 67.4% in small G1 HCCs (Tables 4 and 5). In the latter, the sensitivity for Vorinostat cell line HCC detection was 50% with 100% specificity, and the performance of the 4M panel was much better than that of the 3M panel (Table 4). In addition, we noticed that a consistent fraction of these tumors showed negative staining (6/30, 20%; Table 2) or one marker only (9/30, 30%; data not shown). The most likely (though speculative) explanation is that G1 HCCs greater than 2 cm and G1 HCCs smaller than 2 cm are not the same disease. An international agreement between Eastern and Western pathologists has recently been obtained for a new HCC entity: very well-differentiated, ≤2-cm HCC (which is also called very early HCC).20 This is the earliest described and well-differentiated form of HCC and is likely the morphological link between HGDN (dysplasia) and HCC that has progressed.