Nucera, Nehs, Nagarkatti et al. and demonstrated an average weight loss from baseline of 4.2 g 1.1 g (23% of baseline 6%) (p .001) (Fig. 4). However, the mice in the PLX4720 treatment group main- tained their weight and showed no signs of cachexia (Fig. 4). Five weeks post–tumor implantation, all animals were sacrificed and there was a dramatically (97%) lower tumor volume in PLX4720-treated animals than in controls (1.9 mm3 versus 72 18 mm3; p .001) (Fig. 4). These results confirm the in vivo effects of PLX4720 with thyroid cancer cells that harbor B-RafV600E [19]. Here, for the first time, we show detailed histological and IHC analyses performed on B-RafV600E-positive orthotopic thyroid carcinomas treated with PLX4720 or vehicle (control). The control tumors grew circumferentially around the trachea with extrathyroi- dal extension, and these tumors demonstrated histologic features of aggressiveness such as tracheal invasion, pleo- morphism, and numerous mitotic figures (Fig. 5A1–5A4). The PLX4720-treated mice, however, had small and dis- crete tumor foci with minimal evidence of local invasion grossly or histologically (Fig. 5A5–5A8). Additionally, the proliferative index (Ki67 nuclear expression) in the PLX4720-treated orthotopic tumors was significantly lower than in the control (p .01) (Fig. 4). None of the mice showed gross evidence of lung metas- tasis, but on fluorescence microscopy there were numerous evident metastases in control mice lungs with a dramati- cally lower number of metastases in treated animals (99% lower) (Fig. 4B). Histologic evaluation showed that eight mice from the control group had multiple metastatic foci (average, 15.7 2.4) (Fig. 5B1, 5B2), whereas no histolog- ical evidence of lung metastasis could be found in the lungs of the PLX4720-treated mice (average, 0) (p .001) (Fig. 5B5, 5B6). Lymph node metastases are not frequently seen in our model of thyroid cancer ( 50% of cases) (Fig. 5B3, 5B4) and no lymph node metastases were seen in the PLX4720-treated group (Fig. 5B7, 5B8). Tumors from mice receiving PLX4720 showed upregu- lation of thyroid-specific cell differentiation markers (i.e., TTF-1 and PAX-8) at the protein level by IHC (Fig. 5C). IHC staining showed moderate immunoexpression of TTF-1 (diffuse nuclear localization) in PLX4720-treated 8505c orthotopic thyroid tumors, compared with weaker immunoexpression in untreated 8505c orthotopic thyroid tumors (control). Similarly, PAX-8 IHC showed weak cy- toplasmic localization staining and absence in the nuclei in control orthotopic carcinomas, whereas the PLX4720- treated orthotopic carcinomas showed greater focal nuclear positivity (with normal thyroid follicular cells that were strongly positive in the nuclei) (Fig. 5C). Other thyroid cell differentiation markers, such as TPO, thyroglobulin (Tg), TSH receptor, and human sodium–iodide symporter www.TheOncologist.com 303 (hNIS), showed neither immunoexpression in the controls nor upregulation after 3 weeks of PLX4720 treatment in this model. Additionally, given that PLX4720 caused a paradoxical increase in phospho-ERK-1/ERK-2 in vitro in wt B-Raf thyroid cancer cells (TPC-1) and wt B-Raf NT cells, we tested in vivo phospho-ERK-1/ERK-2 levels in mouse tis- sues that express wt B-Raf after treatment with PLX4720. We found no significant difference in phospho-ERK-1/ ERK-2 levels in tissue protein extracts from muscles of SCID mice, compared with control animals, 35 days post– tumor injection (supplemental online Fig. 1B). PLX4720 DOWNREGULATES GENES INVOLVED IN CELL PROLIFERATION, MIGRATION, AND INVASION In order to identify genes that are downstream targets of the B-RafV600E signaling cascade, we quantified absolute mRNA expression in vitro in both 8505c and TPC-1 human thyroid cancer cells following treatment with PLX4720 or vehicle. We analyzed mRNA copy number of early and late gene expression with 1 M or 10 M of PLX4720 for many genes crucial to the control of cell proliferation, migration, and invasion mechanisms (supplemental on- line Table 1). In 8505c cells, we found that only high doses (10 M) of PLX4720 led to significantly lower mRNA expression levels at 72 hours in the following: cyclin D1 (CCND1) (12.1 0.4 copies), thrombospondin-1 (TSP-1) (12.6 1.2 copies), and integrin 6 (ITGA6) (2.9 0.33 copies), compared with controls (38.1 1.7 copies, 45.6 1.3 cop- ies, and 9.7 0.4 copies, respectively; p .05) (Fig. 6). In contrast, we found that the TSP-1 copy number was lower in 8505c cells after 24 hours of treatment with PLX4720 at both the 1 M (32.4 0.58 copies) and 10 M (11.4 0.9 copies) levels, compared with controls (67.84 2.27 copies and 48.05 0.9 copies, respectively; p .05) (Fig. 6). In addition, the TSP-1 copy number was lower in TPC-1 cells treated with PLX4720 (at both the 1 M and 10 M levels) than in controls (Fig. 6). In 8505c cells, we found that both 1 M and 10 M PLX4720 resulted in a higher mRNA expression level of the thyroid follicular cell differentiation marker TTF-1 at 72 hours of treatment (9.5 copies and 9.5 0.4 copies, re- spectively) than in controls (5.1 0.2 copies and 5.1 0.3 copies, respectively; p .05) (Fig. 6). The TTF-1 copy number was greater in 8505c cells after 24 hours of PLX4720 treatment with both 1 M PLX4720 (25.5 1.2 copies) and 10 M PLX4720 (14.7 0.3 copies) than in controls (19.6 0.9 copies and 11.4 0.5 copies, respec- tively; p .05) (Fig. 6). In contrast, PAX-8 showed low mRNA basal levels ( 1 copy number). Furthermore, TPO,