006, Fig. 2A). The copy numbers for FGF3/FGF4 were 10.2 ± 0.8/6.7 ± 0.8, 26.7 ± 0.4/35.1 ± 3.1, and 162.5 ± 9.0/165.0 ± 12.5 copies in the amplified samples, whereas the copy numbers of FGF3 for all the other samples were below 5 copies. The correlation between the FGF3 locus and the FGF4 locus copy numbers was very high (R

= 0.998), indicating that the DNA copy number assay for FGF3/FGF4 was a sensitive and reproducible method. We examined the messenger RNA (mRNA) expression levels of FGF3/FGF4 in nine HCC samples that were available as frozen samples among the this website 48 sorafenib-treated samples, as shown in Fig. 2A. One amplified sample expressed extremely high mRNA levels of FGF3/FGF4 compared with nonamplified samples (Fig. 2B). The results demonstrated that FGF3/FGF4 gene amplification mediates the overexpression of FGF3/FGF4 mRNAs and proteins (Figs. 2B and 1D). We used FISH analysis to examine FGF3/FGF4 amplification and to verify the results of the above-described PCR-based DNA copy number assay. All FGF3/FGF4-amplified clinical samples were confirmed as exhibiting high-level FGF3 amplification using FISH analysis (Fig. 3). One patient showed multiple scattered signals, whereas two patients showed large clustered signals. Nonamplified HCC

yielded a negative result for gene amplification. These results clearly demonstrate the presence of FGF3/FGF4-amplified HCC among the clinical samples, and the FISH

analysis results were consistent with those for the copy number assay. To determine the frequency check details of FGF3/FGF4 gene amplification in HCC, we performed a copy number assay for HCC samples without sorafenib treatment in a series of surgical specimens. Two of the 82 (2.4%) HCC samples exhibited FGF3/FGF4 gene amplification, with copy numbers medchemexpress of 10.7/15.3 and 133.3/112.7 copies, respectively (Fig. 4). One amplified HCC was a poorly differentiated tumor, whereas the other was a moderately differentiated tumor. The clinico-pathological features of the sorafenib responders are shown in Table 1. A comparison of clinical factors (age, sex, viral status, alpha-fetoprotein level, PIVKA-II, clinical stage, primary tumor size, metastatic status, histological type, and tumor response between responders and nonresponders) is given in Table 2. Notably, multiple lung metastases over five nodules was significantly higher among responders to sorafenib (responders, 5/13 [38%]; nonresponders, 2/42 [5%]; P = 0.006). Although the difference was not significant, poorly differentiated HCC tended to be more common among responders to sorafenib (responders, 5/13 [38%]; nonresponders, 6/42 [14%]; P = 0.13). These results suggest that multiple lung metastases and a poorly differentiated histology may be clinical biomarkers for sorafenib treatment in patients with HCC.