Concurrent versus Sequential Sorafenib Therapy in Combination with Radiation for Hepatocellular Carcinoma

<div><p>Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both <i>in vitro</i> and <i>in vivo</i>. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines <i>in vitro</i> by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed <i>in vivo</i> by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. <i>In vitro</i>, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G₁-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. <i>In vivo</i>, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both <i>in vitro</i> and <i>in vivo</i>. These results may have implications for clinical decision-making and prospective trial design.</p></div

Mechanism of sorafenib-mediated radioprotection <i>in vitro</i>.

<p>HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G₁-S versus G₂-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay in both cell lines and reduced the number of cells in G₂-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's <i>t-</i>test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2</a>. Data for unsynchronized HepG2 cells are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2</a>. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2C</a>.</p

Active clinical trials currently registered on ClinicalTrials.gov that involve the administration of sorafenib and radiation for hepatocellular carcinoma (HCC).

<p>If available, the treatment schedule is specified in the right-most column; all sequential schedules consisted of radiotherapy first followed by sorafenib.</p

A sequential radiation-sorafenib regimen is most efficacious against HCC <i>in vivo</i>.

<p>A HepG2 hind-flank xenograft model was utilized to measure the efficacy of (A) 5 different treatment arms: control (sham injection of vehicle control on days 1–5), sorafenib alone (SOR; injection of 6 mg/mL sorafenib on days 1–5), radiation alone (RT; irradiation at a dose of 3 Gy on days 1–3), concurrent radiation-sorafenib (CONC; sorafenib injection on days 1–5 and irradiation at a dose of 3 Gy on days 2–4), and sequential radiation-sorafenib (SEQ; irradiation at a dose of 3 Gy on days 1–3 and sorafenib injection on days 4–8). The number of tumors per arm was: <i>n</i> = 13 for control, <i>n</i> = 12 for SOR, <i>n</i> = 15 for RT, <i>n</i> = 17 for CONC, and <i>n</i> = 19 for SEQ. Data for each arm are plotted as (B) tumor volume ratio over time (<i>left</i>) and as Kaplan-Meier curves with attainment of quadruple the pre-treatment tumor volume as the event of interest (<i>right</i>). Using two methods of statistical analysis (Mann-Whitney U-test for <i>left</i> and log-rank test for <i>right</i>), SEQ was shown to achieve a significantly longer time to quadruple the pre-treatment tumor volume than any of the other treatment arms. The CONC and RT arms were not significantly different from one another. (C–F) Immunofluorescence staining from xenografts harvested from all treatment arms show significantly more downregulation of vascularity (CD31) (C – <i>left column</i>, D) and decreased mitotic index (Ki-67) (C – <i>right column</i>, E) in arms that received sorafenib treatment, with the most pronounced reductions occurring in the SEQ arm. Immunohistochemical staining for γ-H2AX (C – <i>middle column</i>, F), however, revealed a significantly greater percent of nuclei with high or moderate numbers of foci, as well as a lower percent of nuclei with no foci, for the SEQ and RT arms compared to the CONC arm, similar to our <i>in vitro</i> results above. Column graphs summarizing the data for CD31, γ-H2AX, and Ki-67 are shown in D-F. Asterisks represent significant differences between columns ascertained by Student's <i>t-</i>test (CD31) or by Fisher's exact test (γ-H2AX and Ki-67) as indicated by the accompanying brackets.</p