Liver volume-based prediction model stratifies risks for hepatocellular carcinoma in chronic hepatitis B patients on surveillance

<div><p>Background and aim</p><p>The aim of this study was to determine whether dynamic computed tomography (CT)-measured liver volume predicts the risk of hepatocellular carcinoma (HCC) when the CT scans do not reveal evidence of HCC in chronic hepatitis B (CHB) patients on surveillance.</p><p>Methods</p><p>This retrospective multicentre cohort study included 1,246 patients who received entecavir and regular HCC surveillance in three tertiary referral centres in South Korea. Liver volumes were measured on portal venous phase CT images. A nomogram was developed based on Cox independent predictors and externally validated. Time-dependent receiver operating characteristic (ROC) analysis was performed for comparison with previous prediction models.</p><p>Results</p><p>Patients who received dynamic CT studies during surveillance had significantly higher risk for HCC compared to patients without CT studies (hazard ratio [HR] = 3.1; p < 0.001). Expected/measured liver volume ratio was an independent predictor of HCC (HR = 4.2; p = 0.002) in addition to age, sex and cirrhosis. The nomogram based on the four predictors discriminated risks for HCC (HR = 4.1 and 6.0 in derivation and validation cohort, respectively, for volume score > 150; p < 0.001). Time-dependent ROC analysis confirmed better performance of the volume score compared to HCC prediction models with conventional predictors (integrated area under curve = 0.758 vs. 0.661–0.712; p < 0.05).</p><p>Conclusions</p><p>CT-measured liver volume is an independent predictor of future HCC, and nomogram-based liver volume score may stratify the risks of HCC in CHB patients who showed negative CT findings for HCC during surveillance.</p></div

Kaplan-Meier analysis of HCC incidence in the derivation cohort.

<p>Kaplan-Meier analysis of HCC incidence in the derivation cohort.</p

Flow chart of the study population.

<p>*HCV or HIV coinfection, other malignancy or organ transplantation. ^†Dynamic imaging (+) subgroup received at least one dynamic CT study during surveillance which revealed no evidence of HCC: this subgroup served as the derivation dataset for liver volume analysis. No dynamic imaging subgroup did not receive dynamic CT studies during surveillance, except for the confirmative imaging tests in case of HCC. ETV, entecavir; HCC, hepatocellular carcinoma; NA, nucleos(t)ide analogue.</p

Predictors of HCC development by Cox proportional hazard model.

<p>Predictors of HCC development by Cox proportional hazard model.</p

Stratification of HCC probability by nomogram-based liver volume score.

<p>Kaplan-Meier probabilities of HCC incidence were plotted according to the liver volume score cut-off of 150 in the derivation and validation cohort.</p

Anticancer activity of a novel small molecule tubulin inhibitor STK899704

<div><p>We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC₅₀ values ranging from 0.2 to 1.0 μM. STK899704 prevented the polymerization of purified tubulin <i>in vitro</i> and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αβ-tubulin heterodimer overlapping with the colchicine-binding site. Our <i>in vivo</i> carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.</p></div

STK899704 inhibited tubulin polymerization and mitotic spindle organization.

<p>(A) Tubulin polymerization assay. The effect of STK899704 (5 μM) on polymerization of purified tubulin <i>in vitro</i> was examined in a GTP-containing buffer. DMSO was used as a negative control. Tubulin-targeting agents Taxol (5 μM) and vinblastine (5 μM) were also used as controls for tubulin-stabilizing and tubulin-destabilizing agents, respectively. Assembly of tubulin into microtubules was determined by the degree of turbidity at 340 nm. (B) Immunofluorescence staining of microtubules in HeLa cells. Cells were treated with DMSO, Taxol (100 nM), nocodazole (200 ng/ml), or indicated concentrations of STK899704 for 17 h. Cells were then fixed and stained with Alexa Fluor 488-conjugated anti-tubulin antibody and Hoechst 33342 to visualize α-tubulin and DNA, respectively. Scale bar, 10 μm. (C) Fraction of mitotic cells. At least 100 cells from (B) were counted from the different regions. Percentages of normal metaphase, misaligned, multipolar, and tubulin aggregate phenotypes were shown. (D) Proposed binding model of STK899704 on tubulin. The αβ-tubulin heterodimer from PDB entry 1SA0 is shown as ribbon (gray, α-tubulin; cyan, β-tubulin). STK899704 is presented in red stick while colchicine is shown in green. (E) Effect of STK899704 on tubulin binding. Tubulin binding was tested with a SPA-based competition assay. Error bars represent mean ± SDs from three independent experiments.</p

STK899704 inhibited tubulin polymerization and mitotic spindle organization.

<p>(A) Tubulin polymerization assay. The effect of STK899704 (5 μM) on polymerization of purified tubulin <i>in vitro</i> was examined in a GTP-containing buffer. DMSO was used as a negative control. Tubulin-targeting agents Taxol (5 μM) and vinblastine (5 μM) were also used as controls for tubulin-stabilizing and tubulin-destabilizing agents, respectively. Assembly of tubulin into microtubules was determined by the degree of turbidity at 340 nm. (B) Immunofluorescence staining of microtubules in HeLa cells. Cells were treated with DMSO, Taxol (100 nM), nocodazole (200 ng/ml), or indicated concentrations of STK899704 for 17 h. Cells were then fixed and stained with Alexa Fluor 488-conjugated anti-tubulin antibody and Hoechst 33342 to visualize α-tubulin and DNA, respectively. Scale bar, 10 μm. (C) Fraction of mitotic cells. At least 100 cells from (B) were counted from the different regions. Percentages of normal metaphase, misaligned, multipolar, and tubulin aggregate phenotypes were shown. (D) Proposed binding model of STK899704 on tubulin. The αβ-tubulin heterodimer from PDB entry 1SA0 is shown as ribbon (gray, α-tubulin; cyan, β-tubulin). STK899704 is presented in red stick while colchicine is shown in green. (E) Effect of STK899704 on tubulin binding. Tubulin binding was tested with a SPA-based competition assay. Error bars represent mean ± SDs from three independent experiments.</p

STK899704 triggered programmed cell death.

<p>(A) Effect of STK899704 on DNA fragmentation. HeLa cells were treated with 200 ng/ml nocodazole (Noc), 100 nM colchicine (Col), or indicated concentrations of STK899704. Treated cells were then stained with PI and processed for cell cycle analysis at 24 and 48 h. (B) Antagonistic effect of Z-VAD-FMK on STK899704-induced cell death. HeLa cells were treated with DMSO or STK899704 (STK, 1 or 5 μM) in the presence or absence of Z-VAD-FMK (50 μM). Cells were then stained with PI and processed for cell cycle analysis at 24 and 48 h. (C) Effect of STK899704 on the levels of activated caspases. HeLa cells were treated as in (A) and then subjected to immunoblot analysis with antibodies against caspase-3, caspase-7, caspase-8, caspase-9, and PARP. GAPDH was used as a loading control. Each bar indicates mean ± SD of subG₁ population from three independent experiments.</p

STK899704 suppressed the growth of a variety of human cancer cell lines.

<p>(A) Chemical structure of STK899704. (B) Antiproliferative effect of STK899704 on HeLa cells. Cells were seeded at 2 x 10³ cells in 96-well plate and treated with various concentrations of STK899704 for 4 days. Cell growth was determined by MTT assay. (C) Inhibitory effects of STK899704 on the growth of various cancer cell lines. Data were fitted with dose-response curve by using Graphpad Prism software.</p