Single-Dose Neoadjuvant AKT Pathway Inhibitor Reduces Growth of Hepatocellular Carcinoma after Laser Thermal Ablation in Small-Animal Model.

BackgroundLocal recurrence following thermal ablation of hepatocellular carcinoma (HCC) larger than 2-3 cm remains a challenging clinical problem. Prior studies suggest that phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)-dependent protein kinase B (AKT) signaling mediates HCC cell survival caused by moderate heat stress in vitro, but these findings need in vivo validation.PurposeTo test the hypothesis that neoadjuvant inhibition of PI3K/mTOR/AKT signaling reduces HCC tumor growth in vivo after laser ablation and to evaluate the effects of moderate heat stress on molecular signaling and cellular function in HCC cells in vitro.Materials and MethodsHCC tumor-bearing mice were randomized to neoadjuvant PI3K/mTOR inhibitor (BEZ235) or control groups followed by an intentional partial laser ablation or sham ablation; there were at least nine mice per group. Postablation tumor growth was monitored up to 7 days. Tumor volumes were compared for drug or ablation groups by using two-way analysis of variance. N1S1 HCC cells pretreated with BEZ235 or control and subjected to moderate heat stress (45°C for 10 minutes) or control (37°C for 10 minutes) were analyzed by using mass spectrometry. Protein interaction networks were derived from protein expression analysis software, and cellular function activation state (Z-score) and fold-change in AKT phosphorylation were calculated.ResultsThere was a 37%-75% reduction in HCC tumor volume by day 7 after ablation in the BEZ235 plus ablation group (713 mm3 ± 417) compared with vehicle plus sham (1559 mm3 ± 552), vehicle plus ablation (1041 mm3 ± 591), and BEZ235 plus sham (1108 mm3 ± 523) groups (P < .001, P = .04, and P = .005, respectively). PI3K/mTOR inhibition prevented moderate heat stress-induced AKT signaling (Z-score, -0.2; P < .001) and isoform-specific AKT phosphorylation compared with the vehicle plus heat stress group. PI3K/mTOR inhibition prevented moderate heat stress-induced global effects on HCC molecular signaling and cellular function, including decreased cell survival, growth, and proliferation (Z-score, -0.3 to -3.2; P < .001) and increased apoptosis and cell death (Z-score, 0.4-1.1; P < .001).ConclusionModerate heat stress induces PI3K/mTOR/AKT-dependent global effects on hepatocellular carcinoma (HCC) cell survival, function, and death. Neoadjuvant PI3K/mTOR/AKT inhibition reduces postablation HCC tumor growth.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by White in this issue.

Heat Stress and Hepatic Laser Thermal Ablation Induce Hepatocellular Carcinoma Growth: Role of PI3K/mTOR/AKT Signaling.

Purpose To determine if heat stress and hepatic laser thermal ablation induce hepatocellular carcinoma (HCC) growth and to identify growth factors induced by heat stress. Materials and Methods Non-heat-stressed HCC cells were cocultured with HCC cells or hepatocytes that were heat stressed at 37°C (physiologic), 45°C (moderate), or 50°C (severe) for 10 minutes and proliferation monitored with bioluminescence imaging for up to 6 days after heat stress (three experiments). Rats bearing orthotopic N1S1 HCC were randomly assigned to undergo immediate sham or laser thermal (3 W for 60 or 90 seconds; hereafter, 3W×60s and 3W×90s, respectively) ablation of the median (local) or left (distant) hepatic lobe, and tumor growth was monitored with magnetic resonance imaging for up to 18 days after ablation (six or more rats per group). Experiments were repeated with rats randomly assigned to receive either the adjuvant phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor (NVP-BEZ235) or the vehicle control. Heat-stressed HCC cells and hepatocytes were analyzed by using microarray or quantitative real-time polymerase chain reaction analysis for growth factor expression (three or more experiments). Groups were compared by using one- or two-way analysis of variance, and post hoc pairwise comparison was performed with the Dunnett test. Results There were more non-heat-stressed HCC cells when cells were cocultured with cells subjected to moderate but not physiologic or severe heat stress (P < .001 for both). Local intrahepatic N1S1 tumors were larger at day 18 in the 3W×60s (mean, 3102 mm3 ± 463 [standard error]; P = .004) and 3W×90s (mean, 3538 mm3 ± 667; P < .001) groups than in the sham group (mean, 1363 mm3 ± 361) but not in distant intrahepatic tumors (P = .31). Adjuvant BEZ235 resulted in smaller N1S1 tumors in the BEZ235 and laser thermal ablation group than in the vehicle control and laser thermal ablation group (mean, 1731 mm3 ± 1457 vs 3844 mm3 ± 2400, P < .001). Moderate heat stress induced expression of growth factors in HCC cells and hepatocytes, including heparin-binding growth factor, fibroblast growth factor 21, and nerve growth factor (range, 2.9-66.9-fold; P < .05). Conclusion Moderate heat stress and laser thermal ablation induce hepatocellular carcinoma growth, which is prevented with adjuvant PI3K/mTOR/protein kinase B inhibition.

Heat Stress and Thermal Ablation Induce Local Expression of Nerve Growth Factor Inducible (VGF) in Hepatocytes and Hepatocellular Carcinoma: Preclinical and Clinical Studies.

The purposes of this study were to test the hypothesis that heat stress and hepatic thermal ablation induce nerve growth factor inducible (VGF) and to determine intrahepatic versus systemic VGF expression induced by thermal ablation in vivo and in patients. Hepatocytes and HCC cells were subjected to moderate (45°C) or physiologic (37°C) heat stress for 10 min and assessed for VGF expression at 0-72 h post-heat stress (n ≥ 3 experiments). Orthotopic N1S1 HCC-bearing rats were randomized to sham or laser thermal ablation (3 W × 90 s), and liver/serum was harvested at 0-7 days postablation for analysis of VGF expression (n ≥ 6 per group). Serum was collected from patients undergoing thermal ablation for HCC (n = 16) at baseline, 3-6, and 18-24 h postablation and analyzed for VGF expression. Data were analyzed using ordinary or repeated-measures one-way analysis of variance and post hoc pairwise comparison with Dunnett's test. Moderate heat stress induced time-dependent VGF mRNA (3- to 15-fold; p < 0.04) and protein expression and secretion (3.1- to 3.3-fold; p < 0.05). Thermal ablation induced VGF expression at the hepatic ablation margin at 1 and 3 days postablation but not remote from the ablation zone or distant intrahepatic lobe. There was no detectable serum VGF following hepatic thermal ablation in rats and no increase in serum VGF following HCC thermal ablation in patients at 3-6 and 18-24 h postablation compared to baseline (0.71- and 0.63-fold; p = 0.27 and p = 0.16, respectively). Moderate heat stress induces expression and secretion of VGF in HCC cells and hepatocytes in vitro, and thermal ablation induces local intrahepatic but not distant intrahepatic or systemic VGF expression in vivo.

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma.

PURPOSE: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling. MATERIALS AND METHODS: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining. RESULTS: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling. CONCLUSIONS: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

PSMA as a Theranostic Target in Hepatocellular Carcinoma: Immunohistochemistry and 68 Ga-PSMA-11 PET Using Cyclotron-Produced 68 Ga.

Prostate-specific membrane antigen (PSMA) is a validated target for molecular diagnostics and targeted radionuclide therapy. Our purpose was to evaluate PSMA expression in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatic adenoma (HCA); investigate the genetic pathways in HCC associated with PSMA expression; and evaluate HCC detection rate with 68 Ga-PSMA-11 positron emission tomography (PET). In phase 1, PSMA immunohistochemistry (IHC) on HCC (n = 148), CCA (n = 111), and HCA (n = 78) was scored. In a subset (n = 30), messenger RNA (mRNA) data from the Cancer Genome Atlas HCC RNA sequencing were correlated with PSMA expression. In phase 2, 68 Ga-PSMA-11 PET was prospectively performed in patients with treatment-naïve HCC on a digital PET scanner using cyclotron-produced 68 Ga. Uptake was graded qualitatively and semi-quantitatively using standard metrics. On IHC, PSMA expression was significantly higher in HCC compared with CCA and HCA (P < 0.0001); 91% of HCCs (n = 134) expressed PSMA, which principally localized to tumor-associated neovasculature. Higher tumor grade was associated with PSMA expression (P = 0.012) but there was no association with tumor size (P = 0.14), fibrosis (P = 0.35), cirrhosis (P = 0.74), hepatitis B virus (P = 0.31), or hepatitis C virus (P = 0.15). Overall survival tended to be longer in patients without versus with PSMA expression (median overall survival: 4.2 vs. 1.9 years; P = 0.273). FGF14 (fibroblast growth factor 14) mRNA expression correlated positively (rho = 0.70; P = 1.70 × 10-5 ) and MAD1L1 (Mitotic spindle assembly checkpoint protein MAD1) correlated negatively with PSMA expression (rho = -0.753; P = 1.58 × 10-6 ). Of the 190 patients who met the eligibility criteria, 31 patients with 39 HCC lesions completed PET; 64% (n = 25) lesions had pronounced 68 Ga-PSMA-11 standardized uptake value: SUVmax (median [range] 9.2 [4.9-28.4]), SUVmean 4.7 (2.4-12.7), and tumor-to-liver background ratio 2 (1.1-11). Conclusion: Ex vivo expression of PSMA in neovasculature of HCC translates to marked tumor avidity on 68 Ga-PSMA-11 PET, which suggests that PSMA has the potential as a theranostic target in patients with HCC.

Automatic and Reliable Quantification of Tonic Dopamine Concentrations In Vivo Using a Novel Probabilistic Inference Method.

Dysregulation of the neurotransmitter dopamine (DA) is implicated in several neuropsychiatric conditions. Multiple-cyclic square-wave voltammetry (MCSWV) is a state-of-the-art technique for measuring tonic DA levels with high sensitivity (<5 nM), selectivity, and spatiotemporal resolution. Currently, however, analysis of MCSWV data requires manual, qualitative adjustments of analysis parameters, which can inadvertently introduce bias. Here, we demonstrate the development of a computational technique using a statistical model for standardized, unbiased analysis of experimental MCSWV data for unbiased quantification of tonic DA. The oxidation current in the MCSWV signal was predicted to follow a lognormal distribution. The DA-related oxidation signal was inferred to be present in the top 5% of this analytical distribution and was used to predict a tonic DA level. The performance of this technique was compared against the previously used peak-based method on paired in vivo and post-calibration in vitro datasets. Analytical inference of DA signals derived from the predicted statistical model enabled high-fidelity conversion of the in vivo current signal to a concentration value via in vitro post-calibration. As a result, this technique demonstrated reliable and improved estimation of tonic DA levels in vivo compared to the conventional manual post-processing technique using the peak current signals. These results show that probabilistic inference-based voltammetry signal processing techniques can standardize the determination of tonic DA concentrations, enabling progress toward the development of MCSWV as a robust research and clinical tool.

Uterine fibroids: correlations between MRI appearance and stiffness via magnetic resonance elastography.

RATIONALE AND OBJECTIVES: Magnetic resonance elastography has proven to be a valuable tool in the diagnosis of liver fibrosis, breast and cervical cancer, but its application in uterine fibroids requires further characterization. The aim of the present study was to examine the relationship between uterine fibroid stiffness by MRE and MR imaging characteristics. MATERIALS AND METHODS: An IRB-approved, HIPAA compliant review was performed of prospectively collected pelvic MRI and 2D-MRE data in patients with symptomatic uterine fibroids (N = 102). T1 and T2 weighted pelvic MRI with gadolinium enhancement were performed. In a small patient subset, fibroid stiffness was assessed by both 2D and 3D MRE. Fibroid stiffness by modality or imaging characteristics was analyzed using one-way analysis of variance followed by Student t test. RESULTS: Four fibroid groups were identified based on T2 appearance: Isointense (N = 7), bright (N = 6), dark with minimal heterogeneity (N = 69), and dark with substantial heterogeneity (N = 20). Mean fibroid stiffness was 4.81 ± 2.12 kPa. Comparison of fibroid stiffness by T2 signal intensity showed that T2 bright fibroids were significantly less stiff than fibroids appearing T2 dark with minimal heterogeneity (mean stiffness difference = 2.38 kPa; p < 0.05) and T2 dark fibroids with substantial heterogeneity were significantly less stiff than T2 dark fibroids with minimal heterogeneity (mean difference = 1.25 kPa; p < 0.05). There was no significant association between fibroid stiffness and T1 signal characteristics or gadolinium enhancement. There was no significant difference in stiffness values obtained by either 2D vs. 3D MRE. CONCLUSIONS: These data suggest differences in fibroid stiffness are associated with different T2 imaging characteristics with less stiff fibroids being T2 bright and more stiff fibroids being T2 dark. Further studies are needed to determine if fibroid stiffness by MRE may serve as an imaging biomarker to help predict MR-guided treatment response.

MRI-guided Wire Localization Surgical Biopsy in an Adolescent Patient with a Difficult to Diagnose Case of Lymphoma.

A 17-year-old previously healthy female presented with a progressive soft tissue infiltrative process involving the neck and thorax. Extensive diagnostic evaluation including multiple imaging, laboratory, and biopsy studies was nondiagnostic. Due to an urgent need to establish a diagnosis and several previous nondiagnostic biopsies, she was referred to interventional radiology for MRI-guided wire localization immediately prior to open surgical biopsy. Under general anesthesia, wires were placed in the areas of increased T2 signal within the bilateral splenius capitis muscles using intermittent MRI-guidance followed by immediate surgical biopsy down to the wires. Pathology confirmed the diagnosis of diffuse large B-cell lymphoma.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.