Home Hypericin as a Marker for Determination of Tissue Viability After Intratumoral Ethanol Injection in a Murine Liver Tumor Model
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Hypericin as a Marker for Determination of Tissue Viability After Intratumoral Ethanol Injection in a Murine Liver Tumor Model

Rationale and Objectives

In this preclinical proof-of-principle study, the necrosis avid agent hypericin was investigated as a potential early indicator for therapeutic response after ethanol-mediated chemical ablation in murine liver tumors.

Materials and Methods

Seven mice bearing intrahepatic radiation-induced fibrosarcoma-1 tumors were intravenously injected with hypericin 1 hour before ( n = 3) or 24 hours after ( n = 4) intratumoral ethanol injection. Mice were euthanized 24 hours after hypericin injection and, taking advantage of the fluorescent property of the compound, the excised livers were investigated qualitatively and quantitatively by means of fluoromacroscopic and fluoromicroscopic examinations, colocalized with conventional histomorphology.

Results

Significant differences in hypericin fluorescence were found in necrosis, viable tumor and normal liver tissue in decreasing order ( P < .05) (ie, in necrosis, mean fluorescence densities were about 4.5 times higher than in viable tumor and approximately 14 times higher than in normal liver). When hypericin was injected 1 hour before, maximal blood concentrations were achieved at the time of ethanol treatment, so that on ablation an outstanding extravasation took place in the entire necrotic area in comparison with accumulation of hypericin only at the peripheral zone of necrosis when it was injected 24 hours after ablation.

Conclusions

Hypericin specifically enhanced the imaging contrast between necrotic and viable tissues and nonspecifically distinguished viable tumor from normal liver. Injection of hypericin shortly before ablation is more favorable than after ablation, because it circumvents difficulties with no-entry zones for hypericin and requires shorter intervals between ethanol ablation and imaging.

Percutaneous ethanol injection (PEI) is a minimally invasive treatment, which has been clinically applied for a couple of decades as an effective alternative to surgery for patients with small hepatocellular carcinoma. Instead of excising the entire tumor from the patient as in standard surgery, PEI serves as a therapy of chemical ablation to instantaneously kill the tumor in situ by intratumoral injection of absolute ethanol. Complete necrosis of lesions smaller than 3 cm in diameter has been achieved ( ). However, as a result of difficulties in obtaining homogeneous intratumoral alcohol distribution, incomplete therapeutic effect may occur because of residual viable neoplastic tissue in tumors larger than 3 cm ( ). This may lead to tumor recurrence particularly along the periphery of the treated area ( ).

A troublesome flaw related to PEI treatment is the difficulty to confirm complete necrosis early after treatment. Early detection of a residual or locally recurrent tumor has a major effect on the prognosis and adjuvant treatment, whereas late diagnosis is associated with peripheral regrowth and remote metastasis making retreatment difficult ( ). Because biopsies and serum tumor markers are only of limited usefulness for assessing the response to PEI, evaluation of the therapeutic effect of PEI is based mainly on findings at imaging studies ( ). However, the visual effects created with all currently applied in vivo imaging techniques (eg, magnetic resonance imaging [MRI], computed tomography, ultrasound [US] imaging) are nonspecific, indirect, and inaccurate. On contrast enhanced MRI, a thin and regular enhancing halo, from an inflammatory reaction, is often observed at the periphery of the hypointense necrotic lesion ( ). Because this strongly enhanced halo may mimic residual tumor, interpretation of enhancing areas at the periphery of the treated lesion can be difficult. Only at later follow-up imaging studies, a few months after treatment, the halo disappears ( ). The use of color Doppler US and contrast-enhanced gray-scale US imaging is only recommended to monitor the response of the tumor during the course of PEI treatment from a substantial risk for false-negative results in postprocedural imaging ( ). Therefore, with the current diagnostic techniques, it is difficult to make a clear-cut distinction between the ablated dead tissues and unablated viable tissues early after PEI treatment.

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Materials and methods

Animals and Tumor System

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Figure 1, Monitoring tumor growth (arrow) of radiation-induced fibrosarcoma (RIF-1) in mice with T2-weighted magnetic resonance imaging at 7, 9, 11, and 13 days after intrahepatic implantation. Tumor size about 5 mm (d) in diameter was considered appropriate for the treatment with intratumoral ethanol injection.

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Intratumoral Ethanol Injection

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Hypericin Preparation and Administration

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Fluoromacroscopic and Fluoromicroscopic Examinations

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Imaging Quantitative Analyses

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Results

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Figure 2, Macroscopic photographs taken under ultraviolet light and corresponding photographs under tungsten light of livers bearing ethanol-treated RIF-1 tumors. Mice were intravenously injected with 10 mg/kg hypericin at 24 hours after (a,b) or 1 hour before (c,d) ethanol treatment. All mice were sacrificed at 24 hours after hypericin injection and excised livers were digitally photographed. Ablated tumor (*), viable tumor (o), normal liver (x), ablated liver (**). Scale bar = 0.5 cm.

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Figure 3, Fluorescence photomicrographs and corresponding hematoxylin and eosin stains of 5-μm slices of livers bearing ethanol-treated RIF-1 tumors. Mice were intravenously injected with 10 mg/kg hypericin at 24 hours after (a,b) or 1 hour before (c,d) ethanol treatment. All mice were sacrificed at 24 hours after hypericin injection. Ablated tissue (NE), dehydrated ablated tissue (NE*), viable tumor (T), normal liver (L). Scale bar = 100 μm.

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Table 1

Ratios of Mean Fluorescence Densities in Different Regions of Fluoromicroscopic Images Taken From 5-μm Tumor Sections

Δ T NE Tissue Viable T NE Tissue Normal L Viable T Normal L 24 h 5.53 ± 2.29 (ns) 15.20 ± 8.89 (ns) 3.84 ± 1.24 (ns) 1 h 3.54 ± 1.12 13.13 ± 5.82 3.05 ± 1.86

Tumors were excised from mice that received 10 mg/kg hypericin either 24 h after (Δ T = 24 h), or 1 h before ethanol treatment (Δ T = 1 h). Statistical analysis, comparing (Δ T = 24 h) with (Δ T = 1 h) was performed using Prism 4.00, GraphPad Software (San Diego, CA). A two-way analysis of variance with Bonferroni test was performed and P values of P < .05 were considered statistically significant. NE: necrotic; T: tumor; L: liver; ns: non-significant.

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Discussion

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