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. Author manuscript; available in PMC: 2023 Apr 25.
Published in final edited form as: Med Phys. 2023 Jan 30;50(3):1728–1735. doi: 10.1002/mp.16232

Hydralazine-augmented contrast ultrasound imaging improves the detection of hepatocellular carcinoma

Laith R Sultan 1,2, Mrigendra B Karmacharya 2, Maryam Al-Hasani 1, Theodore W Cary 2, Chandra M Sehgal 1
PMCID: PMC10128060  NIHMSID: NIHMS1893837  PMID: 36680519

Abstract

Background:

Hepatocellular carcinoma (HCC) detection with B-mode and contrast-enhanced ultrasound (CUS) imaging often varies between subjects, especially in patients with background cirrhosis. Various factors contribute to this variability, including the tumor blood flow, tumor size, internal echoes, and its location in livers with diffuse fibro-cirrhotic changes.

Objective:

Towards improving lesion detection, this study evaluates a vasodilator, hydralazine, to enhance the visibility of HCC by reducing its blood flow relative to the surrounding liver tissue.

Methods:

HCC were analyzed for tumor visibility measured for B-mode, CUS, and hydralazine-augmented-contrast ultrasound (HyCUS) in an autochthonous HCC rat model. 21 tumors from 12 rats were studied. B-mode and CUS images were acquired before hydralazine injection. Rats received an intravenous hydralazine injection of 5 mg/kg, then images were acquired 20 min later. Four rats were used as controls. The difference in echo intensity of the lesion and the surrounding tissue was used to determine the visibility index (VI).

Results:

The visibility index for HCC was found to be significantly improved with the use of HyCUS imaging compared to traditional B-mode and CUS imaging. The visibility index for HCC was 16.5 ± 2.8 for HyCUS, compared to 5.3 ± 4.8 for B-mode and 4.1 ± 3.8 for CUS. The differences between HyCUS and the other imaging modalities were statistically significant, with p-values of 0.001 and 0.02, respectively. Additionally, when compared to control cases, HyCUS showed higher discrimination of HCC (VI = 6.4 ± 1.2) with a p-value of 0.003, while B-mode (VI = 6.7 ± 1.4, p = 0.5) and CUS (VI = 6.4 ± 1.2, p = 0.3) showed lower discrimination.

Conclusion:

Vascular blood flow modulation by hydralazine enhances the visibility of HCC. HyCUS offers a potential problem-solving method for detecting HCC when B-mode and CUS are unsuccessful, especially with background fibro-cirrhotic liver disease. Future evaluation of the approach in humans will determine its translatability for clinical applications.

Keywords: hepatocellular carcinoma, cancer imaging, tumor detection, contrast-enhanced ultrasound, hydralazine, imaging biomarkers

1 |. INTRODUCTION

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and represents one of the highest cancer-incidence and fatalities worldwide.1,2 Patients with liver cirrhosis are considered a particularly high-risk group for developing HCC, prompting the surveillance of adults with cirrhosis for early detection of HCC to improve management outcomes and overall survival.3–8 The preferred imaging modality for HCC surveillance across all major professional liver organizations worldwide has been abdominal ultrasound (US).9–11 US imaging is a safe, noninvasive, and less expensive modality that is frequently performed to detect liver nodules during cirrhotic patients’ surveillance. However, the accuracy of HCC detection with conventional B-mode US varies widely. In cirrhotic patients, its sensitivity is 33%–86%.12 This wide variation is related mainly to the image quality and experience of the operator, patient-related factors such as body habitus and cooperativeness, and tumor-related factors including tumor size, internal tumor echo-intensity pattern, and tumor location in the liver with diffuse fibro-cirrhotic.13

Contrast-enhanced ultrasound (CUS) was introduced in the early 2000s to characterize nodules arising in cirrhotic livers.14 CUS is an advanced form of US imaging utilizing tiny microbubble contrast agents with diameters like that of red blood cells.15 With microbubble contrast agents, CUS provides details of the hemodynamics useful for detecting and analyzing liver tumors.16,17 Currently, CUS is included as a part of the suggested diagnostic work-up of focal liver lesions, resulting in better patient management and cost-effective delivery of therapy.18 However, the CUS findings of HCCs reported in the literature show inconsistency among different institutions in detecting HCC and its use is still controversial.19–25 CUS shares many image quality limitations with the B-mode US and is affected by underlying background liver disease and other patient and tumor-related factors.23 Additionally, the inflow of an echogenic contrast agent in the tumors makes them isoechoic and tissue-lesion margins challenging to detect. The lack of differentiation is pronounced in HCC because of its highly vascular nature.26 This study aims to enhance tissue-lesion margin contrast by affecting tumor blood flow with a vasomodulator.

Hydralazine is a vasodilator that transiently reduces blood flow to the tumor by siphoning it to the surrounding tissue due to the inability of the tumor blood vessels to dilate.27,28 Hydralazine effects on tumor flow have been reported in literature.27–34 Our literature search showed that although studies did report “the steel phenomena” which selectively affect tumor vessels and divert flow toward the peripheral tissue, yet the use of hydralazine for improving the visibility of HCC has not been evaluated previously. This study hypothesizes that modulation of blood flow in the HCC and liver by hydralazine in opposite directions would enhance the visibility of HCC. This study uses a preclinical autochthonous HCC rat model to assess the feasibility of enhancing HCC-liver margins in contrast-ultrasound imaging.

2 |. METHODS

2.1 |. Animal model and tumor growth monitoring

The University Institutional Animal Care and Use Committee approved all animal protocols. Sixteen Wistar male rats (Charles River Laboratories, Wilmington, MA, USA) weighing between 300 and 400 g, with an average of 6 months of age, were acquired and kept under controlled environmental conditions (25°C and a 12 h light/dark cycle). After 1 week of acclimation, the rats were initiated on an established hepatotoxin, 0.01% diethylnitrosamine (DEN, Sigma Aldrich, St. Louis, MO, USA),35 in their drinking water. The animals ingested hepatotoxin ad libitum for 12 weeks. Normal drinking water was returned at the beginning of the 13th week. Following DEN ingestion for 11 weeks, the rats were monitored for DEN-induced liver fibrotic-cirrhotic changes and development of HCC with grayscale B-mode ultrasound. Tumors with sizes ranging from 5 to 15 mm were imaged with contrast (CUS) and with and without an infusion of hydralazine.

After completing ultrasound imaging, Evans blue (EB) solution (5% w/v) was used to assess the tumors’ vascular flow changes induced by hydralazine. EB dye was prepared by dissolving 0.5 g of the dye powder (CAS Number 314-13-6, Sigma Aldrich, St. Louis, MO, USA) in 10 ml of 0.9% normal saline. The solution was filter-sterilized and stored in sterile 1.5 ml polypropylene tubes. EB solution (150 μl) was injected through the lateral tail vein in rats after completing the imaging sessions. After waiting for 10 min for the dye to circulate, the animals were sacrificed, and an autopsy was performed. Liver tissue and tumor samples were collected and processed for histological studies.

Two groups of animals were studied. They consisted of a test group (N = 12) and a control group (N = 4) receiving hydralazine and saline infusion before contrast ultrasound imaging.

2.2 |. Imaging studies for HCC

2.2.1 |. Contrast-enhanced ultrasound (CUS) imaging

A baseline contrast-enhanced ultrasound imaging was performed to image the tumor microvasculature. It involved baseline B-mode imaging of the HCC, followed by microbubbles contrast agent injection, and monitoring the inflow and outflow of microbubbles through a fixed image plane. The images were recorded at 10 fps in a video clip during the contrast enhancement of the tumor till the contrast agent washout. Video clips were analyzed quantitatively for image enhancement.

The baseline B-mode and contrast ultrasound imaging were performed using VisualSonics Vevo 2100 system (13–24 MHz— VevoLAZR, Fujifilm VisualSonics, Toronto, ON, Canada). The image settings and gain were kept the same for all animals. Imaging was performed under anesthesia gaseous anesthetic system [VetE-quip Inc, Livermore, CA, USA], and general anesthesia was induced with 4% isoflurane (IsoSol; VEDCO Inc, St Joseph, MO, USA). Animals were positioned supine on a heated bed, the abdomen was depilated, and continuous vital signs, including body temperature, heart rate, and respiratory rate, were recorded. 0.05 ml of contrast-enhancing perflutren lipid microspheres (Definity, Lantheus Medical Imaging, North Billerica, MA, USA) were injected using a 26G catheter [Covidien, Dublin, Ireland].

2.2.2 |. Hydralazine-augmented contrast ultrasound (HyCUS) imaging for HCC

Following the baseline CUS imaging, the rats received intravenous hydralazine (5 mg/kg; CAS # 304-20-1, Sigma, St. Louis, MO, USA). The choice of the 5 mg/kg hydralazine dose was based on previous studies in mice.36 CUS serial images were acquired 20 min following hydralazine injection to assess the changes in tumor enhancement. Imaging presets (gain = 18 dB; high sensitivity; 100% power; transmit frequency 21 MHz; high line density) and time-gain-compensation was kept fixed for pre- and post-Hyd imaging for all tumors. Twelve rats received hydralazine and four rats did not and were used as controls.

2.3 |. Image analysis of HCC tumor echo-intensity

The quantitative echo-intensity analysis of B-mode, CUS, and HyCUS images was performed using an IDL (Interactive Data Language)-based image analysis platform.37,38 For CUS and HyCUS, videoclips of serial images representing the contrast arrival and washout were analyzed. The borders of the tumors were manually outlined to define a region of interest (ROI). The computer program used this margin to draw a concentric ROI of an equal area in the surrounding liver tissue. The software computed the echo-intensities (brightness) of tumors and surrounding liver tissue for serial images. The brightness (echogenicity) of the lesion Blesion and the surrounding tissue Btissue of the peak-enhanced image of the serial data were used to determine visibility index, VI, by the equation39:

VI=|(Blesion−Btissue)SE| (1)

SE is the above equation represents the standard error between the two means. Vertical brackets in the equation designate the absolute value of the difference without a sign.

2.4 |. Statistical analysis

The mean [±standard error of the mean (SEM)] values of VI were compared between the different groups. Statistical analyses were performed using the student’s two-tailed paired t-test. A p-value less than 0.05 is typically considered to be statistically significant.

2.5 |. Histological assessment of hydralazine effects on HCC by Evans blue injection

Digital microscopic fluorescence images of liver sections were acquired with a microscope (Zeiss Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany). The EB fluorescence of the tumors and the adjacent liver tissue were analyzed using the software ImageJ.40 EB binds to albumin and undergoes a conformational shift producing fluorescence in the red to far-red spectrum (excitation at 620 nm, emission at 680 nm).41 The blue, red, and green colors of the fluorescence images were split for quantification. The intensity of the red color corresponding to EB fluorescence was measured from five different parts of tumors and the adjacent liver tissue. The values for each subject were averaged and presented as means (± standard error of means, SEM).

3 |. RESULTS

3.1 |. Development of HCC and their detection by B-mode ultrasound

All animals developed liver disease and multiple HCC foci in response to DEN treatment. These lesions were seen on B-mode ultrasound imaging and confirmed during autopsy. Images of twenty-one liver tumors from twelve rats in the test group were evaluated. The tumors were first detected between 12 and 15 weeks from the start of the DEN. HCCs ranged between 5 and 12 mm. The tumors were widely spread among different liver lobes and locations within each lobe.

The mean echo intensity of HCC on B-mode images was 42.7 ± 4.6, almost equivalent to the surrounding liver tissue echo intensity (41.1 ± 3.9), p = 0.31. Qualitative image assessment showed 16 of 21 tumors had echo-intensity (isoechoic) similar to the surrounding liver tissue. Four tumors were hypoechoic, and one was hyperechoic relative to the surrounding liver tissue.

3.2 |. Detection of HCC by contrast-enhanced ultrasound (CUS) imaging

In the contrast-enhanced images (CUS), the mean peak enhancement of the HCCs was 62.4 ± 4.7 compared to 64.9 ± 4.6 for the surrounding liver tissue. The difference between tumor and liver enhancement was not statistically significant, p = 0.37. Qualitatively 17 tumors showed comparable enhancement of to surrounding liver tissue. Three tumors were less enhanced, while one tumor was more enhanced than the surrounding liver tissue.

3.3 |. Detection of HCC by hydralazine-augmented ultrasound (HyCUS)

In the HyCUS images, the mean peak enhancement of HCCs was 37.8 ± 5.1, which was lower than 55.3 ± 5.7 for the surrounding liver tissue. The difference was statistically significant p = 0.0002. The visual inspection of HyCUS images showed that all tumors decreased in enhancement following Hyd injection and eventually enabled a better visualization of the tumors than B-mode and CUS images. Fifteen tumors from a total of 21 studied were smaller than 10 mm and located in different lobes at different depths of the liver tissue.

3.4 |. Comparison of lesion visibility in B-mode, CUS and HyCUS

The visibility index (VI) was higher in HyCUS images, 16.51 ± 2.8 (mean ± SE) when compared to 5.3 ± 4.8 for B-mode, and 4.1 ± 3.8 for CUS (Figure 1). The difference was statistically significant, p = 0.001 and p = 0.02, for the two imaging modalities respectively when compared with HyCUS. In comparison to control cases, HyCUS showed higher discrimination of HCC (VI = 6.4 ± 1.2, p = 0.003), than that for B-mode (VI = 6.7 ± 1.4, p = 0.5) and for CUS (VI = 6.4 ± 1.2, p = 0.3). Figure 2 shows HCCs demonstrating an improvement in the visibility of the tumors with hydralazine injection. In B-mode, the tumor was isoechoic (equal brightness) to that of surrounding liver tissue. Following contrast injection, both tumors and liver showed a similar peak enhancement making it hard to identify the tumor accurately. However, tumor detectability improved substantially due to a reduction in the lesion echo intensity when Hyd was used.

FIGURE 1.

FIGURE 1

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A bar graph showing the differences in visibility indexes between B-mode, contrast-enhanced ultrasound (CUS), and hydralazine-augmented contrast ultrasound (HyCUS). The visibility index (VI) was substantially higher in HyCUS images than in CUS and B- mode images. In contrast, VI of HCC did not show a statistically significant difference between CUS and B-mode. It is also notable that the variability of VI is largest with B-mode and CUS but decreases considerably with HyCUS. CUS; contrast-enhanced US and HyCUS; Hydralazine enhanced US. Ns: not statistically significant, ** Statistically significant difference between B-mode and HyCUS, *** Statistical significant difference between HyCUS and CUS.

FIGURE 2.

FIGURE 2

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Examples of HCC that showed a characteristic pattern of increasing enhancement by HyCUS imaging. B-mode images show HCC to be isoechoic to the surrounding liver tissue. Following contrast injection, both tumors and tissue showed similar enhancement at peak level point, making it hard to identify the tumor accurately. However, post-Hyd injection, the tumor echo-intensity was remarkably lower than that of the liver, leading to a major improvement in detectability. The white arrow in the image indicates the tumor. E1:example 1, E2 example 2, CUS: contrast-enhanced US, HyCUS, hydralazine augmented US.

3.5 |. Hydralazine effects on HCC demonstrated by Evans blue dye injection

A qualitative evaluation of micrographs of the liver tissue demonstrated equal Evans blue fluorescence from the tumors and the adjacent liver tissue in the controls. On the other hand, the hydralazine-treated group showed much less Evans blue fluorescence in the tumors than from the adjacent liver tissue (Figure 3). Quantitatively, Evans blue fluorescence in the tumors and the surrounding liver tissue were 94.7 ± 2.8 (a.u.) and 91.2 ± 3.2 (a.u.) in the controls;and 97.4 ± 3.6 (a.u.) and 45.2 ± 0.9 (a.u.) in the Hyd groups. The difference in Evans blue fluorescence between the tumors and the surrounding liver tissue was statistically significant (p < 0.05)

FIGURE 3.

FIGURE 3

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Micrographs showing Evans blue fluorescence in control (left) versus hydralazine injected (Hyd, right) HCC and the adjacent liver tissue. Arrows demarcate the boundary between the tumor and the adjacent liver tissue. Scale bar = 50 μm.

4 |. DISCUSSION

Imaging detection of HCC can be challenging, especially in patients with advanced cirrhosis, in which structural and physiological alterations of the background liver parenchyma can impair HCC detection and diagnosis. Ultrasound is an ideal imaging modality for monitoring and detecting HCC in cirrhotic patients due to its safety, portability, and affordability. A major disadvantage with the B-mode US is that its interpretation is affected by the image quality and patient and tumor factors, including internal tumor echo-intensity patterns, tumor size, and tumor location in the liver. CUS improves HCC detection,19 but the improvements are marginal. Still, it requires substantial experience and expertise to characterize nodules, especially the small tumors located deep in a cirrhotic liver. Despite continued improvements in ultrasound imaging and CUS technology, there is an unmet need for new advances to overcome the current limitations. This study exploits the tumor’s unique physiology to enhance lesion-tissue contrast.

The highly vascular nature of HCC and the aberrant characteristics of tumor blood vessels provides a unique paradigm for vasomodulation of tumor blood flow that is yet to be exploited for enhancing ultrasound imaging. This study used a vasodilator, hydralazine (Hyd), to enhance the contrast resolution of HCC by modulating its blood flow relative to the surrounding liver tissue. Hydralazine reduces tumor blood flow,27,28 but its use for improving ultrasound detection of HCC has not been previously demonstrated. This study hypothesized that pretreatment with hydralazine before CUS would enhance lesion-tissue contrast by siphoning blood flow from the lesion to the surrounding liver tissue. The drain of blood flow from the tumor’s vasculature results in its inability to dilate in response to the hydralazine treatment. The results show that HCCs’ visibility improved substantially after administration of Hyd compared to that of B-mode and CUS images (Figure 1). The decrease in tumor blood flow with Hyd injection resulted in a marked reduction in tumor internal echo intensity while enhancing the echo signal from the surrounding liver tissue. The differential increase between the liver tissue and the lesion results in improved visibility of the lesion (Figure 2). Visually, HCC visibility by HyCUS was higher and more consistent compared to B-mode and CUS images.

With HyCUS images, we particularly improved the visualization of small size tumors (<10 mm). Furthermore, these tumors were in different liver lobes and, at times, deeply located in the liver tissue with an advanced degree of background fibrosis. It is usually hard to visualize the smaller size and deeply located tumors by B-mode or CUS with high accuracy.

Histologic studies with Evans blue dye solution known to flow freely through capillaries confirmed the hydralazine effects observed by imaging. HCCs showed a drastically reduced level of Evans blue in samples from the animals treated with Evans blue before sacrifice (Figure 3). The tissue samples from the control group that did not receive Hyd showed a large presence of Evans blue in the tumors. The difference in Evans blue between the two groups was statistically significant and consistent with the imaging studies.

The low visibility of HCC with B-mode imaging is related to several factors. One factor is the comparable echo intensity levels of the tumors to that of the surrounding tissue. Our results showed the mean echo intensity of HCC on B-mode images was comparable to the surrounding liver tissue. Literature reports different patterns of internal echoes of HCCs on B-mode imaging varying between 17% and 38% for isoechoic, 12% and 38% for hyperechoic, and 23% and 54% for hypoechoic.42–45 The echoic patterns also depend on the tumor’s size.46 HCCs smaller than 10 mm are isoechoic or hypoechoic, and the number of such low-level echoes increases with cell density. Most tumors in our study were less than 10 mm in size, which fits the isoechoic pattern observed and the low detectability on B-mode. The other two factors related to the low detectability of HCCs are their small size and their deep location in the liver. HCCs with these characteristics are hard to visualize and lack clear demarcations from surrounding liver tissue. Likewise, CUS has lower HCC visibility since most tumors and liver show a comparable peak enhancement, resulting in an unchanged or reduced contrast resolution.

HyCUS offers a new approach for improving HCC detection. It has several advantages. It is an inexpensive and well-tolerated drug that can be safely used in patients.47 This study shows that it allows real-time assessment and characterization of tumors. The hydralazine-related effect lasts long enough to enable better HCC characterization than B-mode and CUS. However, HyCUS use is not without limitations. It involves an additional intervention to CUS. This study demonstrates the effect of hydralazine at a fixed dose in a preclinical model. A ‘standard dose’ of 50 mg/kg is used for lowering blood pressure, and the dosage of 7.5 mg/kg/day is used clinically to treat chronic hypertension in children and adolescents.47 Although the dose of 5 mg/kg is within the realm of its previous use, the results should not be extrapolated to clinical practice without additional dosage-effect and safety studies.

5 |. CONCLUSION

This study demonstrates the use of hydralazine for modulating HCC and liver blood flow to improve lesion visualization. HCC visibility improves substantially with the help of hydralazine compared to B-mode and CUS alone. Future successful validation of the technique may promise physicians greater precision and accuracy in HCC surveillance for early detection of small tumors and better treatment success for cancer patients.

ACKNOWLEDGMENT

The financial support from NIH grants CA204446-01 and EB022612-01 is gratefully acknowledged.

Footnotes

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the evolving nature of the project.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the evolving nature of the project.

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