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DOI: 10.1148/radiol.2352040271
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(Radiology 2005;235:509-516.)
© RSNA, 2005

Gastrointestinal Imaging

Effect of Thalidomide in Hepatocellular Carcinoma: Assessment with Power Doppler US and Analysis of Circulating Angiogenic Factors1

Chiun Hsu, MD, PhD, Chiung-Nien Chen, MD, PhD, Li-Tzong Chen, MD, PhD, Chen-Yao Wu, BS, Fon-Jou Hsieh, MD and Ann-Lii Cheng, MD, PhD

1 From the Departments of Oncology (C.H., C.Y.W., A.L.C.), Surgery (C.N.C.), Internal Medicine (C.H., A.L.C.), and Obstetrics and Gynecology (F.J.H.), National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan 100; Graduate Institute of Clinical Medicine (C.H.) and Institute of Toxicology (A.L.C.), National Taiwan University College of Medicine, Taipei, Taiwan; Division of Cancer Research, National Health Research Institutes, Taipei, Taiwan (L.T.C., A.L.C.); and Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan (L.T.C.). Received February 12, 2004; revision requested April 20; revision received June 9; accepted July 21. Supported by grant NHRI-EX90-S829P from National Health Research Institutes, Taiwan, and grant 91–2314-B-002–176 from National Science Council, Taiwan. Address correspondence to A.L.C. (e-mail: andrew@ha.mc.ntu.edu.tw).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To prospectively evaluate the feasibility of using power Doppler ultrasonography (US) and measurement of circulating angiogenic factors to assess the antiangiogenic effect of thalidomide in hepatocellular carcinoma.

MATERIALS AND METHODS: The Ethics Committee of the National Taiwan University Hospital approved the study, and all patients gave prior written informed consent. Evaluation of response to thalidomide treatment was based on findings at computed tomography (CT) and change in serum {alpha}-fetoprotein level. Tumor vascularity index was evaluated with power Doppler US in patients with advanced hepatocellular carcinoma treated with 200–300 mg/d thalidomide. Serum levels of vascular endothelial growth factor, basic fibroblast growth factor, and placental growth factor were measured with enzyme-linked immunoassay. The {chi}2 test or Fisher exact test was used for categorical variables, and the nonparametric Mann-Whitney test was used for numeric variables. A P value of less than .05 was considered to indicate a statistically significant difference.

RESULTS: Of 47 patients enrolled in the study who had disease that was bidimensionally assessable on CT scans, 44 were assessable for tumor response. Of the 44 evaluated, five were classified as showing objective response (responders): One each showed a complete and a partial response according to World Health Organization criteria, and three had a decrease in {alpha}-fetoprotein level by more than 50% and stable disease for 10.4, 5.3, or 3.5 months. The pretreatment vascularity index was significantly higher in responders (median, 7.42; range, 2.99–13.90) than in nonresponders (median, 2.15; range, 0–25.36) (P = .03). Four of five responders had a significant decrease in vascularity index with thalidomide. Serum levels of angiogenic factors did not differ significantly between responders and nonresponders.

CONCLUSION: Higher vascularity index may be associated with a better chance of response to thalidomide in patients with advanced hepatocellular carcinoma. Serum levels of circulating angiogenic factors do not appear to be clinically useful as an indicator of response.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Cancer-induced angiogenesis, first described by Folkman (1) in the 1970s, plays a pivotal role in the development and progression of cancer (2). The high prevalence of cancer-induced angiogenesis makes the latter a logical target for cancer therapy. Compared with conventional cytotoxic therapy targeted at cancer cells, antiangiogenesis therapy aimed at the tumor-induced neovasculature has several theoretical advantages (3). First, the tumor vasculature is pharmacokinetically more homogeneous than the tumor or tumors, which may be located in different parts of the body. Second, the primary target cells, the endothelial cells, are genetically stable diploid cells, and acquired drug resistance may be rare (4). Third, partial damage to the endothelium may be enough to block blood supply to the tumors and result in growth inhibition or even in shrinkage of the tumors.

Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of cancer death in many parts of the world. HCC is characterized by an inherent resistance to all available chemotherapeutic agents. A search for more effective modalities of treatment is mandatory. HCC is a hypervascular tumor. Angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and matrix metalloproteinases, are overexpressed in HCC tumor cells and the surrounding stroma cells. Elevated serum levels of angiogenic factors also have been found in patients with liver cirrhosis or HCC (5,6). It appears that these conditions facilitate tumor proliferation and invasion in HCC. Antiangiogenesis therapy is thus a reasonable choice for control of this cancer.

Thalidomide has been intensively investigated as an antiangiogenesis agent. Using a corneal micropocket assay, D’Amato et al (7) and Kenyon et al (8) demonstrated that angiogenesis induced by VEGF and bFGF can be inhibited by thalidomide. The effectiveness of thalidomide for treatment of neoplastic disorders has been confirmed in patients with refractory multiple myeloma (9) and patients with human immunodeficiency virus–associated Kaposi sarcoma (10). Hsu et al (11) previously showed that thalidomide administered in low dosages (200–300 mg/d) induced objective tumor response in a minority of patients with advanced HCC.

Among the various methods that have been used to estimate the biologic efficacy of antiangiogenic therapy, Doppler ultrasonography (US) has the advantage of noninvasiveness and ease of use for serial follow-up examinations. Angiogenesis detected with Doppler US has been shown to provide important prognostic information for patients with colon (12), gastric (13), ovarian (14), and cervical (15) cancers. Evaluation of tumor vascularity with power (amplitude mode) Doppler US, in which the integrated power of the Doppler signals is displayed with color mapping, has been shown to be superior to conventional color Doppler US, in which the mean Doppler frequency shift is estimated (16,17). The usefulness of power Doppler US for monitoring change in tumor vascularity after antiangiogenic therapy, however, remains unclear.

Circulating angiogenic factors have been shown to provide important prognostic information about a variety of cancers (18). Elevated serum levels of VEGF or bFGF have been associated with increased invasiveness of HCC (19,20). Placental growth factor (PlGF), a homolog of VEGF, has been shown to have a synergistic effect with VEGF in pathologic angiogenesis, such as angiogenesis induced by cancer or ischemia (21), but its prognostic value in human cancers remains unclear. Monitoring of circulating angiogenic factors is a relatively noninvasive and convenient method that is more precise than the semiquantitative evaluation of microvessel density with immunohistochemical study.

In consideration of the foregoing, the purpose of our study was to prospectively evaluate the feasibility of using power Doppler US and serum measurement of circulating angiogenic factors to assess the antiangiogenic effect of thalidomide in HCC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Patient Population
A prospective study of thalidomide for the treatment of advanced HCC was started in April 2000. The diagnosis of HCC was based on histologic and/or clinical findings and on the presence of all of the following criteria: chronic viral hepatitis infection and liver cirrhosis, hepatic tumor with imaging (US, computed tomography [CT]) characteristics compatible with a diagnosis of HCC and without evidence of gastrointestinal or other primary tumor, and a persistent elevation of the serum level of {alpha}-fetoprotein (AFP) to 400 ng/mL or higher (22,23). To be eligible for this study, patients were required to have disease that was bidimensionally assessable on CT scans and that was not suitable for curative surgery or local treatment such as transarterial chemoembolization or percutaneous ethanol injection. Patients were required to have acceptable hepatic and renal function (bilirubin level ≤ 4.0 mg/dL, creatinine level ≤ 2.0 mg/dL). Disease staging was performed according to the TNM Classification System of the American Joint Committee on Cancer (24) and the Cancer of the Liver Italian Program scoring system (25). The patients’ liver function reserves were categorized according to the Child classification system (26). The Ethics Committee of the National Taiwan University Hospital approved the study. All patients provided written informed consent before entry into the study.

Evaluation of Tumor Vascularity with Power Doppler US
Power Doppler US was performed before the start of thalidomide therapy. Patients in whom tumor vascularity was considered assessable with power Doppler US were then recruited into the study. The main reasons for nonassessability included tumor size that exceeded the field of view at US; ill-defined tumor margin as a result of diffuse infiltration, severe cirrhosis, or prior local therapy; and tumor location that was poorly depicted with US (eg, the lung or intraabdominal lymph nodes). The clinical characteristics of patients in whom tumor vascularity was assessable and of patients in whom it was not assessable (eg, age, sex, types of chronic viral infection, disease stage, severity of liver cirrhosis, and survival) were compared to identify possible selection bias.

US examination was performed every 1–2 weeks during treatment. All US examinations were performed by one of the investigators (C.N.C.), by using previously described methods (12,13). The examiner, who had 5 years of experience with the use of power Doppler US at the start of this study, was blinded to other clinical and laboratory data of the patients. A power Doppler US unit (HDI 5000; Advanced Technology Laboratories, Bothell, Wash) with a 2–5-MHz curved-array transducer and a 5–10-MHz broadband linear-array transducer was used. The curved-array transducer was used for evaluation of liver tumors, and the linear-array transducer, for that of superficial tumors such as metastases to lymph nodes. The US examinations were standardized by using a medium wall filter, a color gain of 79%, an ultrasound pulse with a frequency of 1000 Hz and with moderate-to-long persistence, and a slow and steady movement of the transducer to achieve the highest sensitivity without apparent background noise. Focusing depth was set at 1–12 cm, according to the tumor location. Only one representative (index) lesion was chosen for follow-up imaging. We chose the tumor that was most readily visualized with power Doppler US as the index lesion. The index lesion was a liver tumor in 44 patients, a metastasis to a neck lymph node in two patients, and a metastasis to the skin in one patient.

For the examination of liver tumors, routine abdominal US was performed first to identify the index lesion, before power Doppler US. For the examination of superficial tumors, power Doppler US was the only modality used. The tumor was scanned carefully in all directions, and the image of the tumor section with maximal color signals, as determined subjectively by the examiner, was captured and stored for later analysis. Each tumor was scanned three times. After the examination, the previously stored images were retrieved and displayed on the monitor. The contour of the tumor margin was marked with the cursor by the examiner (C.N.C.). Quantification of the vascular color signal in the demarcated tumor area was performed with software (Encomate; Electronic Business Machine, Taipei, Taiwan). The vascularity index was defined as the quotient obtained by dividing the number of colored (vascular) pixels in a well-demarcated tumor area by the number of total pixels in that area, similar to the method used for determining microvessel density at immunohistochemical analysis. For each tumor, the mean of the vascularity indexes from each of three representative tumor images (one vascularity index from each scan) was used for statistical analysis.

Thalidomide Treatment
Oral thalidomide (TTY BioPharm, Taipei, Taiwan) in 50-mg capsules was used. The starting dose of thalidomide was 100 mg twice daily, in the morning and in the evening. The daily thalidomide dose was increased in 100-mg increments every 2–3 weeks if the patient did not show objective response and did not experience treatment-related toxic effects of grade 2 or higher. Toxic effects of thalidomide were evaluated according to the common toxicity criteria of the National Cancer Institute (27). Thalidomide treatment was discontinued if the patient developed any hematologic or nonhematologic toxic effects of grade 3 or higher or showed any signs of hepatic decompensation due to tumor progression. Patients also could withdraw from thalidomide treatment if they could not subjectively tolerate the side effects.

Follow-up Assessment
During thalidomide treatment, patients were followed up every 1–2 weeks with routine updating of the medical history and with physical examination. A complete blood cell count, liver and kidney function tests, and serum AFP test were performed at least every 2 weeks. Patients who completed at least 1 month of thalidomide treatment were considered assessable for treatment response. The evaluation of response to thalidomide treatment was based on CT and clinical findings. World Health Organization criteria were used to evaluate the imaging response (28). Briefly, complete response was defined as the complete disappearance of all clinically detectable lesions for a minimum of 4 weeks. Partial response was defined as a decrease of 50% or more in the sum of the products of perpendicular diameters of all measurable lesions for a minimum of 4 weeks. Stable disease was defined as a decrease of less than 50% or an increase of less than 25% in the sum of the products of perpendicular diameters of all measurable lesions, with no development of new lesions for at least 4 weeks. Progressive disease was defined as the occurrence of new lesions or increase of 25% or more in the sum of the areas of baseline tumor measurement. The patient was considered to have experienced clinical benefit if all of the following criteria were met: stable disease at CT, decrease of more than 50% in the serum AFP level for more than 4 weeks, and improvement in Karnofsky performance score by 10 points or more (29).

CT Examination and Image Analysis
CT was performed before the start of thalidomide therapy, 1 month after the start of therapy, and every 2 months thereafter. Three different CT scanners were used in this study (HiSpeed Advantage, GE Medical Systems, Milwaukee, Wis; PQ 6000, Picker International, Highland Heights, Ohio; Evolution XP [C150], Imatron, South San Francisco, Calif). Unenhanced CT was performed first, followed by contrast-enhanced CT in arterial and portal venous phases. Contiguous 5-mm-thick sections were obtained with a pitch of 1.5. For each examination, about 100 mL of a nonionic contrast material containing 370 mg of iodine per milliliter (iopromide, Ultravist 370; Schering, Berlin, Germany) was injected intravenously at a rate of 2–3 mL/sec.

Hard copies of CT scans were interpreted by a diagnostic radiology team at National Taiwan University Hospital. Each of the radiologists had at least 5 years of experience in interpreting CT scans. The CT scans also were reviewed by the attending physicians. Disagreement in interpretation was minimal and was resolved with consensus.

Serum Collection and Measurement of Circulating Angiogenic Factors
The patient’s blood was sampled before the start of thalidomide treatment and every 2 weeks during thalidomide treatment. Five milliliters of blood was drawn each time and placed in a labeled vacuum-sealed glass tube. The samples were chilled to 4°C and centrifuged (2000g, 20 minutes at 4°C) as soon as possible. Serum samples were then divided into aliquots of 500 µL, transferred via pipette to labeled polypropylene tubes, and stored at –70°C until further analysis. Levels of VEGF, bFGF, and PlGF were determined by using an enzyme-linked immunoassay kit (Quantikine; R&D System, Minneapolis, Minn) according to the manufacturer’s guidelines. The enzyme-linked immunoassay was performed by one of the authors (C.H.).

Statistical Analysis
The clinical characteristics of patients with an objective tumor response were compared with those of patients without an objective tumor response by using statistical software (SPSS, version 10.0; SPSS, Chicago, Ill). For categorical variables such as sex, types of viral infections, and staging (according to the Cancer of the Liver Italian Program scoring system), a {chi}2 test or Fisher exact test was used. For numeric variables such as age and levels of AFP and of angiogenic factors, the nonparametric Mann-Whitney test was used. The Spearman correlation coefficient was calculated to evaluate the correlation between the vascularity index and levels of angiogenic factors. A P value of less than .05 was considered to indicate a statistically significant difference. Overall survival was calculated with the Kaplan-Meier method from the date of start of thalidomide therapy to the date of death or last follow-up.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Screening with Power Doppler US
From April 2000 to March 2003, 144 patients with advanced HCC met our eligibility criteria and underwent power Doppler US examination. Tumor vascularity was assessable in only 56 of these patients. There was no significant difference (P > .05) in age, sex, type of chronic viral infection, disease stage, severity of liver cirrhosis, or survival between patients in whom tumor vascularity was assessable and those in whom it was not assessable.

Clinical Response of Patients
From April 2000 to March 2003, 47 of the 56 patients with assessable tumor vascularity were enrolled in this study. There were 38 men and nine women, and the median age was 62.2 years. HCC in 24 patients (51%) was diagnosed at histologic analysis, and HCC in the other 23 patients (49%) was clinically diagnosed. Tumors in 44 of the 47 patients were assessable for response to thalidomide treatment. All 47 patients were evaluated for toxic effects of treatment. Three of 47 did not complete 1 month of treatment because of subjective intolerance of side effects, mainly fatigue. On the basis of CT findings, there was a complete response in one patient and a partial response in another patient. The CT finding of response in these patients was supported by a decrease of more than 50% in AFP level and an improvement in Karnofsky performance status by 10 points or more. Three other patients, in whom CT findings indicated stable disease, also had a decrease of more than 50% in AFP level and an improvement in Karnofsky performance status by 10 points or more after thalidomide treatment and thus fulfilled the criteria for demonstration of clinical benefit. These five of 44 patients were considered to demonstrate objective tumor response to thalidomide (response rate, 11%; 95% confidence interval: 1.6%, 21.2%). Among the other 39 patients, 15 had stable disease and 24 had progressive disease.

All patients with an objective tumor response (responders) had received thalidomide at a dosage of 200–300 mg/d. Escalation of dosage in patients without objective tumor response (nonresponders) did not affect further response. The clinical characteristics of responders (n = 5) and nonresponders (n = 39) are compared in Table 1. Toxic effects of thalidomide were generally mild. Seven patients experienced toxic effects of grade 3 or 4, which included effects in the liver (three patients) and skin (one patient), as well as leukopenia, anemia, and constipation (one patient each). Common toxic effects of grade 1 or 2 included skin effects (24 patients), constipation (19 patients), fatigue (13 patients), dizziness (11 patients), and stomatitis (seven patients). Six of the 47 patients stopped thalidomide therapy because of subjective intolerance to side effects, mainly fatigue, and the other patients stopped thalidomide therapy after documentation of disease progression. Forty of the 47 patients died of progressive disease during the study period. As of October 31, 2003, the median follow-up of patients was 20.0 months (95% confidence interval: 10.0, 30.0) and median overall survival was 4.3 months (95% confidence interval: 2.7, 5.9).


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  		TABLE 1. Clinical Characteristics of 44 Patients with HCC

 
Evaluation of Vascularity Index and Circulating Angiogenic Factors
In all 44 patients who were assessable for tumor response, baseline data for vascularity index were obtained at power Doppler US prior to thalidomide treatment. The tumors in three of these patients, however, were nonassessable for vascularity index at follow-up examination for determination of response (ie, after 4 weeks of thalidomide therapy); all three patients had progressive disease. The tumor margins in these patients were ill defined because of progressive diffuse tumor infiltration, which rendered the evaluation of tumor vascularity with power Doppler US unfeasible at the time of response assessment. Serum samples obtained at the start of thalidomide therapy and at follow-up assessment were available for the determination of response to treatment in 37 patients. A comparison of serum values for determination of response was not possible in seven of the 44 patients because of technical difficulty in obtaining enough blood for analysis.

Pretreatment (baseline) values.—The median pretreatment value of the vascularity index was 2.73 (range, 0–25.36). The median pretreatment serum levels of VEGF, bFGF, and PlGF were 75.32 pg/mL (range, 38.71–208.92 pg/mL), 25.01 pg/mL (range, 19.57–36.07 pg/mL), and 43.17 pg/mL (range, 26.90–109.46 pg/mL), respectively. The pretreatment vascularity index was significantly correlated with bFGF and PlGF levels (Table 2). Pretreatment bFGF levels were also significantly correlated with VEGF and PlGF levels.


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  		TABLE 2. Correlation of Pretreatment Vascularity Index and Levels of Circulating Angiogenic Factors

 
Comparisons between responders and nonresponders.—Table 3 shows the baseline values for vascularity index and levels of circulating VEGF, bFGF, and PlGF measured in patients with objective tumor response to thalidomide versus those without objective response. Responders had a significantly higher pretreatment vascularity index than did nonresponders (median, 7.42 vs 2.15; P = .03), while the levels of VEGF, bFGF, and PlGF were not significantly different between the two groups of patients. At the time of documentation of tumor response, four of the five responders had a decrease in vascularity index (Fig 1). However, 20 of the 36 nonresponders also had a decrease in vascularity index. As for change in circulating levels of angiogenic factors, most patients had an increase in the level of PlGF after thalidomide therapy, without consistent changes in the levels of VEGF and bFGF (Fig 1). The clinical characteristics of the five patients with objective tumor response to thalidomide are summarized in Table 4.


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  		TABLE 3. Comparison of Pretreatment Vascularity Index and Levels of Circulating Angiogenic Factors Measured in Responders versus Nonresponders

 


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  		Figure 1. Graphs show percent change in vascularity index (VI) and in levels of circulating angiogenic factors after thalidomide treatment for nonresponders versus responders. Four of five patients with response had a decrease in vascularity index, but 20 of 36 patients without response to thalidomide also had a decrease in vascularity index. Most patients had an increased level of PlGF after thalidomide therapy, without consistent changes in levels of VEGF and bFGF.

 

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  		TABLE 4. Clinical Characteristics of Patients with Objective Tumor Response to Thalidomide

 
Serial Follow-up in an Illustrative Case
The findings at CT and Doppler US imaging and the AFP levels measured during serial follow-up in a patient with objective partial tumor response to thalidomide (patient B, Table 4) are shown in Figures 2 and 3. This patient underwent atypical hepatectomy for HCC 42 months before the start of thalidomide therapy. He previously had undergone transarterial chemoembolization three times because of disease progression. Multifocal recurrence of HCC in the liver was subsequently diagnosed, and oral thalidomide treatment was initiated at a dosage of 200 mg/d. Two weeks after the start of treatment, a decline in the vascularity index was noted. A parallel reduction in the serum AFP level was noted at the same time. The vascularity index remained low until thalidomide therapy was discontinued because of unacceptable lethargy, on day 126. The serum AFP level then gradually increased. A CT scan on day 184 showed disease progression.



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  		Figure 2a. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 2b. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 2c. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 2d. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 2e. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 2f. (a, c, e) Serial CT scans and (b, d, f) power Doppler US images in patient with partial response to thalidomide show HCC (a, b) before treatment, (c, d) at day 27 of treatment, and (e, f) at day 97 of treatment. Imaging level was varied slightly between CT examinations to depict the maximal dimensions of the tumor. Arrows in b, d, and f indicate the vascular signal.

 


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  		Figure 3. Plot shows approximately parallel trends in serum AFP level and vascularity index values obtained at baseline and at various follow-up intervals in patient with partial tumor response to thalidomide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results of this study demonstrate that evaluation of tumor vascularity with power Doppler US is feasible in a subset of patients with advanced HCC and that a higher pretreatment vascularity index in these patients may predict a better chance of response to thalidomide. Measurement of circulating angiogenic factors, on the other hand, was not useful either for prediction of therapy response or for follow-up of patients undergoing thalidomide treatment.

Several imaging methods have been used to estimate cancer-induced angiogenesis (30,31). Tumor vascularity at conventional color Doppler US correlates well with vessel density determined with immunohistochemical staining (12–15,32). Preclinical studies have shown the potential of follow-up examination with Doppler US as a surrogate end point in antiangiogenesis therapy (33,34). Power Doppler US has the advantages of low noise variance, relative angular independence, and increased dynamic signal range, which make it more sensitive and specific than conventional Doppler US (16,17,35). Therefore, power Doppler US may provide a convenient real-time method for observing the biologic effects of novel agents targeted at tumor vasculature. New US techniques such as contrast-enhanced US and tissue harmonic imaging have been shown to improve the sensitivity and resolution of conventional US (36–39). The potential of these new techniques for use in monitoring antiangiogenic therapy for HCC should be further investigated.

The results of correlation of the pretreatment vascularity index with the serum levels of angiogenic factors bFGF and PlGF suggest that the vascularity index may reflect the activity of tumor angiogenesis. A precise correlation between vascularity index, serum levels of angiogenic factors, and response to thalidomide could not, however, be performed in this study. Although the median vascularity index before thalidomide treatment was significantly higher in patients who responded to thalidomide treatment, a clear-cut threshold vascularity index value that may be used to predict response to treatment in the majority of patients was not found. This may partly be due to the relatively small sample size of the study and the low response rate of HCC to thalidomide. Further, a decrease in the vascularity index after thalidomide treatment was found in nonresponsive tumors, and one of the five patients with objective tumor response even had a paradoxical increase in vascularity index during thalidomide treatment. It has been argued that microvessel density may not represent the true angiogenic activity of tumors. Change in microvessel density reflects change in the ratio of the vascular component of the tumor to its tumor cell component, rather than vascular inhibition per se (40). Besides, the possibility that thalidomide may exert its antitumoral effect through mechanisms other than angiogenesis inhibition should be considered as a potential explanation for the anomalous vascularity index increase during thalidomide treatment in one of our patients. For instance, thalidomide has been shown to inhibit the activity of tumor necrosis factor {alpha} by enhancing the messenger RNA degradation (41) and to stimulate cytotoxic T cells to produce interferon {gamma} and interleukin-2 (42). These possibilities remain to be clarified.

Our study had several limitations. The first was the reproducibility of the techniques. In this study, all of the US examinations were performed by the same examiner and with the same US unit. The same transducer and the same field of view were used at each examination in each patient. Many investigators suggested individual calibration of the color gain setting to obtain optimal results. We did this in our previous studies and found that a setting of 78% or 79% appeared not to cause significant problems with regard to noise control (12,13). Therefore, a single setting of color gain was used in this study to minimize variability between patients and examinations.

The second limitation is the patient population. The application of Doppler US generally has been successful in patients with small HCC tumors (35–37). In this study, however, only about 40% of patients with advanced HCC who were otherwise eligible for thalidomide therapy were considered assessable with power Doppler US. The vascularity index cannot be calculated in patients with a very large tumor or diffuse tumor infiltration in the liver because tumor contours in these patients cannot be defined with US. Tumors in patients who have undergone transarterial chemoembolization also may not be assessable because of interference from the retained embolization material with US depiction of the tumor vasculature. Metastatic tumors in the lung or retroperitoneal lymph nodes are not visualizable with US. Although in this study no significant differences in clinical characteristics were found in comparisons between assessable and nonassessable patients, screening with power Doppler US may introduce a selection bias against patients with antiangiogenic therapy.

The relationship between tumor response to thalidomide and changes in circulating angiogenic factors has been extensively investigated in patients with multiple myeloma (43–47). Levels of VEGF, bFGF, tumor necrosis factor {alpha}, and interleukin-6 have been found in some studies to be associated with tumor response to thalidomide, while the key regulators of tumor angiogenesis remain undetermined. In a phase II trial of thalidomide for patients with recurrent high-grade glioma, patients whose serum bFGF decreased after thalidomide treatment had the longest survival and time to disease progression, whereas patients with an increase in bFGF level fared the worst (48). Most of the angiogenic factors are proteins secreted by the tumor or the stromal cells, and, in theory, the levels of the angiogenic factors may reflect the angiogenic activity of the tumors. In this study, however, we did not find any consistent pattern of change in levels of angiogenic factors that was correlated with tumor response. The clinical implications of an increase in PlGF level after thalidomide therapy and its correlation with VEGF change, regardless of tumor response, are unknown.

In conclusion, our data suggest that evaluation of tumor vascularity with power Doppler US is feasible in a subset of patients with advanced HCC. In this group of patients, a higher vascularity index may be associated with a better chance of response to thalidomide. Further studies in a larger group of patients and with more specific antiangiogenesis agents are needed.


   ACKNOWLEDGMENTS
 
The authors thank Fu-Chin Chen, BS, for technical assistance in the processing of US data.


   FOOTNOTES
 
Abbreviations: AFP = {alpha}-fetoprotein, bFGF = basic fibroblast growth factor, HCC = hepatocellular carcinoma, PlGF = placental growth factor, VEGF = vascular endothelial growth factor

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, C.H., C.N.C., A.L.C.; study concepts and design, all authors; literature research, C.H., C.N.C., L.T.C.; clinical studies, C.H., C.N.C., L.T.C., C.Y.W.; experimental studies, C.H., C.N.C.; data acquisition and analysis/interpretation, all authors; statistical analysis, C.H., C.Y.W.; manuscript preparation and definition of intellectual content, all authors; manuscript editing, C.H., A.L.C.; manuscript revision/review and final version approval, all authors

C.H. and C.N.C. contributed equally to this work.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
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