Study of Systemic Therapy for Hepatocellular Carcinoma
Date Issued
2004
Date
2004
Author(s)
Hsu, Chiun
DOI
zh-TW
Abstract
Hepatocellular carcinoma (HCC) is one of the most common cancers in Taiwan. Surgery is curative in only a minority (about 20 to 30%) of patients. Most patients die from locally advanced or metastatic diseases within a relatively short period of time. Systemic chemotherapy is difficult for HCC patients to tolerate because hepatic function is often impaired due to underlying cirrhosis, which is often accompanied by hypersplenism and peripheral cytopenia. New modalities of treatment with a more favorable therapeutic index are needed for patients with advanced HCC.
In this thesis we explore two approaches to the treatment of advanced HCC. The first is anti-angiogenesis therapy. Cancer-induced angiogenesis plays a pivotal role in the development and progression of cancer. Compared with conventional cytotoxic therapy, which targets cancer cells, anti-angiogenesis therapy aiming at the tumor-induced neovasculature has a pharmacokinetic advantage because the tumor vasculature has better drug accessibility than the tumors per se. Besides, the endothelial cells are genetically stable diploid cells, and acquired drug resistance may be rare. HCC is typically a hypervascular tumor. Anti-angiogenesis therapy may thus be a reasonable choice for patients with advanced HCC.
The second is modulation of cellular signal transduction pathways. Cellular signal transduction pathways are involved in the control of cell cycle, cell motility, and apoptosis threshold. Modulators of various cellular signal transduction pathways have been intensively studied for their anti-cancer activity, either alone or in combination with cytotoxic agents, and success has been seen in the treatment of lung and breast cancers and in non-Hodgkin’s lymphoma. In earlier studies we have shown that high-dose tamoxifen may enhance chemotherapy-induced apoptosis of HCC cells by modulating cellular protein kinase C activation. The use of signal transduction modulators in the treatment of advanced HCC is worthy of further evaluation because of the generally favorable toxicity profile of these agents.
Part I. Study of Anti-angiogenesis Therapy for the Treatment of Advanced HCC
HCC tumor cells and their surrounding stroma cells may over-express pro-angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and matrix metalloproteinases. Some of these pro-angiogenic factors are closely associated with high histological grade, portal venous thrombosis, and tumor capsular invasion. Elevated serum levels of angiogenic factors have also been found in patients with liver cirrhosis or HCC. It appears that a pro-angiogenic milieu exists to facilitate tumor proliferation and invasion in HCC and anti-angiogenesis therapy may be able to control this tumor.
Thalidomide was used as a sedative and anti-emetic agent for pregnant women in the 1950’s in Europe, but was soon withdrawn from this indication after the discovery of serious teratotoxicity (amelia and phocomelia) associated with its use. Using a corneal micropocket assay, D’Amato et al demonstrated that angiogenesis induced by VEGF and bFGF can be inhibited by thalidomide. Its potential therapeutic efficacy for neoplastic disorders has been reported in patients with refractory multiple myeloma and human immunodeficiency virus-associated Kaposi’s sarcoma. Preliminary data also suggest beneficial activity of thalidomide in advanced HCC.
We first started a compassionate treatment program for patients with unresectable and not embolizable HCC to analyze the efficacy of thalidomide in the treatment of advanced HCC. Thalidomide was given at a starting dose of 200 mg per day, and the dose was gradually escalated in 100-mg steps up to 600 mg per day if no limiting toxicities developed. Sixty-three of the 68 patients were evaluable for response. One complete and 3 partial responses, defined by WHO criteria, were seen, with a response rate of 6.3 % (95% C.I. 0 to 12.5%). The duration of response was 50+, 24.6, 11.6+ and 8.7+ weeks, respectively. All 4 responders had dramatic decrease of alpha-fetoprotein (a-FP) levels. Another 6 of the 42 patients with elevated pre-treatment a-FP had more than 50% decrease of their a-FP levels. Totally 10 patients had an objective response to thalidomide. The median overall survival for all of the 68 patients was 18.7 weeks (95% C.I. 11.8 to 25.6 weeks) with a 1-year survival rate of 27.6%. The median overall survival of the 10 patients with an objective response to thalidomide was 62.4 weeks (95% C.I. 31.2 to 93.6 weeks). All responders responded at a dose equal to or less than 300 mg per day. Toxicities of thalidomide were generally manageable, and only 16, 6, and 0 patients developed grade 2, 3, and 4 toxicities, respectively.
This study has demonstrated that low-dose thalidomide induces an unequivocal beneficial tumor response in a minority of patients with advanced HCC. Patients with an objective response to thalidomide treatment had improvement in cancer-related symptoms without developing significant treatment-related toxicities. However, if the tumors are refractory to low-dose (200 to 300 mg per day) thalidomide, escalation to higher dose is not beneficial. This relatively dose-independent effect of thalidomide appears to be a common feature of non-cytotoxic biologic agents. In contrast to the relatively dose-independent anti-cancer effect of thalidomide, the treatment-related toxicities of thalidomide, such as somnolence, fatigue, and constipation, are largely dose-dependent. It is intriguing to note that patients with chronic hepatitis C-related HCC appear to be more likely to benefit from thalidomide therapy in this study, although the possibility of false-positive finding resulting from multiple comparisons can not be excluded. Previous studies have compared the clinicopathological features of hepatitis B- and C- related HCC, but the results are inconclusive [35-39]. However, more and more evidence has suggested that HCC associated with hepatitis B and C infection may involve different genetic mechanisms in the regulation of angiogenesis and tumor microenvironment. It remains to be clarified whether these differential mechanisms of angiogenesis and microenvironment regulation may partly account for the differential response to thalidomide between hepatitis B- and C-related HCC.
We then tried to evaluate the feasibility of using power Doppler sonography (PDS) and circulating angiogenic factors (CAF) to monitor the anti-angiogenic effect of thalidomide in HCC. Doppler sonography has the advantage of non-invasiveness and ease to use in serial follow-up. Angiogenesis detected by Doppler sonography has been shown to provide important prognostic information for patients with colon, gastric, ovarian, and cervical cancers. In this study patients with advanced HCC were treated with thalidomide 200-300 mg/day. Tumor vascularity index (VI), evaluated by PDS, and CAF, including VEGF, bFGF, and placental growth factor (PlGF), were measured at baseline and periodically during thalidomide treatment. Fifty-six of the 144 patients with advanced HCC who were screened by PDS were considered evaluable for tumor vascularity. The main reasons for non-evaluability included (1) huge tumor size that could not be encompassed within the field of the sonographic examination; (2) indiscrete tumor margin resulting from diffuse tumor infiltration, severe cirrhosis, or prior local therapy; (3) location of the index tumor that could not be approached by PDS (e.g., lung, retroperitoneal lymph nodes). There was no significant difference between patients whose tumor vascularity was evaulable or not evaluable in terms of age, sex, types of chronic viral infection, disease stage, severity of liver cirrhosis, and survival. Of the 47 patients with bi-dimensionally evaluable disease enrolled in this study, 44 were evaluable for tumor response. Among these, 5 were classified as having objective response, including 1 complete and 1 partial response by WHO criteria, and 3 other patients with decreased a-FP level for more than 50%, together with stable disease for 10.4, 5.3, and 3.5 months, respectively. The pre-treatment median VI was significantly higher in responders (7.42, range: 2.99 to 13.88) than non-responders (2.15, range: 0 to 25.35) (p=0.03). Four of the 5 responders had a significant decrease of VI (45% to 100%) after starting thalidomide treatment. However, none of the CAF was significantly different between responders and non-responders.
This study demonstrated that evaluation of tumor vascularity by power Doppler sonography is feasible in a subset of patients with advanced HCC, and that higher pre-treatment VI level in these patients may predict a better chance of response to thalidomide. Tumor vascularity detected by conventional color Doppler sonography has shown good correlation with vessel density determined by immunohistochemical staining. Preclinical studies have shown the potential of Doppler sonography as a surrogate endpoint in anti-angiogenesis therapy. Power Doppler sonography has the advantages of low noise variance, relative angular independence, and increased dynamic signal range, which make it more sensitive and specific than the conventional Doppler sonography. However, the use of power Doppler sonography for HCC has several limitations. The first is the reproducibility of the techniques. Inter-observer variation, difference in instrument, angle of evaluation, and setting of color gain may make comparison among patients difficult.. The second limitation is the patient population. The application of Doppler sonography was generally successful for patients with small HCC. However, in this study only about 40% of patients with advanced HCC who were otherwise eligible for thalidomide therapy were considered evaluable by power Doppler sonography. Although in this study no significant differences were found in the clinical characteristics between evaluable and non-evaluable patients, screening by power Doppler sonography may impose a selection bias on patients with anti-angiogenic therapy.
The correlation of pre-treatment VI with the angiogenic factors bFGF and PlGF suggests that VI may reflect the activity of tumor angiogenesis. However, a precise correlation between levels of VI or angiogeneic factors and response to thalidomide could not be concluded in this study. Although the median VI before thalidomide treatment was significantly higher in patients who responded to thalidomide treatment, a clear-cut threshold VI value that may predict the 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, decrease in VI after thalidomide treatment was found in un-responsive patients, and one of the 5 patients with objective tumor response even had a paradoxical increase of VI during thalidomide treatment. It has been argued that microvessel density may not represent the true angiogenic activity of tumors. Change in microvessel density reflects the change of ratio of the vascular component of the tumor to its tumor cell component rather than vascular inhibition per se. Besides, the possibility that thalidomide may exert its anti-tumor effect through mechanisms other than angiogenesis inhibition should be considered as a potential explanation for the anomalous VI increase during thalidomide treatment in one of our patients.
Measurement of circulating angiogenic factors was not useful in either prediction or follow-up of patients undergoing thalidomide treatment.
The relationship between tumor response to thalidomide and changes in circulating angiogenic factors has been extensively investigated in patients with multiple myeloma. VEGF, bFGF, TNFa and interleukin-6 have all been found in some but not other series to be associated with tumor response to thalidomide, while the key regulators of tumor angiogenesis remain undetermined. This study did not find any consistent patterns of change of angiogenic factors correlated with tumor response. The clinical implications of elevation of PlGF level after thalidomide therapy and its correlation with VEGF change, regardless of tumor response, are unknown.
Part II. Study of the relationship between chronic hepatitis C infection and angiogenesis in HCC
Previous studies have suggested that chronic HCV infection may be more angiogenic than chronic HBV infection. It is not yet know whether HCV- and HBV-related HCC is different in terms of the extent of angiogenesis and the underlying mechanisms of angiogenesis induction. We first sought to clarify if HCV- and HBV-related HCC may respond differently to thalidomide. We collected the clinical data from 3 clinical trials conducted for patients with advanced HCC in National Taiwan University Hospital and the National Health Research Institute in which thalidomide was given by a similar protocol. One hundred and twelve patients were enrolled. Patients with positive serum HBV surface antigen (HBsAg) and negative anti-HCV antibody were categorized as HBV-related HCC (B-HCC, n=61), while patients with negative HBsAg and positive anti-HCV were categorized as HCV-related HCC (C-HCC, n=33). Response to thalidomide was defined by either a complete or partial response according to WHO criteria or a more than 50% decrease of serum a-FP level for more than 8 weeks for patients with elevated a-FP level at the start of thalidomide therapy. All major clinicopathological features were comparable between the two groups except for age (C-HCC, 67.5 ± 7.6 years; B-HCC, 53.6 ± 13.7 years, p< 0.001). Eight patients with B-HCC and 9 patients with C-HCC responded to thalidomide treatment. The total response rate to thalidomide was 13.1% (95% C.I. 4.4 to 21.8%) for B-HCC and 27.3% (95% C.I. 11.2 to 43.3%) for C-HCC (p=0.09). Patients with C-HCC appeared to have better median time to disease progression (14.1 weeks vs. 8.3 weeks, p=0.03) and overall survival (32.6 weeks vs. 21.4 weeks, p=0.08) than patients with B-HCC. In multivariate analysis, staging (CLIP score), performance status and types of viral infection were independent predictors for overall survival (p<0.01), while only types of viral infection was independent predictor for time to disease progression (p=0.027). There is a trend favoring better anti-tumor activity of thalidomide in patients with C-HCC. Whether this result is related to angiogenesis inhibition remains to be determined.
We then tried to explore the possible mechanisms of angiogenesis induced by chronic hepatitis C infection. HCV genome encodes a single polyprotein. The core protein lies at the N-terminal end and is cleaved by host cell proteases. Core protein has been shown to regulate the expression of multiple viral and cellular genes. It has also been implicated in control of apoptosis and cellular transformation. The direct effect of core protein or other HCV proteins on angiogenesis regulation remains elusive.
A two-chamber co-culture system was used to examine the effects of core protein-expressing cancer cells on endothelial cells and macrophage. Expression of HCV core protein in HeLa cancer cells was done by using a bicistronic retroviral vector. (Dr Lih-Hwa Hwang, Graduate Institute of Microbiology, National Taiwan University College of Medicine). HUVEC (human umbilical vein endothelial cells) were prepared from umbilical cords obtained from nature or caesarean birth. The macrophage cell line THP-1 was kindly provided by Prof Pan-Chyr Yang. The function of endothelial cells and macrophage were evaluated by proliferation assay and cell migration assay. Briefly, HUVEC or macrophage were plated in the inner chamber and HeLa cells transfected with HCV core gene or mock vectors were plated in the outer chamber. A polycarbonate membrane with different pore size and coated with 0.1% gelatin, which is used as a substitute for basement membrane, were placed between the upper and lower well plates. In proliferation assay the diameter of the pores in the bottom of the inner chamber was 0.4 mm, while in migration assay the pore diameter was 8 mm. We found that co-culture with the core protein-expressing HeLa cells (HeLa-C190 cells) caused significant increase of proliferation of both HUVEC and macrophage, whereas co-culture with the vector-containing HeLa cells caused no evident change. Increase of migration ability of HUVEC after co-culture with HeLa-C190 cells was also noted, although the difference was not statistically significant from co-culture with HeLa-S2 cells.
Part III. Study of Modulation of Signal Transduction Pathways for the Treatment of Advanced HCC
Over-expression of HER (human epidermal growth factor -related receptor) has been found in a variety of human cancers, and the clinical and biological significance of HER-related signal transduction pathways have been extensively studied. For example, in many cancers over-expression of HER-2/neu is correlated with aggressive tumor behavior, advanced stages of disease, and a worse outcome. However, the role of HER-2/neu over-expression in HCC remains inconclusive.
HER-2 is a novel target for cancer therapy. A humanized form of anti-HER-2 antibody, trastuzumab (herceptin), exerts significant activity in the treatment of breast cancer expressing high levels of HER-2/neu. Importantly, trastuzumab has a significantly synergistic effect with multiple cytotoxic agents in breast cancer, even in patients who are refractory to previous chemotherapy. Whether the therapeutic efficacy of trastuzumab or other drugs targeting HER-2/neu signal transduction pathways can be extended to other human malignancies is currently under intense investigation.
In the present study we sought to examine the significance of HER-2/neu expression in HCC and explored the possibility of using trastuzumab in the treatment of HCC. Tissue specimens from 36 HCC patients who had been enrolled in 3 separate prospective clinical trials of systemic chemotherapy were studied by immunohistochemical staining. A polyclonal antibody (A0485, DAKO, Copenhagen) against HER-2/neu and a horseradish peroxidase-based visualization system (Envision+, DAKO) was used. Scoring criteria was in accordance with the manufacturer’s guidelines. Twelve HCC cell lines were examined for HER-2/neu over-expression by Western blotting. Single-agent growth regulatory activity of the anti-HER-2/neu antibody, trastuzumab (Genentech, South San Francisco), and its combinative cytotoxicity with chemotherapeutic agents (doxorubicin, gemcitabine, cisplatin, irinotecan) were determined by a tetrazolium-based colorimetric assay (MTT test).
In the immunohistochemical study, all but one of the HCC tumor tissues were negative for HER-2/neu expression. The only patient with positive (1+) HER-2/neu expression was a 57-year-old man who achieved stabilization of disease for 2 months after chemotherapy. Eight of the 35 patients with negative HER-2/neu expression had had partial remission after chemotherapy (p=0.78). In the cell line study, only one (Tong cells) out of the 12 HCC cell lines had a significant level of HER-2/neu expression. However, trastuzumab up to 10 mg/ml had no discernible growth inhibitory or chemosensitizing effect on Tong cells or any other cell lines.
Previous studies of HER-2/neu expression in HCC have yielded discrepant results. Most of these studies did not use the standardized immunohistochemistry or fluorescent in situ hybridization methods. Our study employed a new visualization technique and standardized scoring criteria that are used in the FDA-approved HercepTest to avoid these problems. Although the disease in patients in our study may not represent HCC in general, the very low percentage of HER-2/neu expression in our patients indicates that HER-2/neu overexpression is not an important prognostic factor for patients with advanced or metastatic HCC.
Previous in vitro study demonstrated a correlation between HER-2/neu expression and susceptibility to antibody-induced growth inhibition in breast cancer cells. In contrast, HER-2/neu overexpressing gastric and colon cancer cell lines were resistant to anti-HER-2 antibodies, i.e., the growth inhibitory and chemosensitizing effects of anti-HER-2 regulation may be tumor-specific. Variation of the response of downstream signaling pathways to anti-HER-2 regulation in different cancer cells may explain the differential effects of anti-HER-2/neu antibodies. Our data suggest that HER-2/neu expression in HCC cells may not affect cell growth and chemosensitivity because of the moderate level of expression and possible disparity in downstream signal transduction.
The peroxisome proliferator-activated receptor g (PPARg), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor and plays an important role in several signaling pathways, including lipid metabolism, glucose homeostasis, and inflammation. PPARg dimerizes with retinoid X receptor (RXR) after binding with either PPARg or RXR ligand. Synergistic activation of downstream genes may occur when both ligands are present.
The anti-cancer activity of PPARg agonists was first demonstrated in a liposarcoma model. PPARg agonists, at concentrations of 1 to 10 mM, may also induce growth inhibition in a variety of cancers, including cancers of breast, colon, and prostate. Synergistic or additive effects of growth inhibition between PPARg and RXRa agonists have been found in liposarcoma and breast cancer cells. Recent studies have demonstrated that PPARg agonists may induce cell cycle arrest and growth inhibition in HCC cells at high concentration. The utility of PPARg agonists for the treatment of HCC remained undetermined.
In this study the nuclear expression of PPARg
Subjects
藥物治療
肝細胞癌
血管新生
特殊分子標的治療
targeted therapy
angiogenesis
systemic therapy
thalidomide
hepatocellular carcinoma
SDGs
Type
other
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