| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 95 === Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It is a hypervascular tumor mainly supplied by hepatic arteries and characterized by a propensity for vascular invasion. HCC is associated with a high metastatic potential. Early post-operative recurrence is a common phenomenon following resection of HCC. To achieve a better outcome after HCC resection, choosing optimal peri-operative management beneficial for each individual patient is of utmost importance. Of course, this depends on the development of an effective method that can predict clinical outcome for an individual patient.
Conventionally, clinical factors, such as vascular invasion (VI), serum α-FP level (AFP), tumor size and tumor number, are used as the possible predictive factors for clinical outcomes after HCC resection; However, they cannot accurately predict the outcome of all HCC patients. A complementary way is to analyze molecular markers for their prognostic significance with reference to cancer recurrence.
Angiogenesis is now recognized as playing a pivotal role in hepatocarcinogenesis and regarded as one of the marker for invasiveness and metastasis in HCC. Tumor angiogenesis is regulated by a variety of pro- and anti-angiogenic factors. Among them, vascular endothelial growth factor(VEGF) has been studied most extensively and is considered to be the most important proangiogenic factor in HCC. The correlation between VEGF expression and the prognosis of HCC, however, remains unclear. Li et al. (1998) and Ng et al. (2001) both reported that expression of VEGF is related to invasion and metastasis of HCC. Yamaguchi et al. (1998), however, found that VEGF expression was lower in advanced HCC with increased tumor vasculatures. These observations suggest that other angiogenic factors may also contribute to angiogenesis stimulation in HCC.
Placenta growth factor (PlGF) is a dimeric glycoprotein, structurally and functionally related to VEGF. PlGF appears to be crucial for pathological angiogenesis in adults and is not produced by the majority of normal human tissues. A prognostic implication of tumor PlGF expression level independent of conventional pathologic prognosticators has been demonstrated in a variety of cancers. To date, PlGF expression in HCC and any correlation between PlGF and prognosis remains unknown. Therefore, in the first part of this thesis, we evaluated the expression of PlGF in post-operative HCC patients following primary resection of the liver to determine if a correlation between PlGF expression and early postoperative recurrence of HCC existed.
Previous reports provided epidemiological evidence that VI has been involved in early intra-hepatic recurrence after hepatectomy for HCC. Treatments focusing on VI such as anti-angiogenesis of HCC are under development. However, the survival advantages of these regimens are uncertain. Identifying the gene expression patterns in HCC specimens with and without VI might help to predict response to these anti-VI treatments.
VI of HCC has been reported to be associated with molecules of various biological functions. Genome-wide gene expression analysis by microarray offers a systematic approach to unfold comprehensive information about the transcription profile of HCC. Previous studies have used microarrays to address the changes in gene expression of HCC. In the second part of this thesis we aimed at seeking a signature associated with VI from the genome-wide expression profile of HCC as generated from microarray study. With this signature, we tried to predict early recurrence after HCC resection. Hopefully, this will provide information for each individual patient to guide the implementation of adjuvant therapy. Thus, early recurrence after HCC resection can be reduced and survival prolonged.
Materials and methods
Placenta growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma
In the first part of this thesis we evaluate the relationship between the expression of PlGF in tumor tissue and clinical outcomes in 71 HCC patients who underwent primary liver resection for HCC at the National Taiwan University Hospital. Tumor PlGF and vascular endothelial growth factor (VEGF)-A and VEGF-C mRNA were analyzed using qRT-PCR. Concentrations of PlGF in tumor tissues were quantified using enzyme linked immunoabsorbant assay, and CD34 was used to evaluation of intratumorous microvessel density. The clinical and pathologic characteristics of the patients in relation to PlGF, VEGF-A and VEGF-C were compared. Overall, cumulative recurrence free survival rates were obtained using the Kaplan–Meier method. The differences in recurrence free survival between the groups were compared using the log-rank test. To evaluate the prognostic influence of PlGF, VEGF-A and VEGF-C in the entire cohort and in subgroups of patients stratified by AJCC stage, the median value in each group was used as the cutoff value for comparison. Pearson Product Moment Correlation was used to show the correlation between PlGF, VEGF levels and degree of vascularization in HCC.
A gene expression profile for vascular invasion can predict the recurrence after resection of hepatocellular carcinoma: A microarray approach
Eighteen patients receiving complete HCC resection were included as a “training group”. Genome-wide gene expression profile was obtained for each tumor using a microarray technique. Datasets were subjected to clustering analysis supervised by the presence or absence of VI to obtain 14 discriminative genes. We then applied those genes to execute pattern recognition using the k-Nearest Neighbor (KNN) classification method, and the best model for this VI gene signature to predict recurrence-free survival in the training group was obtained. The resulting model was then tested in an independent “test group” of 35 patients.
Results
Placenta growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma
Our results demonstrated that no significant differences were identified when evaluating PlGF expression according to the clinicopathologic parameters, but PlGF expression levels correlate significantly with the degree of vascularization in advanced HCC. Patients with PlGF expression levels higher than median tended to have early recurrence compared to patients with PlGF expression lower than median (P = .031). In patients with AJCC stage Ⅱ-Ⅲ disease, this difference was even more significant (P =.002). In contrast, VEGF-A and VEGF-C could not predict early recurrence-free survival.
A gene expression profile for vascular invasion can predict the recurrence after resection of hepatocellular carcinoma: A microarray approach
In the second part of this thesis, a 14-gene profile was extracted which could accurately separate ten patients with VI and eight patients without VI in the “training group”. The expression levels of four human malignancy related genes obtained from cDNA microarray were confirmed by qRT-PCR. Significant correlation was found in 3 of them. In the “test group”, significant difference in disease-free survival was found between patients predicted to have and not to have recurrence (P = .02823). In patients with stage_I disease, this model can also predict outcomes (P = .000205).
Conclusions
Since PlGF expression correlated with early recurrence of HCC, and is not correlated with any clinicopathologic parameters or VEGF expression level. PlGF may be an important prognostic indicator in HCC, especially in advanced HCC. Therefor, PlGF may be used as a postoperative prognostic indicator and may guide selection and administration of adjuvant therapy in HCC patients following the surgical resection of HCC. This study provides evidence that various angiogenesis related factors play different roles in the metastasis of HCC with different backgrounds and stages. High throughput technologies, especially -omic technologies may help to unravel many complex mechanisms associated with the tumor biology of different HCCs. Biomarkers developed through these technique will immense potentials in HCC screening, diagnosis, prognosis and therapeutic targets.
For this reason, in the second part of this thesis we showed that using the 14-gene expression profile extracted from microarrays based on the presence of VI can not only discriminate the recurrence free survival of 35 independent patients as a whole but was also helpful in separating AJCC stage_I patients into groups with different disease-free survival. This implies that negative vessel invasion, as revealed by pathology examination, cannot precisely reflect the behavior of an early HCC. The patients classified as VI positive or at high risk of recurrence should be followed up more closely for recurrence, and they may be the subjects who will benefit from the future effective adjuvant treatment now under development. Also, this strategy for stratification of HCC patients after surgical resection might provide an avenue to more rational clinical trials for HCC post-operative adjuvant therapy in the future. Hopefully, this multigene profile will offer further insights into the extremely complex molecular process of cancer recurrence.
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