| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 89 === Angiogenesis is required for growth and progression of solid tumors, and is important for their metastasis. Neovascularization supplies tumors with nutrients and oxygen to maintain their growth, and provides a route for tumor cells to metastasize distantly. There are two phases in tumor development: the prevascular phase and the vascular phase. In the prevascular phase, tumors have limited growth and little or no metastasis. In contrast, in the vascular phase, tumors grow rapidly and have potential for systemic metastasis. Several studies found increased intratumor microvessel density to correlate positively with the incidence of metastasis and negatively with patient survival. Moreover, intratumor microvessel density appears to be an independent prognostic factor in several types of human cancer. In lung cancer, the microvessel count may correlate with tumor stage, and might be an important prognostic indicator of metastasis and death. A number of angiogenic factors up-regulate angiogenesis in physiologic and pathologic conditions, including neoplastic disease. In this investigation, we evaluated VEGF, VEGF isoform, IL-8 expressions in NSCLC, and investigated the relationship between expression of these angiogenic factors and the progression of tumor and outcome of the patients, and to verify the role of these factors in angiogenesis process in NSCLC. Besides, we also studied the impact of mutation of p53 tumor suppressor gene in regulating the expression of these angiogenic factors in NSCLC.
Part I.
Total VEGF mRNA and protein expression correlates with histologic type, tumor angiogenesis, patient survival, and timing of relapse in non-small-cell lung cancer
We have, for the first time, quantified the expression of all four isoforms of vascular endothelial growth factor (VEGF) mRNA in non-small cell lung cancer (NSCLC) using a new kinetic quantitative polymerase chain reaction method, real-time quantitative reverse transcription-PCR (RTQ RT-PCR), and investigated the association between VEGF expression at the mRNA and protein levels and clinicopathologic variables, tumor angiogenesis, patient survival, and timing of relapse. Surgical tumor specimens from 72 NCSLC patients (37 squamous cell carcinomas, 35 adenocarcinomas) were examined. Twenty-eight patients had stage I, 10 stage II, and 34 stage IIIA or IIIB disease. Total VEGF mRNA (all four isoforms) was quantified by RTQ RT-PCR, while VEGF protein expression and microvessel number in tumors were assessed immunohistochemically. VEGF mRNA was detected in all 72 tumor samples (100%) at significantly higher levels than in adjacent normal tissue. Tumoral VEGF mRNA levels correlated strongly with the VEGF protein staining score and microvessel count. Adenocarcinomas showed significantly higher VEGF mRNA expression and a higher protein staining score than squamous cell carcinomas. High tumoral VEGF mRNA expression was associated with advanced (IIIA or IIIB) tumor stage, lymph node metastasis, high tumoral microvessel counts, short patient survival (<24 mo), and early relapse (<12 mo), while a high VEGF protein staining score was associated with high tumoral microvessel counts, short patient survival, and early relapse. Patients with high tumoral levels of both VEGF mRNA and protein had significantly shorter survival and earlier relapse. In multivariate analysis, the VEGF protein staining score and nodal status were the most important independent predictors of survival and recurrence. We conclude that RTQ RT-PCR is a sensitive method for detecting and quantifying VEGF mRNA expression in NSCLC, and that the expression levels of total VEGF mRNA and protein in NSCLC are strongly associated with histologic type, tumor angiogenesis, survival, and timing of relapse. High VEGF expression in adenocarcinomas may, in part, contribute to their greater metastatic potential.
Part II.
Vascular endothelial growth factor (VEGF) 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small cell lung cancer
This study was also to evaluate the correlation between the expression of four different VEGF mRNA isoforms (VEGF121, VEGF165, VEGF 189, and VEGF206) and the clinicopathologic characteristics, tumor angiogenesis, and patients’ outcome in non-small cell lung cancer.
We examined the expression of four different VEGF mRNA isoforms in 57 non-small cell lung cancers using reverse transcription polymerase chain reaction (RT-PCR), and the tumor angiogenesis using immunohistochemical staining.
All 57 lung cancer samples expressed the VEGF121, VEGF165, and VEGF189 mRNA isoforms, and 3 expressed the VEGF206 mRNA isoform. A high tumoral VEGF189 mRNA isoform expression ratio was associated with a high intratumoral microvessel count (p = 0.013), short survival (less than 24 months; p = 0.001), and early postoperative relapse (less than 12 months; p =0.001). Survival and post-operative relapse time were significantly shorter in patients with a high, compared with a low, tumor VEGF189 mRNA isoform expression ratio (p = 0.0001, p=0.0086 respectively, log rank tests). In contrast, the VEGF165 and VEGF 206 mRNA isoform expression ratios showed no statistical correlation with tumor angiogenesis, post-operative relapse time, or survival. A high VEGF121 mRNA isoform expression ratio was associated with short survival (<24 Mo) and early relapse (<12Mo). Multivariate analysis showed that VEGF 189 mRNA isoform expression, microvessel count, and nodal status were the most important independent prognostic factors for patient survival and post-operation recurrence.
The VEGF189 mRNA isoform expression ratio shows a greater correlation with tumor angiogenesis, post-operative relapse time, and survival than do the expression ratios for the VEGF121, VEGF165 and VEGF206 mRNA isoforms, and can be used as a prognostic indicator for patients with non-small cell lung cancers.
Part III.
Interleukin-8 mRNA expression correlates with tumor progression, tumor angiogenesis, patient survival, and timing of relapse in non-small-cell lung cancer
Interleukin-8 (IL-8), a well known chemokine, was recently reported to be an important angiogenic factor in in vitro and in vivo models. In this study, we have, for the first time, evaluated IL-8 mRNA expression in non-small cell lung cancer (NSCLC) using real-time quantitative reverse-transcription (RT) PCR, and correlated tumoral IL-8 mRNA expression with the clinicopathologic characteristics, angiogenesis, and outcome. Surgical tumor specimens from 58 NSCLC patients (29 squamous cell carcinoma and 29 adenocarcinoma) were examined. Twenty patients had stage I, 10 stage II, and 28 stage III disease according to the TMN staging system. IL-8 mRNA expression in tumor and non-tumor lung samples from the same patient was evaluated by real-time quantitative RT-PCR. IL-8 protein expression and microvessel counts in tumors were assessed by immunohistochemical staining. IL-8 mRNA expression was detected in all 58 tumor samples and at significant higher levels than in the adjacent normal tissue (p=0.012). IL-8 protein expression was mainly located in the cytoplasm of cancer cells. High tumoral IL-8 mRNA expression (using the median value for the threshold cycle as the cut-off point) was highly associated with advance tumor stages (p=0.03), distant lymph node metastasis (p=0.02), high tumor microvessel counts (MC>115) (p=0.00003), short survival (<26 mo) (p<0.00001), and early relapse (<16 mo) (p<0.00001). Microvessel counts in the tumor correlated strongly with IL-8 mRNA expression (r=0.56, p<0.001). Patients with high tumoral IL-8 mRNA expression had significantly shorter survival and earlier post-operative relapse than patients with low IL-8 mRNA expression (p<0.0001, log-rank test). Multivariate analysis showed that IL-8 mRNA expression and intratumor microvessel counts were the most important predictors for patient survival and relapse. We conclude that, in NSCLC, IL-8 mRNA expression is strongly associated with tumor progression, tumor angiogenesis, survival, and time of relapse, and that IL-8 mRNA expression can be used as a prognostic indicator in NSCLC.
Part IV.
P53 mutation correlates with expression of vascular endothelial growth factor (VEGF) mRNA, interleukin-8 mRNA and neoangiogenesis in non-small cell lung cancer
This study evaluated the interaction between tumor suppressor gene p53 and the expression of angiogenic factors of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and their effect on tumor angiogenesis in non-small cell lung cancer (NSCLC).
Surgical specimens of 61 NSCLC were included for evaluation of p53 gene mutation, VEGF mRNA and IL-8 mRNA expression, and intratumor microvessel counts. The immunohistochemical method (IHC) was used to stain the nuclear mutated p53 protein accumulation, and to stain the endothelium of microvessels. The VEGF mRNA and IL-8 mRNA expression was quantified using real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). Clinicopathologic variables including surgical-pathologic stages, tumor status, lymph node metastasis, histologic type, age, sex and survival time were also correlated with p53 gene mutation.
The percentage of cells with positive mutated p53 protein expression ranged from 0 to 92%, with a mean value of 39.1% ± 36.7%, and medium value of 15%. The VEGF mRNA expression (-ΔCT) ranged form 2.45 to 14.0, with a mean of 9.44 ± 2.28, while the IL-8 mRNA expression ranged from 5.0 to 15.7, with a mean of 9.6 ± 2.25. Using 15% as cut-off point for mutated p53 protein expression, the tumors with high mutated p53 expression had significantly higher VEGF mRNA expression as compared to tumors with low mutated p53 protein expression (10.57 ± 11.89 vs 8.34 ± 2.04; p<0.0001, independent t-test). The IL-8 mRNA expression (-ΔCT) in tumors with high mutated p53 protein expression was 10.62 ± 2.22, significantly higher than that in tumor with low mutated p53 protein expression (8.66 ± 1.66; p<0.0001, independent t-test). The tumors with high mutated p53 protein expression also had higher microvessel count (151.1 ± 55.7 in 200X field) as compared with tumors with low mutated p53 protein expression (90.9 ± 46.7 in 200Xfield) (p<0.0001, independent t-test). When used as a continuous variable, the percentage of cells with mutated p53 protein expression correlated strongly and positively with VEGF mRNA expression (r=0.68, p<0.0001, linear regression), IL-8 mRNA expression (r=0.71, p<0.0001), and microvessel counts (r=0.64, p<0.0001). The tumor with high mutated p53 protein expression was associated with regional or distant lymph node metastasis (p=0.013), and had a shorter survival time as compared to tumor with low mutated p53 protein expression (18.00 ± 1.51 months vs 40.0 ± 2.13 months; p=0.0115, log-rank test).
We, for the first time, report that p53 mutation was highly correlated positively with both VEGF mRNA and IL-8 mRNA expression in NSCLC. This implied that p53 mutation might play a significant role in regulation of VEGF and IL-8 expression, and take part in controlling the angiogenesis process in NSCLC.
From these serial studies, we can concluded that VEGF, its isoform of VEGF189, and IL-8 expressions were associated with the increased angiogenesis, the progression of tumors, and poor outcome of patients with NSCLC. The tumor suppressor gene p53 might play an important role in the regulation of the expression of these angiogenic factors in NSCLC. The latter also partially explains the association of p53 mutation and the adverse outcome in patients with NSCLC.
|
|---|
