胎盤生長因子於口腔鱗狀細胞癌之表現

Expression of Placenta Growth Factor (PlGF) in Oral Squamous Cell Carcinomas

鄭世榮(Shih-Jung Cheng)

指導教授 ： 江俊斌 ； 郭彥彬

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02251

摘要

背景: 胎盤生長因子(Placenta growth factor，PlGF)是屬於血管內皮生長因子家族(VEGF family)。PlGF可藉由VEGFR-1引發腫瘤血管新生，PlGF過度表現與全身很多癌症有關，亦包括口腔癌。之前，我們利用免疫組織化學染色法研究顯示，口腔鱗狀細胞癌 (OSCC) 組織中，有過度PlGF蛋白表現，則OSCC患者之預後較差。但並無文獻提到有關OSCC患者，其癌組織中PlGF mRNA量及血清中PlGF蛋白量，與OSCC患者預後之相關性。因此，本研究進一步檢測OSCC患者癌組織中PlGF mRNA量及血清中PlGF蛋白量，是否和OSCC患者之腫瘤發展、復發及存活有關。

材料及方法
本研究第一部分利用即時定量聚合酶鏈式反應(quantitative real-time reverse transcription-polymerase chain reaction; qRT-PCR)方法，測量63例OSCC癌組織及癌旁正常口腔黏膜 (non-SCC) 中PlGF mRNA量。Threshold cycle (CT) 定義為 DNA可被監測出之最少PCR cycle 數目。CT數值大表示PlGF mRNA copy number少。OSCC及non-SCC組織中，PlGF mRNA的差異表現為 -∆CT = – (OSCC CT – non-OSCC CT) 。-∆CT數值愈大，表示OSCC組織中PlGF mRNA copy number 愈多。ANOVA、卡方檢定(Chi-square test)、Kaplan-Meier 存活率方法及Cox proportional hazard regression model，來分析癌組織中PlGF mRNA量與OSCC患者臨床病理參數及存活率之相關性。本研究第二部分利用酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)，探討72位OSCC患者術前及術後三個月及30位具正常口腔黏膜者，其血清中 PlGF 蛋白量。再利用 ANOVA、卡方檢定、Kaplan-Meier 存活率方法及Cox proportional hazard regression model，來分析血清中 PlGF 蛋白量與OSCC患者臨床病理參數及存活率之相關性，並試圖尋找預測存活時間的獨立預後因子。

結果
本研究第一部分發現，PlGF mRNA -∆CT的平均值較高和患者有較大腫瘤 (P = 0.03)、有局部淋巴結轉移(P = 0.003)、有較高臨床分期 (P = 0.013) 及有局部復發 (P = 0.039) ，有統計學上有意義之相關。以Cox regression model 進行多變數分析發現，淋巴結轉移情形 (P = 0.019)、PlGF mRNA -∆CT value > 2 (P = 0.016)為影響OSCC患者存活時間的獨立預測因子。Kaplan-Meier存活分析發現， PlGF mRNA -∆CT value > 2比PlGF mRNA -∆CT value ≤ 2之OSCC患者，有較差的無復發存活率 (log-rank test, P = 0.017)。
本研究第二部分發現，術前OSCC患者血清中 PlGF蛋白量的平均值，比具正常口腔黏膜者的平均值高，且具統計學上有意義之差別 (19.1 ± 10.7 pg/ml vs. 10.1 ± 4.5 pg/ml，P < 0.001)。術前OSCC患者血清中 PlGF蛋白量平均值較高和患者有較大腫瘤 (P = 0.015)、有局部淋巴結轉移 (P =0.001)、有較高臨床分期 (P = 0.002)及有局部復發(P = 0.037)，有統計學上有意義之相關。Cox regression model 進行多變數分析發現，血清中 PlGF蛋白濃度為影響存活時間的獨立預測因子(P = 0.014)。Kaplan-Meier存活分析發現，血清中 PlGF蛋白 > 19.1 pg/ml比血清中 PlGF蛋白 ≦ 19.1 pg/ml 者，會有較差的無復發存活率 (log-rank test，P = 0.009)。當以血清中 PlGF蛋白為 19.1 pg/ml (正常平均值加2個標準差) 為切點時, 預測腫瘤復發的敏感度 (sensitivity)、特異度 (specificity)及陽性預測值 (positive predictive value) 分別為80%、56% 和 78%。

結論
本研究發現，OSCC患者組織中PlGF mRNA量及血清中 PlGF蛋白量，可以預測OSCC之腫瘤大小、有無局部淋巴結轉移、臨床分期、有無局部復發，及其無復發存活率，因此OSCC患者組織中PlGF mRNA量及血清中 PlGF蛋白量，可以當作OSCC患者預後的指標。

關鍵字

胎盤生長因子 ； 口腔癌 ； 血管新生

並列摘要

Background/Purpose
Placenta growth factor (PlGF) belongs to vascular endothelial growth factor (VEGF) family. PlGF could induce tumor angiogenesis through binding to VEGFR-1, which was highly expressed in a variety of human cancers, including oral cancer. Our previous study demonstrated a significant association of higher expression of PlGF protein with poor prognosis of patients with oral squamous cell carcinoma (OSCC). This study further examined whether the expression of PlGF mRNA in OSCC tissues and the serum PlGF protein level in OSCC patients could be used to predict the progression and prognosis of OSCCs in Taiwan.

Materials and methods
In the first part of this study, we used quantitative real-time reverse transcription-polymerase chain reaction (quantitative RT-PCR) to detect the PlGF mRNA levels in 63 paired OSCC and adjacent normal-looking oral mucosa (non-OSCC) tissues. Threshold cycle (CT) was defined as the PCR cycle number needed to generate a pre-determined amount of DNA (threshold). For a chosen threshold, a smaller starting copy number of mRNA results in a higher CT value. In this study, the relative expression level of tissue PlGF mRNA in each OSCC patient was expressed as -∆CT = – (OSCC CT – non-OSCC CT). Thus, the higher the -∆CT, the greater the copy number of PlGF mRNA in tissues. In the second part of this study, serum samples were obtained from 72 OSCC patients before and 3 months after surgical cancer excision and from 30 normal control subjects. Serum PlGF protein levels were determined by enzyme-linked immunosorbent assay (ELISA).

Results
In the first part of this study, we found that the higher mean PlGF mRNA -∆CT value was significantly associated with OSCCs with larger tumor size (P = 0.03), positive lymph node metastasis (P = 0.003), more advanced clinical stages (P = 0.013) or the presence of loco-regional recurrence (P = 0.039). Positive lymph node metastasis (P = 0.019) and PlGF mRNA -∆CT value > 2 (P = 0.016) were identified as two independent unfavorable prognosis factors by multivariate analyses with Cox regression model. Moreover, Kaplan-Meier curve showed that OSCC patients with a PlGF mRNA -∆CT value > 2 had a significantly poorer recurrence-free survival than those with a PlGF mRNA -∆CT value ≦ 2 (log-rank test, P = 0.017). In the second part of this study, we found that the mean serum PlGF protein levels were significantly higher in pre-surgery OSCC patients than in normal controls (19.1 ± 10.7 pg/ml vs. 10.1 ± 4.5 pg/ml, P < 0.001). Serum PlGF protein levels dropped to near the normal control levels after surgical cancer removal. Higher pre-surgery serum PlGF protein levels were significantly associated with OSCCs with larger tumor size (P = 0.015), positive lymph node metastasis (P = 0.001), more advanced clinical stages (P = 0.002), and loco-regional recurrence (P = 0.037). The serum PlGF protein level was identified as an independent unfavorable prognosis factor by multivariate Cox regression analyses (P = 0.014). Kaplan-Meier curve showed that OSCC patients with a higher serum PlGF protein level had a significantly poorer cumulative recurrence-free survival than those with a lower serum PlGF protein level (log-rank test, P = 0.009). When we used the serum PlGF protein level of 19.1 pg/ml (mean normal control value plus 2 standard deviations) as a cutoff point, the sensitivity, specificity, and positive predictive value for tumor recurrence was 80%, 56% and 78%, respectively.
Conclusion
This study found that PlGF mRNA level in OSCC tissues and serum PlGF protein level in OSCC patients could be used to predict the tumor size, regional lymph node metastasis, clinical stage, and recurrence of OSCCs and the prognosis of OSCC patients. Therefore, we conclude that PlGF mRNA level in OSCC tissues and serum PlGF protein level in OSCC patients may be valuable biomarkers for prediction of progression, recurrence and prognosis of OSCC in Taiwan.

並列關鍵字

placenta growth factor ； oral cancer ； angiogenesis

參考文獻

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