台灣乳癌病患人類表皮生長因子受體-2基因多型性及其基因表現之研究

The study of the gene polymorphism and gene expression of human epidermal growth factor receptor-2 in patients with breast cancer in Taiwan

謝佩真(Pei-Chen Hsieh)

指導教授 ： 蔡麗玉

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

乳癌在歐美佔婦女癌症的第一位，為國內女性癌症罹患率之第二位，僅次於子宮頸癌，在過去20年呈現穩定的增加且逐年上升。一般多好發於四十歲以後的婦女，但年輕婦女也可能罹患此症。台灣女性乳癌好發年齡更較國外年輕10歲，平均年齡48歲，預後較差，造成此差異之原因仍不明，值得探討。近十年來，在新抗癌藥物的發展，特別是Herceptin®的使用，大大的提升轉移性乳癌的存活率。許多的研究已經證實人類表皮生長因子受體-2(Human Epidermal growth factor Receptor, 以下簡稱HER2)蛋白質的過度表現與預後較差、存活率低、復發機率較大以及與雌性素接受器(Estrogen receptor, 以下簡稱ER)、黃體激素接受器(Progesterone receptor, 以下簡稱PR)陰性表現有極大的相關性，而HER2的過度表現也意味著病患對於荷爾蒙治療的反應較差。另外，HER2的轉活化也與HER2的基因多型性有關，因此本研究探討乳癌病患其HER2 I655V基因的多型性以及基因的表現，並探討目前臨床上乳癌的危險因子，例如：年齡、ER、PR、腫瘤的分期(Tumor stage)、組織學分級(Histological grade)與HER2 I655V 基因多型性及HER基因表現之間的相關性，希望藉此研究結果以為臨床之應用參考。
本研究採用限制酶片段多型性分析(Polymerase chain reaction - restriction fragment length polymorphism, 以下簡稱PCR-RFLP)分析乳癌病人HER2 I655V基因的多型性；並以兩種不同相對即時定量聚合酵素連鎖反應(relative real-time PCR)及免疫組織染色法(Immunohistochemistry, 以下簡稱IHC)，探討HER2基因的表現。
於HER2 I655V 基因多型性與乳癌的相關性研究中我們收集了424位病患以及395位健康女性的血液檢體進行實驗，結果顯示：在乳癌病患中帶有Ile/Ile基因型者佔80.4％，而Val攜帶者則佔了19.6％；至於健康者帶有Ile/Ile基因型者佔86.3％，而Val攜帶者則佔了13.7％，兩者之差別具有統計上的顯著性意義。另外，若定義發病年齡在45歲為早發性乳癌，將受研究群者分為兩群，再做探討。結果發現早發性乳癌病患其基因型為Val攜帶者的頻率高於年齡大於非早發性乳癌者。至於HER2 I655V 基因多型性與HER2、荷爾蒙接受器的蛋白質表現及腫瘤分期等，則沒有顯著的相關性。因此我們的結論是：HER2 I655V 的基因多型性中若為Val攜帶者在乳癌病患的表現頻率高於健康者，尤其是在早發性乳癌病患中扮演重要的角色。
另外，在臨床常規上HER2蛋白質表現以IHC檢測方法為主，但是此種方法較不精確且費時亦不易標準化，因此，在本實驗中，我們另以兩種不同的相對即時定量PCR方法評估50位乳癌病患HER2 的基因表現，同時將其結果與傳統的IHC的檢測結果做比較。第一種相對定量的方法又稱為A 方法，是先建立標的基因(HER2)及內部參考基因(TATA box-binding protein, 以下簡稱TBP)的外標準曲線，再以此標準曲線計算出乳癌病人其腫瘤組織及正常組織的基因套數(copy number)對比值，此對比值稱為”N1”。 第二種相對定量的方法又稱為B 方法，則是先記錄標的基因(HER2)及內部參考基因(TBP)其即時定量PCR反應擴增曲線的交叉點(crossing point, 以下簡稱Cp)，再以此Cp數值之對比值來計算出在乳癌病人其腫瘤組織及正常組織的Cp對比值，稱為”N2”。此研究結果顯示，檢測HER2基因表現量方面，第二種方法比第一種方法為佳，因第二種相對即時定量PCR的方法檢測HER2基因過度表現的結果與晚期的乳癌病患有極高的相關性，不論是A方法或B方法其與IHC均在Grade III呈有意義之相關；然而B方法在Stage III 呈有意義的陽性率(p = 0.04)，A方法均無統計學上有意義的變化，因此，我們建議採用第二種相對即時定量PCR的方法檢測HER2基因表現，尤其是在晚期的乳癌病患。但是若要應用於臨床上藥物治療的監測，還有相當大的距離，因此，在未來的研究中，期望可使用第二種相對即時定量PCR的方法於刺針型切片檢查中，監控經Herceptin®治療後乳癌病患HER2表現變化，以利臨床上的應用。

關鍵字

人類表皮生長因子受體-2 ； I655V 基因型 ； 相對即時定量聚合?連鎖反應 ； 乳癌 ； 限制?片段多型性分析 ； 黃體激素接受器 ； 雌性素接受器

並列摘要

HER2 plays a major role in breast cancer. Several recent epidemiological studies demonstrated that there was a single missense point mutation at codon 655 of HER2 and this might be associated with breast cancer. Therefore, the aim of this study was to investigate the HER2 I655V genotyping in a case-control study of breast cancer at Kaohsiung in Taiwan. We analyzed the genomic DNA I655V polymorphism with a PCR-RFLP assay in 424 patients with breast cancer and 395 controls. In addition, we also evaluated the relationships of the HER2 genotype to the HER2 protein expression, hormone receptors, and tumor stages in patients with breast cancer. The results showed that the frequencies of the Ile/Ile and Val carrier genotype were 80.4% and 19.6%, respectively, in patients with breast cancer and 86.3% and 13.7%, respectively, in the controls. The patients were classified into two groups according to whether they were defined as patients with an early-onset breast cancer, which is by 45 years of age. After analysis, we found that patients who were younger than 45 years of age had a higher frequency of the Val carrier compared with those over 45 years of age. There were no correlations between the HER2 I655V genotype and either of HER2 and hormone receptor protein status and tumor stages in patients with breast cancer. We conclude that the HER2 I655V genotype especially with a Val carrier plays a strong role in breast cancer at a younger age.
Routinely the IHC method is the most frequently used for the detection of the HER2 gene expression at the protein level, however, the IHC method is more time-consuming and it must be precisely quantified. Therefore, we compared the IHC method with two relative real-time PCR methods to assess the gene expression of HER2. The first method (method A) is based on the external standard curve to calculate the ratio of the copy number of the target gene (HER2) and, that of the reference gene (TBP) in each human breast tumor and adjacent normal tissue (the ratio termed “N1”). The second method (method B) is based on the efficiencies and the Cp deviation of each human breast tumor versus the compared adjacent normal tissue, and then it is expressed in comparison with the reference gene (the ratio termed “N2”). After statistical analysis, we found that the patients with HER2 overexpression in the method B analysis were highly associated with advanced-stage breast cancer. We conclude that method B is very useful for use in the identification of the HER2 gene expression for breast cancer, especially stage III of AJCC.

並列關鍵字

HER2 ； PR ； relative real-time PCR ； I655V genotype ； PCR-RFLP ； Breast cancer ； ER

參考文獻

[1] Breast cancer treatment guidelines for patients. American Cancer Society 2003.

[2] Cancer reference information. American Cancer Society 2004.

[3] Bieche I, Onody P, Laurendeau I, Olivi M, Vidaud D, Lidereau R, et al. Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. Clin Chem 1999; 45:1148-1156.

[4] Frankel AE. New HER2-directed therapies for breast cancer. Commentary re: C. I. Spiridon et al., Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity. Clin. Cancer Res., 8: 1720-1730, 2002. Clin Cancer Res 2002; 8:1699-1701.

[5] Taucher S, Rudas M, Mader RM, Gnant M, Dubsky P, Bachleitner T, et al. Do we need HER-2/neu testing for all patients with primary breast carcinoma? Cancer 2003; 98:2547-2553.