馮宇，趙霞，呂淑貞，穆穎，王丹丹，李艷萍

(首都醫(yī)科大學附屬北京世紀壇醫(yī)院，北京100038)

表柔比星聯(lián)合多西他賽新輔助化療對乳腺癌患者癌組織Ki-67表達的影響及意義

馮宇，趙霞，呂淑貞，穆穎，王丹丹，李艷萍

(首都醫(yī)科大學附屬北京世紀壇醫(yī)院，北京100038)

目的 探討表柔比星聯(lián)合多西他賽新輔助化療對乳腺癌患者癌組織Ki-67表達的影響及意義。方法 選擇行乳腺癌根治術的乳腺癌患者86例，術前均予表柔比星聯(lián)合多西他賽新輔助化療?；熐凹盎熀蟛捎枚縋CR法及Western blotting法檢測癌組織(化療前取穿刺活檢組織，化療后取手術切除組織)Ki-67 mRNA及蛋白表達。術后隨訪5年，采用Kaplan Meire生存曲線法統(tǒng)計無病生存期(DFS)、總生存期(OS)，采用Cox比例風險回歸模型分析DFS、OS與乳腺癌組織Ki-67表達的關系。結果 化療后乳腺癌組織Ki-67 mRNA及蛋白表達較化療前均降低(P均<0.05)。根據(jù)化療前后Ki-67 mRNA表達的變化量，以下調10%為臨界值，86例患者中Ki-67 mRNA下調≥10%者57例、下調<10%者39例，其DFS分別為36、22個月，OS分別為45、37個月，二者比較P均<0.01。乳腺癌組織Ki-67表達是乳腺癌患者DFS和OS獨立影響因素(HR分別為1.807、1.768，P均<0.05)。結論 表柔比星聯(lián)合多西他賽新輔助化療可下調乳腺癌患者癌組織Ki-67表達，而Ki-67表達與患者預后有關。

乳腺癌；新輔助化療；表柔比星；多西他賽；Ki-67

近年來，我國乳腺癌的發(fā)病率呈逐年升高趨勢，增長速度超過全球平均水平[1，2]。新輔助化療是指在手術治療或放療前進行的系統(tǒng)性細胞毒性藥物治療，是局部晚期乳腺癌的常用治療方法，旨在縮小病灶、提高手術切除率，消除或減少轉移灶，防止遠處轉移，提高保乳率、延長生存期等[3,4]。目前，新輔助化療已應用于ⅡA或ⅡB期乳腺癌患者。Ki-67是增殖細胞核抗原，在多種惡性腫瘤組織中表達異常。本研究探討表柔比星聯(lián)合多西他賽新輔助化療對乳腺癌患者癌組織Ki-67表達的影響及意義。

1 資料與方法

1.1 臨床資料 選擇2009年1月～2012年12月我院收治的乳腺癌患者86例，均行乳腺癌根治術及新輔助化療；年齡28～67歲，中位年齡45歲；有乳腺癌家族病史16例；均有淋巴結轉移，轉移淋巴結<4個45例，≥4個41例；組織學分級：Ⅰ級4例，Ⅱ級60例，Ⅲ級22例；分子亞型：Luminal A型13例，Luminal B型46例，HER2過表達型15例，三陰型12例。納入標準：年齡≥18歲；經(jīng)組織病理檢查確診為乳腺終末導管小葉單元上皮來源惡性腫瘤；腫瘤分期為局部晚期或未達局部晚期但根治術前行新輔助化療者。排除標準：伴發(fā)其他臟器惡性腫瘤者；接受其他化療藥物者；新輔助化療失敗，遠處轉移無法手術者；未采用本化療方案或曾中斷化療者；乳腺癌根治術外采用其他術式者。

1.2 治療方法 患者入院后完善相關檢查，3天后開始新輔助化療，化療方案中包含表柔比星、多西他賽。4例Luminal A型患者給予TE方案：表柔比星75 mg/m2靜滴，多西他賽75 mg/m2靜滴；9例Luminal A型患者給予TEC方案：表柔比星 60 mg/m2靜滴，多西他賽75 mg/m2靜滴，環(huán)磷酰胺500 mg/m2靜滴；Luminal B型及HER-2過表達型患者均給予EC-T方案：表柔比星 75 mg/m2靜滴，環(huán)磷酰胺600 mg/m2靜滴，多西他賽100 mg/m2靜滴；三陰型患者均給予CEF-T方案：表柔比星 75 mg/m2靜滴，環(huán)磷酰胺600 mg/m2靜滴，5-Fu 600 mg/m2靜滴，多西他賽100 mg/m2靜滴。21天為一療程，均治療4個療程。化療前30 min肌注異丙嗪25 mg、苯海拉明20 mg，靜推地塞米松10 mg及5 mg/mL鹽酸托烷司瓊以對抗化療的毒副反應。每2周行乳腺B超觀察腫瘤體積變化，并選擇適宜時機行乳腺癌根治術。

1.3 相關指標觀察

1.3.1 乳腺癌組織 Ki-67表達 ①Ki-67 mRNA表達：采用實時定量PCR法。取化療前后乳腺癌組織標本(化療前取穿刺活檢組織，化療后取手術切除組織)。采用TRIzol試劑盒提取細胞總RNA，紫外分光光度計檢測樣本濃度和純度，經(jīng)逆轉錄試劑盒逆轉錄為cDNA。Ki-67引物序列：上游引物5′-CCAGAACTAACAGACACTGCCACG-3′，下游引物5′-GCCCTCATCACCGCTTGCT-3′；內參照GAPDH引物序列：上游引物5′-AAATCAAGTGGGGCGATGCTG-3′，下游引物5′-GCAGAGATGATGACCCTTTTG-3′。采用PRISM 7500實時定量PCR儀擴增引物。反應體系25 μL，包括SYBR premix(2×)12.5 μL，目的基因上下游引物各0.5 μL，cDNA模板2.0 μL，ddH2O 9.5 μL；反應條件：94 ℃ 4 min，95 ℃ 40 s、60 ℃ 30 s、72 ℃ 30 s，循環(huán)35次，72 ℃ 1 min。瓊脂糖凝膠電泳檢測PCR擴增產(chǎn)物，采用2-ΔΔCt計算Ki-67 mRNA的相對表達量。② Ki-67 蛋白表達：采用Western blotting法。取化療前后乳腺癌組織標本，提取組織蛋白，經(jīng)SDS-PAGE濃縮膠及分離膠電泳，轉膜，5%脫脂奶粉封閉，Ki-67一抗孵育，洗膜3次，二抗孵育，顯色。以GAPDH為內參照，采用Fluorchem軟件分析蛋白條帶，以目的蛋白條帶灰度值與內參條帶灰度值比值表示目的蛋白的相對表達量。

1.3.2 乳腺癌患者無病生存期(DFS)、總生存期(OS) 術后隨訪5年，中位隨訪時間51.5個月，明確患者腫瘤復發(fā)情況及生存狀況。采用Kaplan Meire生存曲線法統(tǒng)計DFS和OS。

2 結果

化療后乳腺癌組織Ki-67 mRNA及蛋白表達均較化療前降低(P均<0.05)，見表1。根據(jù)化療前后Ki-67 mRNA表達的變化量，以下調10%為臨界值，57例Ki-67 mRNA下調≥10%、39例下調<10%，二者DFS分別為36、22個月，OS分別為45、37個月，二者比較P均<0.01。Cox比例風險回歸模型分析顯示，Ki-67表達是乳腺癌患者DFS和OS獨立影響因素(HR分別為1.807、1.768，P均<0.05)。

表1 化療前后乳腺癌組織Ki-67 mRNA及蛋白相對表達量比較

注：與化療前比較，*P<0.05。

3 討論

蒽環(huán)及紫杉類藥物是乳腺癌一線化療藥物，其中蒽環(huán)類藥物作為乳腺癌聯(lián)合化療的基石，幾乎包含在所有的術前化療方案中；紫杉類藥物可使新輔助化療的有效率提高75%～80%。聯(lián)合應用蒽環(huán)及紫杉類藥物可為乳腺癌新輔助化療提供更為可靠的臨床療效。但目前其具體作用機制及靶向因子仍不明確。

惡性腫瘤的發(fā)生、發(fā)展均離不開細胞的異常增殖。增殖細胞核抗原Ki-67是一種與細胞增殖密切相關的核蛋白，是較為明確的與腫瘤細胞增殖相關的因子[5]，與多種腫瘤的轉移及患者預后相關。萬安等[6]研究證實，Ki-67高表達與子宮頸癌局部侵襲及淋巴結轉移相關。在乳腺癌分子分型中，腫瘤組織中Ki-67陽性表達率是Luminal A型和B型的區(qū)分依據(jù)。有研究指出，Ki-67可能是乳腺癌新輔助化療及內分泌治療的分子靶點之一[7]。對宮頸鱗癌的研究發(fā)現(xiàn)，Ki-67表達水平有助于判定腫瘤細胞的生物學行為[8,9]。Ki-67相對低表達的化療患者化療期間毒副反應發(fā)生率降低[10,11]。

本研究結果顯示，表柔比星及紫杉類方案新輔助化療后乳腺癌組織Ki-67 mRNA和蛋白表達較化療前均下降，與多種實體腫瘤中Ki-67的表達情況一致。Ki-67高表達可促進腫瘤增殖及轉移活性，與腫瘤分子分型、淋巴結轉移、腫瘤分化程度以及ER、PR負性表達相關[12]。本研究Ki-67 mRNA下調≥10%者DFS、OS均長于下調<10%者；進一步研究發(fā)現(xiàn)，Ki-67表達是乳腺癌患者DFS和OS的獨立影響因素；提示Ki-67可作為乳腺癌患者預后的預測因子，Ki-67高表達導致腫瘤增殖、侵襲力升高可能是其作用機制[13]。

綜上所述，表柔比星聯(lián)合多西他賽新輔助化療可下調乳腺癌患者癌組織Ki-67表達，而Ki-67表達與患者預后有關。

[1] Gathirua-Mwangi WG, Zollinger TW, Murage MJ, et al. Adult BMI change and risk of Breast Cancer: National Health and Nutrition Examination Survey (NHANES) 2005-2010[J]. Breast Cancer, 2015,22(6):648-656.

[2] Lei F, Kathrin SW, Li JJ, et al. Breast cancer in China[J]. Lancet Oncol, 2014,15(7):e279-e289.

[3] Hahn SY, Ko EY, Han BK, et al. Role of diffusion-weighted imaging as an adjunct to contrast-enhanced breast MRI in evaluating residual breast cancer following neoadjuvant chemotherapy[J]. Eur J Radiol, 2014,83(2):283-288.

[4] Fowble BL, Einck JP, Kim DN, et al. Role of postmastectomy radiation after neoadjuvant chemotherapy in stage II-III breast cancer[J]. Int J Radiat Oncol Biol Phys, 2012,83(2):494-503.

[5] Hafeez F, Neboori HJ, Harigopal M, et al. Is Ki-67 expression prognostic for local relapse in early-stage breast cancer patients treated with breast conservation therapy (BCT)[J]. Int J Radiat Oncol Biol Phys, 2013,87(2):344-348.

[6] 萬安,周穎,曲皖君,等.子宮頸癌中vimentin 和ki-67 蛋白表達及其影響因素分析[J].中華疾病控制雜志,2014,18(9):805-808.

[7] Kennecke H, Yerushalmi R, Woods R, et al. Metastatic behavior of breast cancer subtypes[J]. J Clin Oncol, 2010,28(20):3271-3277.

[8] Inwald EC, Klinkhammer-Schalke M, Hofstdter F, et al. Ki-67 is a prognostic parameter in breast cancer patients: Results of a large population-based cohort of a cancer registry[J]. Breast Cancer Res Treat, 2013,139(2):539-552.

[9] Wei DZ, Ying Z, Beihua K, et al. Snail family proteins in cervical squamous carcinoma: Expression and signicance[J]. Clin Invest Med, 2013,36(4):E223-E233.

[10] Gandini S, Guerrieri-Gonzaga A, Pruneri G, et al. Association of molecular subtypes with Ki-67 changes in untreated breast cancer patients undergoing pre-surgical trials[J]. Ann Oncol, 2014,25(3):618-623.

[11] Romaniuk A, Lyndin M, Sikora V, et al. Histological and immunohistochemical features of medullary breast cancer[J]. Folia Med Cracov, 2015,55(2):41-48.

[12] Liu Y, Yin W, Yan T, et al. The clinical significance of ki-67 as a marker of prognostic value and chemosensitivity prediction in hormone-receptor-positive breast cancer: a meta-analysis of the published literature[J]. Curr Med Res Opin, 2013,29(11):1453-1461.

[13] Matsubara N, Mukai H, Fujii S, et al. Different prognostic significance of Ki-67 change between pre- and post-neoadjuvant chemotherapy in various subtypes of breast cancer[J]. Breast Cancer Res Treat, 2013,137(1):203-212.

Effect of neoadjuvant chemotherapy with docetaxel and epirubicin on Ki-67 expression in patients with breast cancer

FENGYu,ZHAOXia,LYUShuzhen,MUYing,WANGDandan,LIYanping

(BeijingShijitanHospitalAffialtedtoCapitalMedicalUniversity,Beijing100038,China)

Objective To investigate the effect of neoadjuvant chemotherapy with epirubicin and docetaxel on Ki-67 expression in patients with breast cancer and its significance. Methods Eighty-six cases of patients with breast cancer treated with breast cancer radical surgery were selected in this study, epirubicin combined with docetaxel neoadjuvant chemotherapy were carried out before operation. Quantitative PCR and Western blotting were used to detect the expression of Ki-67 mRNA and protein before and after chemotherapy. After 5-year follow-up, we used Kaplan-meire survival curve method to analyze disease-free survival (DFS) and overall survival (OS), used Cox proportional hazard regression model to analyze the relationship between DFS, OS and Ki-67 expression. Results The expression of Ki-67 mRNA and Ki-67 protein in the breast cancer after chemotherapy were lower than that before chemotherapy (allP<0.05). According to Ki-67 mRNA expression changes, patients were further divided into higher change group (down-regulation of Ki-67 mRNA was more than or equal to 10%, 57 cases) and lower change group (Ki-67 mRNA reduced less than 10%, 39 cases). DFS of the two groups (higher and lower changes group respectively) was 36, 22 months and OS was 37, 45 months, and there were significant differences between the two groups (allP<0.01). Ki-67 was the an independent influencing factor for DFS and OS in patients with breast cancer (HR were 1.807, 1.768, allP<0.05). Conclusion Epirubicin and docetaxel neoadjuvant chemotherapy can down-regulate the expression of Ki-67 in tumor tissues of patients with breast cancer, and the high expression of Ki-67 is related to the poor prognosis.

breast carcinoma; neoadjuvant chemotherapy； epirubicin; docetaxel; Ki-67

李艷萍(E-mail： lyp671102@sina.com)

10.3969/j.issn.1002-266X.2016.24.024

R562

B

1002-266X(2016)24-0063-03

2016-01-12)